Influence of reduced levels or suppression of sodium nitrite on the outgrowth and toxinogenesis of psychrotrophic Clostridium botulinum Group II type B in cooked ham.

Outgrowth and toxinogenesis of Clostridium botulinum Group II (non-proteolytic) type B were studied in cooked ham prepared with different NaNO2 (ranging from 0 to 80 mg/kg) and sodium chloride (NaCl, ranging from 12 to 19 g/kg) incorporation rates. Cured ground pork batters were inoculated with a cocktail of 3 strains of C. botulinum Group II type B at 3.5 log10 CFU/g, portioned and samples of 50 g were vacuum packed then cooked and cooled based on thermal processing employed by the meat processing industry. These cooked ham model samples were stored under reasonably foreseeable conditions of use and storage i.e. for 14 days at 4 °C, followed by a cold chain break for 1 h at 20 °C then up to 33 days at 8 °C. Storage times and temperatures were used to mimic those commonly encountered along the supply chain. Enumeration of C. botulinum and detection of the botulinum neurotoxin type B (BoNT/B) were performed in triplicate at different storage times. Under these experimental conditions, incorporation rates of NaNO2 ≥ 30 mg/kg prevented the outgrowth and toxinogenesis of C. botulinum Group II type B in the cooked ham model, regardless of the NaCl concentrations tested. In contrast, total removal of nitrite allowed outgrowth and toxin production during storage of the processed meat product. Results showed that the maximum ingoing amount of nitrite (i.e. 150 mg/kg) that may be added according to the EU legislation (Regulation (EC) No 1333/2008) can be reduced in cooked ham while still ensuring control of C. botulinum Group II type B. According to the multiple factors that could affect C. botulinum behavior in processing meat products, outgrowth and toxin production of C. botulinum should be evaluated on a case by case basis, depending on the recipe, manufacturing process, food matrix and storage conditions.

Oxygen as a key parameter in in vitro dynamic and multi-compartment models to improve microbiome studies of the small intestine?

In vitro digestion and fermentation models are frequently used for human and animal research purposes. Different dynamic and multi-compartment models exist, but none have been validated with representative microbiota in the distal parts of the small intestine. We recently developed a dynamic and multi-compartment piglet model introducing microbiota in an ileum bioreactor. However, it presented discrepancies compared to in vivo data. Recommendations are available to standardize studies in this field. They target the digestion model but include elements of a fermentation model. But no recommendation is given concerning control of the atmosphere. The gastrointestinal tract is generally associated with anaerobiosis to conduct a good fermentation process. In this study, we attempted to improve the ileal microbiota of the piglet model by testing inoculation: real intestinal content vs feces; the latter being generally used for ethical and economical aspects. Results showed a positive effect of using real intestinal content. Fusobacteriia were less abundant in the model, Bacteroidia were better maintained in the colon. But for the ileum, results showed that anoxic conditions in the ileum bioreactor conditioned the microbial profile probably more than the type of inoculum itself, leading to the general conclusion that in vitro dynamic and multi-compartment models probably have to get oxygenated to improve microbiome studies of the small intestine.

Progression of atherosclerosis in the Apo E-/- model: 12-month exposure to cigarette mainstream smoke combined with high-cholesterol/fat diet.

This study was performed to gain information about the influence of two cardiovascular risk factors, cigarette mainstream smoke (MS) and high-cholesterol/fat diet, on the progression of atherosclerosis in apolipoprotein E-deficient (Apo E-/-) mice. Eight to 12-week-old mice were whole-body exposed for up to 12 months (6h/day, 5 days/week) to diluted cigarette mainstream smoke at total particulate matter (TPM) concentrations of 100 or 200mg/m(3), or to filtered fresh air (sham) in combination with a normal chow diet or a high-cholesterol/fat diet. Cholesterol in the aortic arch was elevated in the high-cholesterol/fat diet groups exposed to 200 mg TPM/m(3) compared to sham at all time points. In the brachiocephalic artery (BA), absolute plaque size and fraction area of plaques was elevated over the 12-month time course in mice exposed to 200 mg TPM/m(3) compared to sham (both diets). Exposure to 100 and 200 mg TPM/m(3) altered the number of elastin-rich layers in the BA in mice fed a high-cholesterol/fat diet, indicating changes in plaque morphology at 6 and 9 months. This study shows for the first time the influence of two different risk factors, MS and high-cholesterol/fat diet, both alone and in combination over a period of 12 months, on the progression of atherosclerosis in Apo E-/- mice. Data suggest that long-term exposure to cigarette mainstream smoke accelerates the development of atherosclerosis in Apo E-/- mice, particularly in combination with a high-cholesterol/fat diet.

