Tumour necrosis factor alpha induces rapid reduction in AMPA receptor-mediated calcium entry in motor neurones by increasing cell surface expression of the GluR2 subunit: relevance to neurodegeneration

The AMPA receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) have both been implicated in motor neurone vulnerability in Amyotrophic Lateral Sclerosis/Motor Neurone Disease. TNF alpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNF alpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/ml, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using Fura-2 AM microfluorimetry we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggests that TNF alpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in ALS

Analysis of void volumes in proteins and application to stability of the p53 tumour suppressor protein

We have developed a new method for the analysis of voids in proteins (defined as empty cavities not accessible to solvent). This method combines analysis of individual discrete voids with analysis of packing quality. While these are different aspects of the same effect, they have traditionally been analysed using different approaches. The method has been applied to the calculation of total void volume and maximum void size in a non-redundant set of protein domains and has been used to examine correlations between thermal stability and void size. The tumour-suppressor protein p53 has then been compared with the non-redundant data set to determine whether its low thermal stability results from poor packing. We found that p53 has average packing, but the detrimental effects of some previously unexplained mutations to p53 observed in cancer can be explained by the creation of unusually large voids. (C) 2004 Elsevier Ltd. All rights reserved.

Synthesis of alpha-galactooligosaccharides with alpha-galactosidase from Lactobacillus reuteri of canine origin

Crude cell-free extracts from Lactobacillus reuteri grown on cellobiose, maltose, lactose and raffinose were assayed for glycosidic activities. When raffinose was used as the carbon source, alpha-galactosidase was produced, showing the highest yield at the beginning of the stationary growth phase. A 64 kDa enzyme was purified by ultra- and gel filtration, and characterized for its hydrolytic and synthetic activity. Highest hydrolytic activity was found at pH 5.0 at 50 degreesC (K-M 0.55 mM, V-max 0.80 mumol min(-1) mg(-1) of protein). The crude cell-free extract was further used in glycosyl transfer reactions to synthesize oligosaccharides from melibiose and raffinose. At a substrate concentration of 23% (w/v) oligosaccharide mixtures were formed with main products being a trisaccharide at 26% (w/w) yield from melibiose after 8 h and a tetrasaccharide at 18% (w/w) yield from raffinose after 7 h. Methylation analysis revealed the trisaccharide to be 6' alpha-galactosyl melibiose and the tetrasaccharide to be stachyose. In both cases synthesis ceased when hydrolysis of the substrate reached 50%.

Modulation of anti-pathogenic activity in canine-derived Lactobacillus species by carbohydrate growth substrate

Aims: To investigate the effect of various carbon sources on the production of extracellular antagonistic compounds against two Escherichia coli strains and Salmonella enterica serotype Typhimurium by three canine-derived lactobacilli strains. Methods and Materials: Cell-free preparations, pH neutralized, were used in antibiotic disc experiments as an initial screening. The bacteria/carbohydrate combinations that showed inhibition of the growth of those pathogens, were further investigated in batch co-culture experiments. The cell-free supernatants of the cultures, that decreased the population number of the pathogens in the co-culture experiments to log CFU ml(-1) less than or equal to 4, were tested for inhibition of the pathogens in pure cultures at neutral and acidic pH. Conclusions: The results showed that the substrate seems to affect the production of antimicrobial compounds and this effect could not just be ascribed to the ability of the bacteria to grow in the various carbon sources. L. mucosae, L. acidophilus and L. reuteri, when grown in sugar mixtures consisting of alpha-glucosides (Degree of Polymerization (DP) 1-4) could produce antimicrobial compounds active against all three pathogens in vitro. This effect could not be attributed to a single ingredient of those sugar mixtures and was synergistic. This inhibition had a dose-response characteristic and was more active at acidic pH. Significance and Impact of the Study: Knowledge of the effect that the carbon source has on the production of antimicrobial compounds by gut-associated lactobacilli allows the rational design of prebiotic/probiotic combinations to combat gastrointestinal pathogens.

