Loss of anchorage primarily induces non-apoptotic cell death in a human mammary epithelial cell line under atypical focal adhesion kinase signaling.

Anchorage dependence of cellular growth and survival prevents inappropriate cell growth or survival in ectopic environments, and serves as a potential barrier to metastasis of cancer cells. Therefore, obtaining a better understanding of anchorage-dependent responses in normal cells is the first step to understand and impede anchorage independence of growth and survival in cancer cells and finally to eradicate cancer cells during metastasis. Anoikis, a type of apoptosis specifically induced by lack of appropriate cell-extracellular matrix adhesion, has been established as the dominant response of normal epithelial cells to anchorage loss. For example, under detached conditions, the untransformed mammary epithelial cell (MEC) line MCF-10 A, which exhibits myoepithelial characteristics, underwent anoikis dependent on classical ERK signaling. On the other hand, recent studies have revealed a variety of phenotypes resulting in cell death modalities distinct from anoikis, such as autophagy, necrosis, and cornification, in detached epithelial cells. In the present study, we characterized detachment-induced cell death (DICD) in primary human MECs immortalized with hTERT ((Tert)HMECs), which are bipotent progenitor-like cells with a differentiating phenotype to luminal cells. In contrast to MCF-10 A cells, apoptosis was not observed in detached (Tert)HMECs; instead, non-apoptotic cell death marked by features of entosis, cornification, and necrosis was observed along with downregulation of focal adhesion kinase (FAK) signaling. Cell death was overcome by anchorage-independent activities of FAK but not PI3K/AKT, SRC, and MEK/ERK, suggesting critical roles of atypical FAK signaling pathways in the regulation of non-apoptotic cell death. Further analysis revealed an important role of TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand) as a mediator of FAK signaling in regulation of entosis and necrosis and a role of p38 MAPK in the induction of necrosis. Overall, the present study highlighted outstanding cell subtype or differentiation stage specificity in cell death phenotypes induced upon anchorage loss in human MECs.

Oral treatment with probiotic Lactobacillus johnsonii NCC533 (La1) for a specific part of the weaning period prevents the development of atopic dermatitis induced after maturation in model mice, NC/Nga.

BACKGROUND: The inhibitory effect of probiotic bacteria on atopic dermatitis has been shown in human infants, but the mechanism is still unclear. OBJECTIVE: This study aimed to show the effects of the administration of a probiotic during the weaning period in mouse models on production of the intestinal secretory IgA (sIgA) and on the development of atopic dermatitis (AD) in later life. METHODS: The effects of the administration of Lactobacillus johnsonii NCC533 (La1) during weaning were evaluated using a mouse model (Balb/c). The weaning period of mice was divided into three phases according to the evolution of faecal IgA. La1 was administered in each phase and the evolution of the faecal IgA was estimated. In the next experiment, the effect of the administration of La1 in phase 2 on host immunity after maturation was further assessed by using the model NC/Nga mouse for human AD. RESULTS: Administration of La1 in each phase showed a distinct effect on the production of sIgA. But sIgA production was only positively stimulated when La1 was administrated in phase 2. The development of AD induced by mite antigen from 6 weeks old was significantly prevented by the primary administration of La1 in phase 2. AD-like lesions were significantly milder than those of the control mice, and histological observations showed an almost normal appearance of the epidermis and upper dermis of the mice treated with La1. CONCLUSION: This study suggested that the primary administration of La1 in a specific part of the weaning period is effective in preventing or inhibiting the development of AD after maturation by modulating or accelerating the gut immune response.

Cell preparation of Enterococcus faecalis strain EC-12 prevents vancomycin-resistant enterococci colonization in the cecum of newly hatched chicks.