Sorption and redox processes controlling arsenic fate and transport in a stream impacted by acid mine drainage.

Reigous acid creek originating from the Carnoulès tailings impoundment supplies high concentrations of arsenic under soluble (up to approximately 4 mg/l) and particulate (up to 150 mgAs/g) phases to the Amous river, situated at the drainage basin of the Rhône river (Southern France). The metalloid is present as As(III) (>95%) in Reigous creek water while As(V) predominates (50-80%) in the solid phase, i.e. schwertmannite. At the confluence between acid (pH<5) creek and alkaline Amous river, As(III) concentrations decrease ten-fold through dilution and formation of As-rich ferrihydrite (As/Fe=0.02-0.1) containing 10-30% As(III). However, these attenuation processes are not efficient in the summer heatwave of 2003 since As concentrations in Amous river water (>or=20 microg/l) and As/Fe ratios in particulate matter (>or=0.07) are closed to those of Reigous creek (

Repair of ochratoxin A-induced DNA damage and modulation of OTA-related genotoxicity by co-incubation with bile acids and methotrexatein vitro.

The mycotoxin ochratoxin A (OTA) induced DNA strand breaks in porcine urinary bladder epithelial cells (PUBEC) and in Madin Darby canine kidney (MDCK) cells. A co-incubation with bile acids or methotrexate reduced or even prevented this adverse effect of OTAin vitro. The protective effect is possibly attributable to a decreased OTA uptake in cells, since bile acids and methotrexate are known to share common transport systems such as organic anion transporters (OAT) and/or organic anion transporting polypeptides (OATP) with the mycotoxin. OTA uptake in cells and its modulation can be one factor which determines the extent of adverse effects in different cell types. Another aspect of interest in this regard relates to repair of DNA damage: PUBEC cells are sensitive to OTA-induced damage which is more pronounced when DNA repair is blocked (by cytosine ß-D-arabino-furanosid/hydroxyurea). On the other hand, when cells are kept in fresh (toxin-free) medium for 3 h, OTA-induced DNA damage decreased to control levels.

Modulation of ochratoxin A induced DNA-damage in urothelial cell cultures.

Despite good evidence for a genotoxic potential of ochratoxin A (OTA), the mechanism of OTA-induced genotoxicity (direct or indirect?) is still unclear. This calls for a further characterization of OTA-related DNA damage, and investigations of factors that may modulate dose-effect relationships in cells.Since bladder epithelium is a target tissue for the toxicity of OTA, its effects were studied in cultures of human bladder carcinoma (H5637) cells. Cytotoxicity of OTA, assessed by Neutral red (NR) uptake or Alamar-Blue assay, is concentration- and time-dependent: Upon 24 h treatment of 5637 cells, NR uptake is reduced by 50% with OTA concentrations of ≥0.2 microM, but not with 3 h treatment of the cells. Since cytotoxicity of OTA was not affected by addition of xenobiotic metabolizing enzymes (S-9 mix), it appears to be unrelated to biotransformation of the mycotoxin. Also, addition of S-9 mix did not significantly affect the genotoxicity of OTA as studied by alkaline single cell gel electrophoresis (Comet assay). DNA damage was detectable after 3 h treatment of cells at OTA concentrations between 0.1 and 1 microM, and increased further at higher concentrations. The magnitude of OTA-induced DNA damage did not increase with longer treatment times (18, 24 h), probably due to repair processes in the cells. Repair of OTA-induced lesions is quite efficient in kidney (Arch Toxicol 2002, 75, 734-741) and in porcine bladder cells (Föllmann and Lebrun, 2005, Mycotoxin Research, this volume). Interestingly, the genotoxicity of OTA is modulated by the pH of the culture medium, with higher damage at pH 5 compared to pH 7.5. In line with this, uptake studies with tritiated OTA show a higher cellular accumulation of the mycotoxin at pH 5 than in buffer of pH 7.5. Thus, bladder cells exposed to OTA in slightly acidic urine (which facilitates reabsorption) may be at higher risk.

Pharmacokinetic evaluation of a new oral sustained release dosage form of tramadol.