Slackia faecicanis sp nov., isolated from canine faeces

Morphological, biochemical and molecular genetic studies were carried out on an unknown non-spore-forming, Gram-positive, rod-shaped bacterium that was isolated from dog faeces. The bacterium grew under strictly anaerobic conditions, was asaccharolytic, and possessed a relatively high G + C content of 61 mol%. Phylogenetic analysis based on comparative 16S rRNA gene sequencing showed that the unidentified bacterium was a member of the family Coriobacteriaceae and represents a hitherto unknown subline within the genus Slackia. Based on the presented findings, a novel species, Slackia faecicanis sp. nov., is described. The type strain of Slackia faecicanis is 5WC12(T) (=CCUG 48399(T)=CIP 108281(T)).

Gut fermentation products of insulin-derived prebiotics beneficially modulate markers of tumour progression in human colon tumour cells

Insulin is a prebiotic food ingredient, which suppresses colon tumour growth and development in rats. In the gut lumen, it is fermented to lactic acid and short chain fatty acids (SCFA). Of these, butyrate has suppressing agent activities, but little is known concerning cellular responses to complex fermentation samples. To investigate the effects of fermentation products of insulin on cellular responses related to colon carcinogenesis. Fermentations were performed in anaerobic batch cultures or in a three-stage fermentation model that simulates conditions in colon-segments (proximal, transverse, distal). Substrate was insulin enriched with oligofructose (Raftilose® Synergy1), fermented with probiotics (Bifidobacterium lactis Bb12, Lactobacillus rhamnosus GG), and/or faecal inocula. HT29 or CaCo-2 cells were incubated with supernatants of the fermented samples (2.5%-25% v/v, 24-72 hours). Cellular parameters of survival, differentiation, tumour progression, and invasive growth were determined. Fermentation supernatants derived from probiotics and Synergy1 were more effective than with glucose. The additional fermentation with faecal slurries produced supernatants with lower toxicity, higher SCFA contents, and distinct cellular functions. The supernatant derived from the gut model vessel representing the distal colon, was most effective for all parameters, probably on account of higher butyrate-concentrations. Biological effects of insulin upon colon cells may be mediated not only by growth stimulation of the lactic acid-producing bacteria and/or production of butyrate, but also by other bacteria and products of the gut lumen. These newly reported properties of the supernatants to inhibit growth and metastases in colon tumour cells are important mechanisms of tumour suppression.

Allobaculum stercoricanis gen. nov., sp nov., isolated from canine feces

Morphological, biochemical, and molecular genetic studies were performed on an unknown anaerobic, catalase-negative, nonspore-forming, rod-shaped bacterium isolated from dog feces. The unknown bacterium was tentatively identified as a Eubacterium species, based on cellular morphological and biochemical tests. 16S rRNA gene sequencing studies, however, revealed that it was phylogenetically distant from Eubacterium limosum, the type species of the genus Eubacterium. Phylogenetically, the unknown species forms a hitherto unknown sub-line proximal to the base of a cluster of organisms (designated rRNA cluster XVI), which includes Clostridium innocuum, Streptococcus pleomorphus, and some Eubacterium species. Based on both phenotypic and phylogenetic criteria, it is proposed that the unknown bacterium be classified as a new genus and species, Allobaculum stercoricanis. Using a specific rRNA-targeted probe designed to identify Allobacultan stercoricanis, in situ hybridisation showed this novel species represents a significant organism in canine feces comprising between 0.1% and 3.7% of total cells stained with DAPI (21 dog fecal samples). The type strain of Allobaculum stereoricanis is DSM 13633(T) = CCUG 45212(T). (C) 2004 Elsevier Ltd. All rights reserved.

