Consumption of partially hydrolysed guar gum stimulates Bifidobacteria and butyrate-producing bacteria in the human large intestine.

Partially hydrolysed guar gum (PHGG) is a water-soluble dietary fibre that is non-digestible in the upper gastrointestinal tract. It is believed that PHGG benefits the health of hosts by altering the colonic microbiota and stimulating short-chain fatty acid (SCFA) production. However, it remains unclear which bacteria ferment PHGG in the human large intestine. In this study, the effect of PHGG on faecal bacteria was analysed to specify the bacteria that contribute to the fermentation of PHGG in the human large intestine. Ten healthy volunteers consumed PHGG (6 g/day) for 2 weeks. Faeces were collected at 2 weeks prior to consumption, at the end of 2 weeks of consumption, and 2 weeks after consumption of PHGG. Bacterial DNA was extracted from these collected faeces and subjected to real-time PCR using bacterial group- or species-specific primers. The copy number of the butyryl-CoA CoA-transferase gene and the 16S rRNA gene copy numbers of Bifidobacterium, the Clostridium coccoides group, the Roseburia/ Eubacterium rectale group, Eubacterium hallii, and butyrate-producing bacterium strain SS2/1 were significantly increased by the intake of PHGG. Other bacteria and bacterial groups were not significantly influenced by the intake of PHGG. It was believed that the Roseburia/E. rectale group bacteria, Bifidobacterium, the lactate-utilising, butyrate-producing bacteria, E. hallii and bacterium strain SS2/1, would contribute to the fermentation of PHGG in the human large intestine. PHGG may benefit health by stimulating Bifidobacterium and butyrate-producing bacteria in the human large intestine.

Short-term exposure to ethanol causes a differential response between nerve growth factor and brain-derived neurotrophic factor ligand/receptor systems in the mouse cerebellum.

Alcohol ingestion affects both neuropsychological and motor functions. We hypothesized that one of the key factors involved in such functions are neurotrophins and their receptors. We have therefore examined the effects of short-term ethanol exposure on the mRNA expression and protein levels of neurotrophin ligands and receptors in the cerebellum using real-time RT-PCR and Western blotting techniques. Male BALB/C mice were fed a liquid diet containing 5% (v/v) ethanol. The pair-fed control mice were fed an identical liquid diet except that sucrose was substituted isocalorically for ethanol. The cerebellum of mice exhibiting intoxication signs of stage 1 or 2 were used in the present study. We found that exposure to ethanol resulted in elevated levels of nerve growth factor (NGF) and TrkA mRNA expression but a decreased level of brain-derived neurotrophic factor (BDNF) mRNA expression. The expression of TrkB and p73 mRNA was unchanged. Changes in the level of these proteins were found to mirror these mRNA expression levels. We conclude that exposure to ethanol for a short period can cause a differential responsive in the various neurotrophin ligand/receptor systems. The functional consequences of these changes are unknown at present.

Do rapid systemic changes of brain temperature have an influence on the brain?

BACKGROUND: The purpose of the present study was to examine the influence of cooling and rewarming conditions using an accurate brain temperature control system. METHOD: The brain temperature of animals was measured with a thermometer while feedback regulation was achieved with a cold (4( degrees )C) and hot (50( degrees )C) water on-off flow system. Brain temperature was well controlled throughout the experiment by using both cold water and hot water simultaneously. Three groups were studied, as follows: 1) the standard group (cooled to 24( degrees )C for 1 hour, kept at 24( degrees )C for 2 hours and rewarmed to 37( degrees )C for 1 hour), 2) the rapid-cooling group (cooled to 24( degrees )C for 30 min, kept at 24( degrees )C for 2 h, and rewarmed to 37( degrees )C for 1 h), 3) the rapid-rewarming group (cooled to 24( degrees )C for 1 h, kept at 24( degrees )C for 2 h, and rewarmed to 37( degrees )C for 30 min) and the normal-control group. FINDINGS: An increase of MAP-2 immunoreactivity of the CA1 neurons in the dorsal hippocampus was observed one week but not one month after hypothermia in the rapid-rewarming group. There was also a significant increase in the glutamate and lactate value at the end of rewarming compared with the baseline in the rapid-rewarming group (p<0.01). INTERPRETATION: Our results suggest that rapid rewarming after hypothermia triggered an uncoupling of cerebral circulation and metabolism, inducing an increase of extracellular glutamate and lactate, consequently reversible neuronal cell damage.