AIMS: To compare the pharmacokinetic profile of a new modified release formulation of tramadol (Tramadol LP 200 mg, SMB Technology, Marche-en-Famenne, Belgium) with that of an immediate release capsule (Topalgic) 50 mg, Grünenthal, Aachen, Germany) after single and multiple dosing and to assess the potential effect of food on its relative bioavailability. METHODS: The first study had an open, single-dose, three-treatment, three-period, six-sequence, randomised, crossover design with at least a five-day wash-out. The second study had an open, steady-state, two-treatment, two-period, two-sequence, randomised crossover design with at least a seven-day wash-out. Both studies contained 30 healthy subjects. Both enantiomers of tramadol and O-demethyl-tramadol (the only active metabolite of tramadol) were assayed in the plasma using an LC-MS/MS method. AUC infinity, AUCt, Cmax, Tmax, and T1/2 were estimated. Statistical analysis was performed using univariate anova, the Wilcoxon nonparametric method or Friedman's nonparametric anova where appropriate. RESULTS: Tramadol had a significantly lower Cmax and longer Tmax than the conventional formulation. Thus, the mean (+/- sd) Cmax of tramadol were 646 +/- 192 and 300 +/- 94 ng ml-1 for Topalgic 4 x 50mg and Tramadol LP 200 mg, respectively (95% confidence interval on the difference expressed as a percentage 42-51). AUC of tramadol from both formulations was comparable (similar AUC infinity and AUCt). Thus, the mean AUC infinity of (+/-)tramadol obtained after multiple dosing were 4611 +/- 1944 and 5105 +/- 2101 ngh ml-1 after Topalgic 4 x 50mg and Tramadol LP 200 mg, respectively (95%CI 102-123%). We also demonstrate that the pharmacokinetics of the drug are not influenced by the intake of food. Thus, the mean AUC infinity of (+/-) tramadol were 5444 +/- 1637 and 5169 +/- 1580 ngh ml-1 after Tramadol LP 200 mg given in the fasting and fed states, respectively (95%CI = 88-103%). CONCLUSIONS: The new sustained release form of tramadol exhibits adequate properties for once a day administration. Furthermore, its pharmacokinetic profile is not affected by the intake of food.

Immobilization of arsenite and ferric iron by Acidithiobacillus ferrooxidans and its relevance to acid mine drainage.

Weathering of the As-rich pyrite-rich tailings of the abandoned mining site of Carnoulès (southeastern France) results in the formation of acid waters heavily loaded with arsenic. Dissolved arsenic present in the seepage waters precipitates within a few meters from the bottom of the tailing dam in the presence of microorganisms. An Acidithiobacillus ferrooxidans strain, referred to as CC1, was isolated from the effluents. This strain was able to remove arsenic from a defined synthetic medium only when grown on ferrous iron. This A. ferrooxidans strain did not oxidize arsenite to arsenate directly or indirectly. Strain CC1 precipitated arsenic unexpectedly as arsenite but not arsenate, with ferric iron produced by its energy metabolism. Furthermore, arsenite was almost not found adsorbed on jarosite but associated with a poorly ordered schwertmannite. Arsenate is known to efficiently precipitate with ferric iron and sulfate in the form of more or less ordered schwertmannite, depending on the sulfur-to-arsenic ratio. Our data demonstrate that the coprecipitation of arsenite with schwertmannite also appears as a potential mechanism of arsenite removal in heavily contaminated acid waters. The removal of arsenite by coprecipitation with ferric iron appears to be a common property of the A. ferrooxidans species, as such a feature was observed with one private and three collection strains, one of which was the type strain.

Uptake and genotoxic effects of ochratoxin A in cultured porcine urinary bladder epithelial cells.

Uptake of radiolabelled ochratoxin A (OTA) into porcine urinary bladder epithelial cells (PUBEC) was measured at neutral (pH 7.5) or acidic (pH 5.0) conditions. Genotoxicity of OTA was evaluated with the Comet assay and cytotoxicity with the neutral red uptake assay.At acidic pH-conditions, the bladder cells were able to take up more OTA than at neutral conditions. Cytotoxic effects were not increased at pH 5.0 compared to pH 7.5, but higher OTA uptake correlated with stronger genotoxic effects in the Comet assay at pH 5.0 compared to pH 7.5.These results demonstrate that uptake of OTA has to be regarded as an important factor for the toxicity of OTA as adverse effects depend on the amount of OTA taken up by the cells.

Effects of ochratoxin A on DNA evaluatedin vitro with the single cell gel electrophoresis (Comet assay).