Clostridium colicanis sp. nov., from canine faeces

Morphological, biochemical and molecular genetic studies were performed on an unknown, anaerobic, rod-shaped organism isolated from faeces of a canine. The organism was tentatively identified as a member of the genus Clostridium based on its cellular morphology and ability to form endospores but, biochemically, it did not appear to correspond to any recognized species of this genus. Comparative 16S rRNA gene sequence analysis showed that the bacterium represents a previously unrecognized subline within Clostridium rRNA group I (Clostridium sensu stricto), which includes Clostridium butyricum, the type species of the genus. The nearest phylogenetic relatives of the unknown bacterium corresponded to Clostridium absonum, Clostridium baratii, Eubacterium budayi, Eubacterium moniliforme, Eubacterium multiforme and Eubacterium nitritogenes, but 16S rRNA sequence divergence values of > 3% demonstrated that it represents a novel species. Based on the findings presented, a novel species, Clostridium colicanis sp. nov., is described, with the type strain 3WC2(T) (=CCUG 44556(T) =DSM 13634(T)).

Sutterella sterconcanis sp nov., isolated from canine faeces

Morphological, biochemical and molecular genetic studies were carried out on an unknown non-spore-forming, Gram-negative, rod-shaped bacterium which was isolated from dog faeces. The bacterium grew under anaerobic conditions, was asaccharolytic, resistant to 20% (v/v) bile and was oxidase- and urease-negative. Phylogenetic analysis based on comparative 16S rRNA gene sequencing showed that the unidentified bacterium clustered with Sutterella wadsworthensis, although a sequence divergence of > 5% indicated that the bacterium from dog faeces represented a previously unrecognized subline within the genus. On the basis of the presented findings, a novel species, Sutterella stercoricanis sp. nov., is described. The type strain of Sutterella stercoricanis is 5BAC4(T) ( = CCUG 47620(T) = CIP 108024(T)).

Antitumour activity of [1,2-di(cyclopentadienyl)-1,2-di(p-N,N-dimethylaminophenyl)-ethanediyl] titanium dichloride in xenografted Ehrlich's ascites tumour

The effects of a new titanocene compound with an ansa ligand in the cyclopentadienyl rings, the 1,2-di(cyclopentadienyl)-1,2-di(p-NNdimethylaminophenyl)-ethanediyl] titanium dichloride (TITANOCENE X), on the growth and differentiation of granulocyte-macrophage progenitor cells [colony-forming unit-granulocyte-macrophage (CFU-GM)] and Natural killer (NK) cell activity in Ehrlich's ascites tumour (EAT)-bearing mice were studied. Myelosuppression concomitant with increased numbers of spleen CFU-GM was observed in tumour-bearing mice. Treatment of these animals with TITANOCENE X (2.5-50mg/kg/day) produced an increase in myelopoicsis, in a dose-dependent manner, and reduced spleen colony formation. In addition, the treatment of EAT-bearing mice with 3 doses of 20 or 50 mg/kg TITANOCENE X restored to normal values the reduced Natural killer cell function observed during tumour growth. In parallel, TITANOCENE X prolonged, in a dose-dependent manner, the survival of mice inoculated with Ehrlich's ascites tumour. The highest dose of 50 mg/kg prolonged in 50% the survival time of EAT-bearing mice, compared to non-treated tumour-bearing controls. In comparison with previous results from our laboratory addressing the effects of titanocenes on haematopoiesis, we observed with TITANOCENE X a similar effective profile as for bis(cyclopentadienyl) dithiocyanate titanium(IV), being both less effective than di(cyclopentadienyl) dichloro titanium(IV), since the latter not only prolonged, but also increased the rate of survival. These differences in efficacy may be due to the nature of the ansa-cyclopentadienyl ligand used in TITANOCENE X, since the C, bridge between the two cyclopentadienyl groups will increase the hydrolytic stability by an organometallic chelate effect. Also, the introduction of two dimethylamino substituents increases the water solubility of TITANOCENE X when compared to titanocene dichloride itself (c) 2006 Elsevier B.V. All rights reserved.