The use of antimicrobials in broilers is considered to be a cause of the appearance of vancomycin-resistant enterococci (VRE). Once VRE penetration occurs, whatever its origin, it is difficult to expel the enterococci from the intestine because of their multiple resistance, whether natural or acquired. In this study, we evaluated the prevention of VRE colonization by the dietary supplementation of a cell-wall preparation of Enterococcus faecalis strain EC-12 (EC-12) in newly hatched broilers that were challenged by experimental infection with VRE. The chicks were fed a basal diet supplemented with 0.05% (wt/wt) EC-12 powder for 15 d. The control group and that administered Lactobacillus sp. were fed the basal diet. The VRE challenge was administered orally when the chicks were 2 d old (d 0). Dietary EC-12 reduced VRE colonization in the intestine from d 3 to 14. Total IgA in the cecal digesta and total IgG in the serum were higher on d 14 in the EC-12 treatment group. However, VRE-specific and EC-12-specific antibodies were not affected in serum. Hence, it appeared that dietary EC-12 stimulated the gut immune system and reinforced the immune reaction against the VRE challenge to accelerate its defecation from the chick intestine.

Novel distance dependence of diffusion constants in hyaluronan aqueous solution resulting from its characteristic nano-microstructure.

Material transports in hyaluronan (HA) aqueous solution were investigated applying two different techniques, i.e., pulsed field gradient NMR (PFG-NMR) and photochemical quenching, to the measurement of diffusion constants to show a sharp contrast resulting from the difference of the spectroscopic observation time while the same probe molecules were commonly used in two experiments. The value from PFG-NMR reflects the relatively long transport along which the majority of the molecules are retarded by the mesh structure of HA solution. In such inhomogeneous fluids, the observable diffusion constant should generally depend on the observation time and, i.e., the averaged distance of diffusion. Quantitative discussion, which compares the obtained characteristic distance of diffusion with the pore size, clarifies the role of the nano-microstructure of HA solution forming small pores surrounded by the polymer chain networks.

Lactobacillus casei strain Shirota-fermented milk stimulates indigenous Lactobacilli in the pig intestine.

The aim of this study was to determine the effect of a probiotic, i.e. fermented milk prepared with Lactobacillus casei strain Shirota, on indigenous Lactobacilli in the pig large intestine. This fermented milk was given as a probiotic to experimental pigs for 2 weeks. The fecal organic acid concentration increased with the fermented milk; acetate and propionate increased significantly (p<0.05). At the same time, lactate and butyrate tended to increase. The fecal pH was significantly reduced by the fermented milk (p<0.05). Although the number of bacteria of strain Shirota in the intestinal contents was much smaller than those of indigenous Lactobacilli, 10(4) vs 10(8) (cfu/g), the numbers of indigenous Lactobacilli and Bifidobacteria in the pig intestine appeared to increase with the fermented milk. In addition, the phenotypic diversity (phenotypic group numbers) of indigenous Lactobacilli increased from 3 to 8 with the fermented milk supplementation. Thus the fermented milk affected the indigenous Lactobacillus population and constitution.

Strain gauge force transducer and its application in a pig model to evaluate the effect of probiotic on colonic motility.

The aim of this study was to investigate the effect of probiotic, i.e., fermented milk prepared with Lactobacillus casei strain Shirota, on colonic motility by the strain gauge force transducer (SGFT) in a pig model. The contractions of the circular muscle layer of the cecum, upper colon, lower colon, and terminal colon in pigs were directly measured in conscious status by this method. This method was useful for quantitatively evaluating the effects of stimuli on colonic motility. Feeding significantly stimulated the motilities of the upper and lower colon. Defecation significantly stimulated the motilities of the upper and terminal colon. Two weeks' feeding of the fermented milk significantly activated the response to feeding in four portions of the large intestine. It increased motility of the terminal colon that did not promote defecation. The frequency of defecation from 9:00 to 10:00 (the period just after the morning meal) increased significantly, but from 0:00 to 1:00 (the midnight period) it decreased as a result of the ingestion of fermented milk. Such effects of the fermented milk on motility of the terminal colon are discussed in relation to the movement of digesta. The effects may relate to the stimulation of colonic fermentation as shown by a decrease in fecal pH.

Organic acid profiles in feces of pigs with pathogenic or non-pathogenic diarrhea.