In cell cultures of Madin Darby canine kidney (MDCK) cells, the mycotoxin ochratoxin A (OTA) induced single strand breaks (ssb) in a concentration dependent manner detected with the single cell gel electrophoresis (Comet assay). When an external metabolizing enzyme system (S9-mix from rat liver) was added, this genotoxic effect was significantly stronger. By addition of methotrexate (MT), a substrate of the hepatic organic anion transporter, the effect of OTA can be completely blocked at concentrations >100 µM methotrexate.When DNA repair was inhibited by addition of cytosine arabinose (araC) and hydroxyurea (HU), the tail length in the Comet assay increased dramatically and all treated cells showed ssb. A further culture of the damaged cells in the absence of any supplement resulted in a complete repair of the damaged DNA within three hours.Compared with MDCK cells, primary cultured porcine urinary bladder epithelial cells (PUBECs) showed weaker effects in the Comet assay if treated with OTA. The presence of S9-mix did not significantly enhance the response. Methotrexate only partially reduced the OTA-induced effects, because in PUBECs methotrexate induced ssb at high concentrations. If DNA repair was inhibited, also in PUBECs clearly more ssb were induced by OTA, an effect which was reversible.These results demonstrate that OTA induces single strand breaks in vitro. The damaged DNA can be repaired more effectively in primary cultured epithelial cells (PUBECs) compared to cells of a cell line (MDCK cells). By competitive inhibition of OTA uptake, DNA damage can be prevented with suitable substrates.

Characterization of methylation site of monomethylflavan-3-ols by liquid chromatography/electrospray ionization tandem mass spectrometry.

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used for the structural characterization and differentiation of four isomeric O-monomethylated catechins (on phenolic positions) by the analysis of the fragmentation behaviour of catechin. The catechin fragmentation routes were rationalized and it is shown that several diagnostic ions such as (1,3)A(+), (1,2)B(+), and (1,4)B(+) allow the unambiguous identification of the methylated ring. The precise position of the methyl group on each ring is determined by the difference in the relative intensities of the diagnostic ions. Isomeric O-methylepicatechins were also differentiated using this methodology.

First synthesis and pharmacological evaluation of benzoindolizidine and benzoquinolizidine analogues of alpha- and beta-peltatin.

The benzoindolizidine and quinolizidine analogues of alpha- and beta-peltatin were designed and synthesized by two different synthetic routes involving as the key step the Bischler-Napieralski cyclization of suitably substituted N-acyl-2-arylmethylpyrrolidine and -piperidine derivatives. The in vitro biological activity of these analogues as well as some of their derivatives was subsequently evaluated.

Cytotoxicity of ochratoxin A and citrinin in different cell typesin vitro.

Cytotoxic effects resulting from the two mycotoxins ochratoxin A (OTA) and citrinin were evaluatedin vitro using cell cultures of different origin. Cytotoxicity was estimated by the neutral red (NR) uptake assay which allows to determine the viability of cells by detecting dye uptake into the cells and its storage in the lysosomes. The assay was performed with primary cell cultures derived from isolated porcine urinary bladder epithelial cells (PUBEC), which are competent in metabolism of xenobiotics, and with V79 hamster fibroblasts, a well established and frequently used cell line. In both systems, OTA was more cytotoxic compared to citrinin. When both mycotoxins were applied simultaneously no additive or synergistic effects were detected. Obviously, the primary cell culture system which represents a target tissue of the mycotoxins was more susceptible to the toxins, expressed in lower IC50-values. These results indicate that origin of a cell system and its competence in metabolism of xenobiotics have to be considered inin vitro investigations particularly when different systems were used.

Interferon-induced upregulation and cytoplasmic localization of Myc-interacting protein Nmi.

Nmi interacts with c-Myc, N-Myc, Max, and fos, as demonstrated by yeast two-hybrid and coimmunoprecipitation assays. Nmi is partially homologous to IFP 35, an interferon (IFN)-inducible protein. In this study, we show that basal expression of Nmi is upregulated by IFN in multiple tumor-derived cell lines. Treatment with IFN results in an increased amount of cytoplasmic Nmi distributed in a punctate granular pattern. We also demonstrate that Nmi is expressed in various fetal and adult tissues. As Nmi does not contain a known DNA-binding motif, it has the potential to form inactive heterodimers with its putative DNA-binding partners. Our studies suggest that Nmi may modulate its binding partners in an IFN-inducible manner.

Hydroxylamine-induced cleavage of the asparaginyl-glycine motif in the production of recombinant proteins: the case of insulin-like growth factor I.