Inhibitor of cyclooxygenase-2 induces cell-cycle arrest in the epithelial cancer cell line via up-regulation of cyclin dependent kinase inhibitor p21.

Cyclooxygenase-2 is the rate-limiting enzyme in synthesis of prostaglandins and other eicosanoids. Prior reports have shown that inhibition of cyclooxygenase-2 activity, either by selective inhibitors or by antisense oligonucleotide, results in suppression of growth of squamous cell carcinoma cell lines which express high cyclooxygenase-2 levels, such as NA, a cell line established from a squamous cell carcinoma of the tongue. To investigate the mechanisms by which cyclooxygenase-2 inhibitors suppressed growth of these cells, the effects of NS-398, the selective cyclooxygenase-2 inhibitor, on cell-cycle distribution were examined. NS-398 induced G0/G1 cell-cycle arrest in NA cells which expressed cyclooxygenase-2. G0/G1 arrest induced by NS-398 was accompanied by up-regulation of cyclin-dependent kinase inhibitor p21, but not by up-regulation of the other cyclin-dependent kinase inhibitors. Transfection with p21 antisense oligonucleotide inhibited cell-cycle arrest induced by NS-398. Accumulation in G0/G1 was also observed in NA cells transfected with cyclooxygenase-2 antisense oligonucleotide. On the other hand, NS-398-treated NA cells showed a loss of plasma membrane asymmetry, a marker of early events in apoptosis. However, NS-398 did not induce other morphological and biochemical changes related to apoptotic cell death. These results suggest that cyclooxygenase-2 inhibitor induces G0/G1 cell-cycle arrest in NA cells by up-regulation of p21. Our results also suggest that NS-398 is not sufficient to complete the whole process of apoptosis in NA cells, although it induces an early event in apoptosis.

Specific inhibition of cyclooxygenase-2 results in inhibition of proliferation of oral cancer cell lines via suppression of prostaglandin E2 production.

Prostaglandins (PGs) are known to play important roles in the proliferation of various types of cancer cells. PGs are produced by the action of cyclooxygenase (COX) enzymes, and two forms of COX, COX-1 and COX-2, have been described. Previous studies have demonstrated that overexpression of COX-2 is associated with colon carcinogenesis, tumor invasion and metastatic potential of colon cancer. In this study, the role of COX-2 on proliferation of squamous cell carcinoma cell lines was investigated. NS-398, a selective COX-2 inhibitor, inhibited proliferation of NA cells, a squamous cell caricinoma cell line that constitutively expresses COX-2 mRNA. NS-398 suppressed the spontaneous production of PGE2 by NA cells, and the antiproliferative effect of NS-398 was abolished by addition of PGE2. Similar results were obtained from experiments using COX-2 antisense oligonucleotide. These results suggest that specific inhibition of COX-2 inhibits proliferation of cancer cells expressing COX-2 mRNA via suppression of PGE2 production.

Clostridium perfringens epsilon toxin causes excessive release of glutamate in the mouse hippocampus.

The mechanism of neurotoxicity of Clostridium perfringens epsilon toxin to the mouse brain was investigated. Intravenous injection in mice with the toxin caused seizure and excited hippocampal neurons. Microdialysis revealed that epsilon toxin induced excessive glutamate release in the hippocampus. Both the seizure and glutamate release were attenuated by prior injection with riluzole, an inhibitor of pre-synaptic glutamate release, suggesting that this toxin enhances glutamate efflux, leading to seizure and hippocampal neuronal damage.

Altered growth response of oral mucosal keratinocytes in p53-deficient mice.

P53 has important regulatory functions in cell growth, differentiation and apoptosis. Here we analyzed the effects of p53 on the growth response of oral mucosal keratinocytes (OMKCs) using p53-deficient (p53-/-) mice. No morphological difference was found between p53-/- and wild-type (p53+/+) oral mucosa. In a long-term culture, p53-/- OMKCs continued to proliferate past the point at which p53+/+ became senescent. The percentage of p53-/- OMKCs in the G0/G1 phase was lower than that of p53+/+ OMKCs. Proliferation of cultured OMKCs induced by epidermal growth factor (EGF) and interleukin-(IL)-1alpha was more strongly enhanced in p53-/- than in p53+/+ mice. Such an enhanced response was not due to increased mRNA expression of growth factor receptors. These data suggest that p53 acts as a modulator of G1 arrest in OMKCs and is also involved in the regulation of responses to EGF and IL-1alpha without affecting the expression of their receptors.