A chemical characteristic of the feces of diarrheal piglets permits differentiation among piglets receiving antibiotic treatment and those with colibacillosis or dyspepsia. A high concentration of lactic or succinic acid was observed in the diarrheic feces of piglets receiving antibiotic treatments and those with dyspepsia; however, no lactic or succinic acids were detected in piglets with colibacillosis. There was, however, little difference in the total concentration of organic acids among the three types of diarrheal illnesses. A quantitative analysis of lactic and succinic acids in diarrheic feces might provide a means for rapidly differentiating between colibacillosis and non-pathogenic diarrheas in piglets.

Effects of animal or plant protein diets on cecal fermentation in guinea pigs (Cavia porcellus), rats (Rattus norvegicus) and chicks (Gallus gallus domesticus).

Monogastric herbivores such as the guinea pig depend on energy supply from enteric fermentation as short-chain fatty acids (SCFA) corresponding to 30-40% of their maintenance energy requirements. They evolved specific digestive system to adapt their indigenous microflora to plant polysaccharides fermentation. No information has been available about the adaptability of microbial fermentation in hindgut of the monogastric herbivorous to an animal protein diet. We investigated if the guinea pig can fully retrieve energy of an animal protein diet by hindgut fermentation compared with a plant protein diet. For comparison, we also studied two omnivores. End products of in vitro cecal fermentation (SCFA, ammonia and gases) were measured to judge how well an animal protein diet could be fermented. The animal protein diet resulted in the less intensive fermentation with increased feed intake and volume of cecal contents than the plant protein diet only in guinea pigs. This may be due to a limited capacity of the hindgut microflora to adapt to the substrate rich in animal protein. We also found that chick cecal contents produced methane at higher emission rate than ruminants.

Volatile sulfur production by pig cecal bacteria in batch culture and screening inhibitors of sulfate reducing bacteria.

We studied the effects of specific inhibitors of methanogenesis (2-bromoethane sulfonate, BES) and sulfate reduction (sodium molybdate) on volatile sulfur production in batch cultures of pig cecal bacteria. The volatile sulfur concentration in headspace gas was determined by flame-photometric detector gas chromatography. BES stimulated production of hydrogen sulfide (H2S) and methanethiol, and sodium molybdate completely inhibited the production of these volatile sulfur compounds. The results indicated that dissimilate sulfate reduction is mainly responsible for volatile sulfur production in the hindgut. Therefore the extracts of herbs, food colors, and aroma chemicals were tested for their inhibitory effects on H2S production by a dissimilatory sulfate-reducing bacteria. Desulfovibrio desulfuricans DSM642. H2S was measured by the chromatography of the headspace gas, using a flame photometric detector. Of 306 herbal extracts tested, 69 extracts from 38 herbs inhibited H2S production at 1.0 mg/mL. Sisymbrium officinale (hedge mustard) was the most potent inhibitor. Six pigments inhibited H2S release. Erythrosine and rose bengal showed inhibitory effects at 0.01 mg/mL. Peppermint oil and 96 aroma chemicals were assayed for their effects on H2S release. Thirty-two aroma chemicals suppressed H2S production at 0.1 mg/mL, and camphene, 1-decanol, and 2-nonanone were effective at 0.01 mg/mL.

Probiotic preparations dose-dependently increase net production rates of organic acids and decrease that of ammonia by pig cecal bacteria in batch culture.

We tested probiotic preparations containing Bifidobacterium, Enterococcus, or Lactobacillus to see if they affect production of organic acids and ammonia by mixed cecal bacteria. Four preparations (Bibalance, Neorakuton, Yakult Seichoyaku, or Yakult Seichoyaku BL) were digested with HC and pancreatin before adding to batch cultures of 50% pig cecal contents. Concentrations of organic acids and ammonia in the culture were quantified. Two preparations stimulated the net production of short-chain fatty acids (SCFA) during the first 8 hr of incubation. From 8 to 24 hr of incubation, all preparations accelerated the net production of SCFA, succinic and lactic acids, and three preparations slowed the net production of isovaleric acid and ammonia, all dose-dependently. The above results indicate that the preparations tested accelerated the breakdown of hard-to-degrade carbohydrate(s) and, possibly thereby, decreased the breakdown of proteinous materials or increased bacterial cell body synthesis in a mixed culture of cecal bacteria.