Hydroxylamine-induced cleavage at the asparaginyl-glycine dipeptide site inserted between the two moieties of recombinant fusion proteins has been used at both the analytical and the preparative scale to obtain the mature protein. In this study a model protein containing a fusion precursor of insulin-like growth factor I was used to investigate the influence of the operating conditions on the cleavage reaction and the formation of undesired side products such as hydroxamate and deamidated analogs. Moreover, the stability of the cleavage site toward deamidation was examined and a chemometric study performed to define the effect of the reaction conditions on the cleavage yield and on the formation of side products.

Clinical and genetic study of chronic (types II and III) childhood onset spinal muscular atrophy.

We have conducted a retrospective study of 63 patients affected by chronic forms of spinal muscular atrophy (SMA) to better document the natural history of this disease. Thirty-nine patients had type II and 24 type III SMA. These patients had manual muscle testing (MMT) and forced vital capacity (FVC) studies done every six to 12 months over follow up period ranging from six to 140 months. A decline in FVC was seen in both types of SMA but there was no significant change in MMT in either group. Genetic studies were also done in a subset of 17 families (23 patients) included in this study. Homozygous deletions in the telomeric survival motor neuron (SMN) and the neuronal apoptosis inhibitory protein (NAIP) genes were observed in 100% and 11.8% of the patients tested respectively.

Faster than the eye can see: blue cones respond to rapid flicker.

Flickering lights that are detected by the blue cones of the human visual system fuse to yield a steady sensation at much lower rates of flicker than do lights that are detected by the red or green cones. Yet, although blue-cone-detected lights flickering at 30-40 Hz appear to be steady, they are still able to interact with red- or green-cone-detected flickering lights to produce clearly detectable beats in the form of an amplitude modulation of the red- or green-cone flicker. Thus the blue cones produce a viable high-frequency flicker signal, as do the red and green cones, but one that is normally lost before it reaches sensation. The temporal-frequency response for the blue-cone beat interaction is similar in shape to the temporal-frequency response for directly detected red- or green-cone flicker. When measured through the same pathway (which we identify as the luminance pathway, since it is able to transmit high-frequency flicker), the response of the blue cones seems to be as fast as that of the other cones.

[Action of betaxolol on ventricular arrhythmias. Control of efficacy and tolerance by repetitive Holter recordings]. / Etude de l'action du bétaxolol sur les troubles du rythme ventriculaire. Contrôle de l'efficacité et de la tolérance par enregistrements de Holter répétés.

The ventricular anti-arrhythmic action of 20 mg of betaxolol per day was evaluated in 14 patients treated in monotherapy for a fortnight, by means of repeated Holter recordings. A significant decrease in the number of ischemic ventricular extrasystoles was observed during the diurnal and nocturnal periods. Repetitive ventricular events were also reduced. Betaxolol therefore appears to have a direct anti-arrhythmic action which is not related to its action on the heart rate. The other effects of beta-blocker treatment were also observed: a decrease in the systolic and diastolic blood pressure and sinus bradycardia.

[Retrospective analysis of a series of 330 Holter recordings. Diagnostic value of the method and correlation with symptomology]. / Analyse rétrospective d'une série de 330 enregistrements de Holter. Valeur diagnostique de la méthode et corrélation avec la symptomatologie.

We have studied a series of 330 Holter recordings (HR) (including 5 double observations) 189 men and 136 women, mean age 58,4 years Old. The analysis of this series shows that: --105 HR were performed on patients with focal ischemia attacks of suputed embolic origin; the HRT was positive in 35 patients (33 p. 100) with 25 supraventricular arrhythmias (SVA) 16 ventricular arrhythmias, associated in 9 cases, and 3 conduction blocks second degrees type 2. These arrhythmias are rare in patients under 40, increasing with age, and reaching 53 p. 100 in patients greater than 70 years. --86 HR were performed in ischemic heart disease (IHD): 52 HR for ST segment analysis, positive in 5 cases, and coexisting with chest pain in 4 cases; 34 HR for detecting arrhythmias in IHD, positive in 18 cases with 14 VA and 4 SVA. In 83 p. 100 the arrhythmias occur without IHD. They were positive in 70 cases, with 49 SVA, 34 VA, associated in 17 cases, and 4 blocks second degrees type 2. --10 cardiomyopathies were recorded; the HR was positive in 6, with 4 SVA, 3 VA associated in 1 case. --8 mitral valve prolapses were recorded with 5 VA and 1 SVA. In conclusion, the HR was positive in 45 p. 100 of the cases, and show especially the great incidence of asymptomatic VA in patients with IHD.