Synergistic induction of ICAM-1 expression by cisplatin and 5-fluorouracil in a cancer cell line via a NF-kappaB independent pathway.

Cisplatin (CDDP) and 5-fluorouracil (5-FU) are common anti-tumour agents, and the anti-tumour effect of CDDP and 5-FU are synergistically enhanced by combined treatment. To clarify the mechanisms of this synergism, we examined the effect of CDDP and 5-FU on the expression of cell adhesion molecules involved in recognition of cancer cells by T lymphocytes. When NA cells, a squamous cell carcinoma cell line, were exposed to CDDP and 5-FU for 18 h, the expression of intercellular adhesion molecule-1 (ICAM-1) was synergistically induced, whereas CDDP or 5-FU alone did not induce the expression of ICAM-1, as determined by flow cytometry. Expression of ICAM-2 and ICAM-3, which are recognized by the same counter receptor on T-cells, were not up-regulated by CDDP and 5-FU. RT-PCR analysis showed that the induction of ICAM-1 on NA cells might be due to transcriptional induction of ICAM-1 mRNA. Treatment with genistein, a protein tyrosine kinase (PTK) inhibitor, inhibited the induction of ICAM-1 on NA cells by CDDP and 5-FU, whereas staurosporin, a protein kinase C inhibitor, did not. Although CDDP and 5-FU induced binding at the nuclear factor kappa B (NF-kappaB) site in the ICAM-1 promoter, pretreatment with genistein did not prevent CDDP and 5-FU-induced binding at the NF-kappaB site. Moreover, a NF-kappaB nuclear translocation inhibitor did not inhibit the induction of ICAM-1 expression by treatment with CDDP and 5-FU. The synergistic effect of CDDP and 5-FU was not specific to NA cells, since ICAM-1 was synergistically induced by CDDP and 5-FU on HSC-4 cells, a squamous cell carcinoma cell line. These findings indicate that treatment with CDDP and 5-FU induces ICAM-1 expression by a NF-kappaB independent regulatory mechanism involving PTK.

Nitric oxide up-regulates the expression of intercellular adhesion molecule-1 on cancer cells.

Nitric oxide (NO) is an unstable free radical that functions as a cytotoxic agent secreted by macrophages to kill cancer cells. Here we report the effect of NO on the expression of intercellular adhesion molecule-1 (ICAM-1) on cancer cells. NO donors such as SNP, SNAP and SIN-1 up-regulated the expression of ICAM-1 on NA cells, a squamous cell carcinoma cell line. Northern blot analysis showed that the induction of ICAM-1 might be due to transcriptional induction of ICAM-1 mRNA. Up-regulation of ICAM-1 mRNA by NO donors was inhibited by carboxy-PTIO, a NO scavenger. Although NF-kappaB activity was induced by NO donors, AP-1 was not induced by them. Staurosporin, a protein kinase C (PKC) inhibitor, inhibited the induction of ICAM-1 on NA cells by NO, whereas genistein, a protein tyrosine kinase inhibitor, did not. These findings indicate that NO up-regulates ICAM-1 expression on cancer cells by a regulatory mechanism involving PKC and suggest that NF-kappaB, but not AP-1, might be involved in induction of ICAM-1 by NO in cancer cells.

Cytotoxic effect of sodium nitroprusside on cancer cells: involvement of apoptosis and suppression of c-myc and c-myb proto-oncogene expression.

Nitric oxide (NO) is an unstable free radical gas known as an effector molecule of macrophage cytotoxicity against cancer cells. Although several mechanisms of NO-mediated cytotoxicity have been proposed, this phenomenon remains to be characterized in detail. To explore the mechanisms by which NO kills cancer cells, we made use of sodium nitroprusside (SNP), which releases NO in the culture medium. SNP showed a dose-dependent cytotoxic effect on NA cells, an epithelial cancer cell line. When NA cells were killed by SNP, high levels of NO2- (stable end product of NO) were detected in the culture medium. The cell death induced by SNP was mediated by apoptosis, as demonstrated by the presence of nuclear condensation and blebbing of the nuclear membrane, and internucleosomal DNA fragmentation quantified by a specific ELISA. Northern blot analysis revealed that c-myc mRNA expression of NA cells was rapidly reduced by treatment with SNP. RT-PCR analysis showed that c-myb mRNA was expressed in untreated NA cells, and c-myb mRNA level of NA cells was dose-dependently reduced by treatment with SNP. These results indicate that SNP exerts its cytotoxic effect on NA cells through spontaneous release of NO. Cytotoxicity induced by SNP is at least partially mediated via the process known as apoptosis. Our results also suggest that down-regulation of c-myc and c-myb proto-oncogenes might be involved in SNP-induced cytotoxicity.