MARCKS functions as a novel growth/tumour suppressor in cells of melanocyte origin

Protein kinase C (PKC) plays a pivotal role in modulating the growth of melanocytic cells in culture. We have shown previously that a major physiological substrate of PKC, the 80 kDa myristoylated alanine-rich C-kinase substrate (MARCKS), can be phosphorylated in quiescent, non-tumorigenic melanocytes exposed transiently to a biologically active phorbol ester, but cannot be phosphorylated in phorbol ester-treated, syngeneic malignant melanoma cells. Despite its ubiquitous distribution, the function of MARCKS in cell growth and transformation remains to be demonstrated clearly. We report here that MARCKS mRNA and protein levels are down-regulated significantly in the spontaneously derived murine B16 melanoma cell line compared with syngeneic normal Mel-ab melanocytes. In contrast, the tumourigenic v-Ha-ras-transfonned melan-ocytic line, LTR Ras 2, showed a high basal level of MARCKS phosphorylation which was not enhanced by treatment of cells with phorbol ester. Furthermore, protein levels of MARCKS in LTR Ras 2 cells were similar to those expressed in Mel-ab melanocytes. However, in four out of six murine tumour cell lines investigated, levels of MARCKS protein were barely detectable. Transfection of B16 cells with a plasmid containing the MARCKS cDNA in the sense orientation produced two neomycin-resistant clones displaying reduced proliferative capacity and decreased anchorage-independent growth compared with control cells. In contrast, transfection with the antisense MARCKS construct produced many colonies which displayed enhanced growth and transforming potential compared with control cells. Thus, MARCKS appears to act as a novel growth suppressor in the spontaneous transformation of cells of melanocyte origin and may play a more general role in the tumour progression of other carcinomas.

Effects of biologically active tumour-promoting and non-promoting phorbol esters on in vivo growth of melanocytic cells

Sapintoxin A (SAP A), a naturally occurring biologically active but non-promoting phorbol ester, acts as an effective in vitro mitogen for freshly derived human melanocytes. Seven days after addition of 50 nM SAP A there was a four to fivefold increase in melanocyte number over that observed in untreated control cultures comparable to that achieved with a 50 nM concentration of 12-0-tetradecanoylphorbol 13-acetate (TPA). The fluorescent stage 2 promoter sapintoxin D (SAP D) also supported the growth of these cells, with a 50 nM dose producing an increase in cell number comparable to that observed with 200 nM TPA. Similar results were obtained with an established, but non-tumorigenic, line of murine melanocytes. The same compounds exerted a potent anti-proliferative effect against transformed melanocyte lines of murine and human origin associated with morphological alterations and an increase in melanin production consistent with induced cytodifferentiation.

Both tumour-promoting and non-promoting phorbol esters inhibit 125I-EGF binding and stimulate the phosphorylation of a 80kd protein kinase C substrate protein in intact Swiss 3T3 cells

Sapintoxin A (SAP A) and 12-deoxyphorbol 13-phenylacetate (DOPP), are two biologically active but non-turnour-promoting phorbol esters that potently bind to and activate the phorbol ester receptor, protein kinase C (PKC). SAP A and DOPP cause a dose-dependent increase in the phosphorylation of an 80 kd (80K) substrate protein for PKC in Swiss 3T3 cells. A similar dose—response effect was seen with sapintoxin D (SAP D), the stage 2 promoting analogue of 12-O-tetradecanoylphorbol-13-acetate and the complete promoter phorbol 12,13-dibutyrate (PDB). The doses resulting in a half maximal phosphorylation of this protein (Ka were 20 nM (SAP A), 45 nM (DOPP), 23 nM (SAP D) and 37 nM (PDB). Both non-promoting and phorbol esters induced a dose-dependent inhibition of [125I]epidermal growth factor (EGF) binding to its receptor in Swiss 3T3 cells. The doses required for 50% inhibition of binding (Ki) were: 8 nM (SAP A), 16 nM (DOPP), 14 nM (SAP D) and 17 nM (PDB). The results clearly demonstrate that induction of phosphorylation of the Pu 80K phosphoprotein and inhibition of [125I]EGF binding in Swiss 3T3 cells following exposure to phorbol esters is independent of the tumour-promoting activity of these compounds. The fact that SAP A, DOPP, SAP D and PDB are mitogenic for a variety of cell types and that exposure to these compounds leads to 80K phosphorylation and inhibition of [125I]EGF binding, suggests that these early biological events may play a role in the mitogenic response induced by these compounds.