Phylogenetic study of methanogens associated with rumen ciliates.

The phylogeny of methanogenic archaea associated with ciliate protozoa in a sheep rumen was investigated. Ruminal ciliate protozoa were exhaustively washed and mixtures of genomic DNA extracted. Archaea-specific nested PCR amplification was conducted with the ciliate genomic mixture. The resultant small subunit (16S) ribosomal RNA gene (ssu rDNA) was cloned into Escherichia coli JM 109. Many methanogens were still observed on and/or in ciliate cells by fluorescent microscopy even after exhaustive washing with buffer. Partial sequences of ssu rDNA close to Methanobrevibacter smithii were dominant in the retrieved sequences. RFLP analyses on the retrieved sequences revealed the absence of Methanobrevibacter ruminantium in the protozoal preparation. The association of Methanobrevibacter spp. with ruminal ciliate protozoa was demonstrated by the isolation of archaeal ssu rDNA phylogenetically close to that of M. smithii.

Cloning, sequencing, and expression of an endoglucanase gene from the rumen anaerobic fungus Neocallimastix frontalis MCH3.

A cDNA clone encoding an endo-1,4-beta-glucanase from a rumen fungus, Neocallimastix frontalis MCH3, was isolated. The nucleotide sequence showed that the gene, celA, encoded a multidomain enzyme containing a family 5 catalytic domain and a reiterated sequence that is involved in the association of a multienzyme complex, the cellulosome. The enzyme expressed in Escherichia coli showed the highest activity against carboxymethylcellulose at 40 degrees C and pH 8.5.

The role of ciliate protozoa in the lysis of methanogenic archaea in rumen fluid.

Predation by ciliate protozoa can account for 90% of the eubacterial protein turnover in the rumen. However, little is known about the factors affecting the lysis of archaea in rumen fluid. Bacterial lysis was followed from the release of acid-soluble 14C from 14C leucine-labelled bacteria. The rumen methanogen Methanobrevibacter MF1 was broken down more rapidly than other non-ruminal archaea in rumen fluid withdrawn from sheep harbouring either a mixed protozoa population or monofaunated with Polyplastron multivesiculatum or Entodinium spp. The removal of protozoa from the rumen fluid had little effect on the breakdown of Methanobrevibacter, while lysis of the non-methanogenic ruminal bacterium Selenomonas ruminantium decreased by over 70%. Substantial lysis of Methanobrevibacter occurred in cell-free rumen fluid and this effect could be abolished by autoclaving. In view of the high number of bacteriophages in rumen fluid and susceptibility of ruminal bacteria to phage-induced lysis it is tempting to suggest that phages have a role in the lysis of archaea in rumen fluid.

Methane production associated with rumen-ciliated protozoa and its effect on protozoan activity.

Methane production by methanogenic bacteria associated with ciliated protozoa in the rumen, and its effect on the metabolic activity of the protozoa, were measured in vitro. Apparent daily methane emission per protozoan cell ranged from a trace amount to 2 nmol. Enhanced substrate disappearance accompanIed methanogenesis.

Sulphate reduction and methanogenesis in the ovine rumen and porcine caecum: a comparison of two microbial ecosystems.

A series of experiments was conducted to study the relationship between methanogenesis and sulphate reduction in ovine rumen and porcine caecum. Effect of 2-bromoethane sulphonate on hydrogen production by digesta suggested that the most important H2-disposal system in the rumen is methanogenesis and that methanogenesis is not predominant H2-disposal system in the porcine caecum. This inference was supported by the difference in predominant H2-utilizers in these two microbial ecosystems; Methanogenic bacteria (MB) were predominant in the rumen and sulphate reducing bacteria (SRB) were predominant in porcine caecum. Free sulphate levels in digesta appear to affect the relationship between MB and SRB. Sulphate levels in the rumen were likely to be insufficient for SRB to outcompete MB.

Effect of defaunation on protein and fibre digestion in sheep fed on ammonia-treated straw-based diets with or without maize.