Prodigiosin 25-C and metacycloprodigiosin suppress the bone resorption by osteoclasts.

Prodigiosin 25-C and metacycloprodigiosin were found to suppress PTH-stimulated pit formation by cultured osteoclasts on bone slices. They also inhibited the acidification of vacuolar organelles in intact osteoclastic cells. Since the acidic pH in these organelles is generated by the action of proton-pumping ATPases of the organelle, these results indicate that the proton-pumping activity of V-ATPase in osteoclastic cells is essential in bone resorption and that the inhibition of the acidification of vacuolar organelles by prodigiosins results in suppression of PTH-stimulated bone resorption.

Inhibitory mechanisms of H(+)-ATPase inhibitor bafilomycin A1 and carbonic anhydrase II inhibitor acetazolamide on experimental bone resorption.

The effects of the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (baf.A1) and the carbonic anhydrase II inhibitor acetazolamide (AZ) on bone resorption and procathepsin L secretion of rat osteoclasts were investigated using the bone slice assay method, pit formation test. Baf.A1 completely suppressed osteoclastic bone resorption stimulated by parathyroid hormone (PTH), but did not affect procathepsin L secretion, while AZ suppressed both bone resorption and procathepsin L secretion. These findings suggest that bone resorption by procathepsin L secretion and its processing are regulated by proton production and proton secretion.

Suppressive effect of N-(benzyloxycarbonyl)-L-phenylalanyl-L-tyrosinal on bone resorption in vitro and in vivo.

The suppressive effect of N-(benzyloxycarbonyl)-L-phenylalanyl-L-tyrosinal on bone resorption was examined in vitro and in vivo. This synthetic peptidyl aldehyde was found to be a potent and selective cathepsin L inhibitor in our screening for cysteine protease inhibitors. In the pit formation assay with unfractionated rat bone cells, 1.5 nM of this compound markedly inhibited parathyroid hormone-stimulated osteoclastic bone resorption. In addition, intraperitoneal administration of this peptidyl aldehyde (2.5-10 mg/kg) for 4 weeks suppressed bone weight loss dose dependently in the ovariectomized mouse, experimental model of osteoporosis. Hydroxyproline measurement of the decalcified femurs from these ovariectomized mice suggested that this compound acts as a bone resorption suppressor through the inhibition of collagen degradation.

Concanamycin B, a vacuolar H(+)-ATPase specific inhibitor suppresses bone resorption in vitro.

Effects of concanamycin B, a specific inhibitor of the vacuolar type H(+)-ATPase (V-ATPase), on the stimulation of bone resorption induced by parathyroid hormone (PTH) were examined in vitro. Concanamycin B was found to inhibit PTH-stimulated osteoclastic pit formation and to suppress the acidification of vacuolar organelles by V-ATPase in the osteoclasts. PTH-stimulated 45Ca release from prelabelled chick embryonic calvariae was also inhibited by concanamycin B in a dose-dependent manner. These results suggest that osteoclastic acidification of lacunae by V-ATPase plays an essential role in mineral dissolution and degradation of the organic matrix during bone resorption.

Secretion and processing mechanisms of procathepsin L in bone resorption.

Secretion of procathepsin L into the culture medium from a bone cell mixture was markedly enhanced by addition of parathyroid hormone (PTH), 1 alpha,25-(OH)2D3 or tumor necrosis factor alpha (TNF alpha). These stimulators of secretion of procathepsin L enhanced bone pit formation, which was inhibited by E-64, but not by CA-074, a specific inhibitor of cathepsin B. Procathepsin L may thus participate in the process of bone collagenolysis during bone resorption. Procathepsin L partially purified from rat long bones under cold conditions was rapidly converted to the mature form under acidic conditions at room temperature. This conversion was inhibited by E-64, suggesting that the procathepsin L secreted into lacunae is catalytically converted to the mature enzyme by cysteine proteinase(s).

Hepatocyte growth factor is involved in formation of osteoclast-like cells mediated by clonal stromal cells (MC3T3-G2/PA6).

Osteoclast formation from hemopoietic precursors has been shown to require the support of stromal cells in bone tissue. In this study, we demonstrated that hepatocyte growth factor (HGF) is one of the stromal cell-derived molecules responsible for osteoclast-like cell formation. For our experiments, we used a coculture system for osteoclastic cell formation and activation in which hemopoietic blast cells are cocultured with calvaria-derived stromal MC3T3-G2/PA6 (PA6) cells on dentine slices in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Addition of anti-HGF neutralizing IgG to the cocultures inhibited the formation of osteoclastic cells and their dentine-resorbing activity. We detected a single 6.0-kb transcript for HGF in PA6 cells, and also recognized immunoreactive M(r) 81,000 and 88,000 forms of HGF in conditioned medium (CM) from PA6 cell cultures, the level of which reached 6 ng/ml. Both the CM and HGF stimulated the proliferation of blast cells synergistically with granulocyte-macrophage colony-stimulating factor, resulting in an increased number of osteoclast precursors that respond to 1,25(OH)2D3 that are tartrate-resistant acid phosphatase-positive multinucleate cells in stromal cell-free blast cell cultures in plastic wells. The effect of the CM was diminished by the addition of anti-HGF IgG. However, neither the CM nor HGF stimulated the formation of osteoclastic cells and pits on dentine slices in the absence of PA6 cells. These results suggest that although HGF cannot completely replace stromal cells, it is one of the paracrine mediators produced by stromal cells that act on proliferation of osteoclastic cell precursors.

Extracellular calcium causes the release of calcium from intracellular stores in chick osteocytes.

We have recently demonstrated that a rise in the extracellular divalent cation concentration induces a rapid elevation of cytosolic calcium in chick osteocytes. Here, we demonstrate that cytosolic calcium elevation that occurs in osteocytes on exposure to elevated extracellular calcium is independent of membrane voltage and is insensitive to modulation by organic calcium channel modulators, namely, BAY K 8644, nicardipine, and nifedipine. However, the calcium elevation was sensitive to modulation by an intracellular calcium antagonist, TMB-8, suggesting that the cytosolic calcium elevation was due to mobilization of this cation from an intracellular store.

Divalent cations elevate cytosolic calcium of chick osteocytes.

Though it is proposed that osteocytes have several functions in bone metabolism, only limited definite information including results of calcium regulation has been forthcoming. We demonstrate here for the first time that the concentration of divalent cations, such as calcium, nickel and cadmium, directly regulates the cytosolic calcium concentration in osteocytes.

The mechanisms and regulation of procathepsin L secretion from osteoclasts in bone resorption.

The secretion mechanisms of cathepsin L from osteoclasts in the process of bone resorption were investigated. The increases in bone pit numbers formed take place by PTH addition in parallel with the increases of cathepsin L and/or L-like proteinase activities in the culture medium of bone cells, and these were suppressed by the addition of calcitonin. The Z-Phe-Arg-MCA hydrolysing activity increased in the medium through the effect of PTH is considered to be a kind of procathepsin L by Western blotting analysis, and was suppressed by calcitonin addition. Furthermore, monensin inhibited not only the PTH-induced pit formation, but also cysteine proteinase activity in osteoclasts. Therefore, the procathepsin L excreted might be transferred from endothelial reticulum via Golgi and/or via lysosomes.

Effect of Intracellular Glutathione Level on the Production of 6-Methoxymellein in Cultured Carrot (Daucus carota) Cells.

To produce phytoalexin, 6-methoxymellein (6-MM) was induced in suspension cultures of carrot (Daucus carota) by buthionine sulfoximine (BSO) and CuCl2. Addition of BSO (a specific inhibitor of glutathione [GSH] synthesis) to the cultures lowered the cellular GSH levels. This depletion of GSH was BSO-concentration dependent, and the extent of 6-MM accumulation was dependent on the GSH depletion. The accumulation of 6-MM induced by BSO was suppressed by exogenous GSH. Exogenous H2O2 stimulated the production of 6-MM when added 1 d after BSO treatment, whereas H2O2 added at time zero or on the 4th d of BSO treatment did not. Moreover, a synergistic effect of simultaneous addition of BSO and CuCl2 was observed. These results suggest that active oxygen species may be involved in the triggering of 6-MM synthesis.