Using a defaunating method which preserved bacteria and fungi in the rumen, the effect of protozoa on protein and fibre digestion was studied in six adult wethers in relation to the nature of the diet. Sheep were given daily, 42 g dry matter (DM)/kg metabolic body-weight (W0.75), one of two isonitrogenous diets: one contained ammonia-treated wheat straw as the only energy source (diet S) and the other was supplemented with maize grain pellets (diet SM). Mean daily intakes (g/d) of nitrogen, neutral-detergent fibre and acid-detergent fibre were respectively 22, 573 and 373 for diet S and 23, 450 and 334 for diet SM. Elimination of protozoa increased duodenal non-ammonia-nitrogen flow. This result was mainly due to an increase in microbial protein flow and, to a lesser extent, to a higher dietary protein flow. Defaunation markedly increased the efficiency of microbial protein synthesis. Maize-grain supplementation had a net positive effect on this variable in defaunated sheep, but not in faunated sheep. Cell-wall carbohydrates were less well digested in the defaunated rumen, and the negative effect of defaunation was greatest with the diet SM. Intestinal fibre digestion increased in the defaunated sheep especially in those fed on diet SM, but not enough to compensate for the decrease in rumen digestion.

Role of rumen protozoa in nitrogen digestion in sheep given two isonitrogenous diets.

1. The effect of protozoa on digestion in the rumen was studied using either defaunated or faunated sheep. 2. Six wethers, each fitted with rumen and simple duodenal cannulas, were given two isonitrogenous diets containing either lucerne (Medicago sativa) hay (diet L) or sodium hydroxide-treated wheat straw (diet S). The diets were given in eight equal portions per day at 3-h intervals. The mean intake of dry matter, 53 g/kg body-weight0.75 per d, was similar for the two diets and each diet had a similar digestible organic matter content. Diet L promoted a large protozoal population and was rich in nitrogen sources of low rumen-degradability, while diet S supported a smaller protozoal population and was rich in rumen-degradable N. 3. Digesta flow at the duodenum was estimated by means of a dual-marker technique using chromium-mordanted lucerne hay and polyethylene glycol as markers. The microbial flow at the duodenum was estimated using diaminopimelic acid (DAPA), nucleic-acid purine bases (PB) and 35S incorporation simultaneously. The different microbial markers were compared in the defaunated sheep. Protozoal N contribution was estimated in faunated sheep. 4. Defaunated sheep had lower rumen ammonia concentrations and molar proportions of butyric acid than faunated sheep, but they had higher molar proportions of propionic acid. 5. Rumen organic matter digestion was reduced by defaunation, but this decrease was compensated for by increased intestinal digestion. 6. There was a net increase of N flow (approximately 10 g/d) between mouth and duodenum in defaunated sheep. This was explained by increases in both microbial and dietary N flows from the rumen compared with faunated sheep. 7. The influence of protozoa on solid- and liquid-phase retention times in the rumen is discussed, as well as the protozoal contribution to microbial N flow in the duodenum of faunated sheep.

Determination of assay parameters for RNA analysis in bacterial and duodenal samples by spectrophotometry. Influence of sample treatment and preservation.

A simple analytical procedure derived from that described by Zinn and Owens (1980), based on the determination of nucleic purine bases (RNA eq), was carried out to measure microbial nitrogen flow in the ruminant duodenum. Several procedures for sample preservation were tested; the efficiency of each step of the analytical method was also determined using high performance liquid chromatography (HPLC). The proposed method (RNA eq) was compared with two other methods considered as references (2-6 diaminopimelic acid and 35S incorporation) and microbial nitrogen flow was measured in defaunated sheep. The recovery of purine bases analysed by the Zinn and Owens method was generally good (101% pure bases; 90% when bases were added to bacterial samples; 96% when added to yeast RNA). The HPLC measurements allowed us to conclude that this spectrophotometric method is specific for purine bases, all pyrimidine bases being eliminated. Moreover, it was found that the method must be used on freeze-dried samples; storage at + 4 degrees C, defatting or freezing gave incorrect results. Using the described assay, we observed that microbial nitrogen flow at the duodenum of defaunated sheep was not significantly different from that obtained using more traditional markers such as sulphur-35 incorporation or diaminopimelic acid.