Prevalence of verotoxigenic Escherichia coli O157 (VTEC O157) and compliance with microbiological safety standards in bovine carcasses from an industrial beef slaughter plant.

UNLABELLED: The presence of Salmonella spp. and levels of Enterobacteriaceae and aerobic plate count were determined in 300 bovine carcasses randomly collected in an industrial cattle slaughterhouse in Catalonia (Spain) as part of a control programme to validate good slaughter practices according to Commission Regulation No 2073/2005. The verotoxigenic Escherichia coli O157 (VTEC O157), although not currently legislated, was also investigated in the same carcasses due to the importance of bovines as a reservoir for this micro-organism. Virulence genes (vtx1, vtx2 and eae), the presence of fliCH 7 and antimicrobial susceptibility were studied in E. coli O157 isolates. Levels of Enterobacteriaceae and aerobic colonies and the presence of Salmonella were within the admissible range stipulated by current legislation. However, VTEC O157 was detected in 14·7% of carcasses. Among the VTEC O157 strains tested for antimicrobial susceptibility, 65% were multiresistant. Overall, the results of this study indicate that even with good manufacturing practices, contamination with VTEC O157 can occur and cattle meat can pose a risk to human health. These results confirm the need for a review of the appropriateness of introducing antimicrobial treatments in the processing of cattle carcasses in Europe. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the prevalence of verotoxigenic and multidrug-resistant E. coli O157 strains in bovine carcasses. These results suggest that despite the good manufacturing practices used in the slaughterhouse studied (the largest in Catalonia slaughtering over 81 000 cattle per year), the absence of verotoxigenic E. coli O157 in bovine carcasses cannot be guaranteed.

Natural flavonoids inhibit the plasma membrane Ca2+-ATPase.

Research on flavonoids from plant sources has recently sparked increasing interest because of their beneficial health properties. Different studies have shown that flavonoids change the intracellular Ca2+ homeostasis linked to alterations in the function of mitochondria, Ca2+ channels and Ca2+ pumps. These findings hint at plasma membrane Ca2+-ATPase (PMCA) involvement, as it transports Ca2+ actively to the extracellular medium coupled to ATP hydrolysis, thus maintaining ion cellular homeostasis. The present study aims to investigate the effect of several natural flavonoids on PMCA both in isolated protein systems and in living cells, and to establish the relationship between flavonoid structure and inhibitory activity on PMCA. Our results show that natural flavonoids inhibited purified and membranous PMCA with different effectiveness: quercetin and gossypin were the most potent and their inhibition mechanisms seem to be different, as quercetin does not prevent ATP binding whereas gossypin does. Moreover, PMCA activity was inhibited in human embryonic kidney cells which transiently overexpress PMCA, suggesting that the effects observed on isolated systems could occur in a complex structure like a living cell. In conclusion, this work reveals a novel molecular mechanism through which flavonoids inhibit PMCA, which leads to Ca2+ homeostasis and signaling alterations in the cell.

Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis.

Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis. We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry. Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3-36-fold) and hemolysis (1-44-fold), partially compensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells. Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43-67%). The un-acylated toxin ProHlyA and the deletion analog HlyA∆914-936 were unable to induce ATP release or hemolysis. For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure. HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPe activation of purinergic P2X receptors reduced swelling by 60-80%. HlyA-RBCs showed an acute 1.3-2.2-fold increase of Ca2+i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media. Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity.

Volumetric response of vertebrate hepatocytes challenged by osmotic gradients: a theoretical approach.

In this study we use a theoretical approach to study the volumetric response of goldfish hepatocytes challenged by osmotic gradients and compared it with that of hepatocytes from another teleost (the trout) and a mammal (the rat). Particular focus was given to the multiple non-linear interactions of transport systems enabling hypotonically challenged cells to trigger a compensatory response known as volume regulatory decrease or RVD. For this purpose we employed a mathematical model which describes the rates of change of the intracellular concentrations of main diffusible ions, of the cell volume, and of the membrane potential. The model was fitted to experimental data on the kinetics of volume change of hepatocytes challenged by anisotonic media. In trout and rat hepatocytes, experimental results had shown that hypotonic cell swelling was followed by RVD, whereas goldfish cells swelled with no concomitant RVD (M.V. Espelt et al., 2003, J. Exp. Biol. 206, 513-522). A comparison between data predicted by the model and that obtained experimentally suggests that in trout and rat hepatocytes hypotonicity activates a sensor element and this, in turn, activates an otherwise silent efflux of KCl - whose kinetics could be successfully predicted - thereby leading to volume down-regulation. In contrast, with regard to the absence of RVD in goldfish hepatocytes the model proposed suggests that either a sensor element triggering RVD is absent or that the effector mechanism (the loss of KCl) remains inactive under the conditions employed. In line with this, we recently found that extracellular nucleotides may be required to induce RVD in these cells, indicating that our model could indeed lead to useful predictions.

Effect of continuous feeding of CO2 and pH in cell concentration and product titers in hIFNγ producing HEK293 cells: Induced metabolic shift for concomitant consumption of glucose and lactate.

Although pH control at physiological levels is generally considered as the optimal culture condition, in some cases other strategies should be taken into account for their beneficial effects on process performance. pH and CO2 levels are chemical variables that have a major impact in cell growth and product titers in cell culture since their effect on key metabolic routes. HEK293 cells expressing recombinant hIFNγ showed different metabolic behavior when cultured in shake flask compared to pH-controlled bioreactors, in which a decrease in cell density and product titer were observed. This yield loss observed in bioreactor cultures could be reverted by adding 1% CO2 to air inlet flow in a non-controlled pH bioprocess. With this strategy, a significant outcome of 4-fold increase in terms of maximum cell density and 2-fold increase in volumetric concentration of recombinant protein (hIFNγ) when compared to the pH-controlled culture in bioreactor (standard culture conditions) has been obtained. Results evidenced the importance of pH and CO2 concentration in this case, in order to reproduce the behavior observed in optimization experiments performed in shake flasks. Thus, it was demonstrated that not always constant controlled variable setpoint (like pH or CO2 addition) becomes the best bioprocess performance strategy.

Identification of two distinct E-NTPDases in liver of goldfish (Carassius auratus L.).

We have recently reported the existence of ATPase activity capable of hydrolyzing extracellular ATP and localized at the external cell membrane of goldfish hepatocytes [Am. J. Physiol. (1998) 274 R1031]. In the present study, we investigated whether one or more enzymes of the ATP diphosphohydrolase family (called E-NTPDases) are responsible for the hydrolysis of extracellular ATP and other nucleotides. Using soluble extracts from goldfish liver, enzyme activity was detected in the presence of ATP (32.1+/-4.0 nmol Pi liberated mg protein(-1) min(-1)), ADP (20.7+/-3.3 nmol Pi liberated mg protein(-1) min(-1)) and UTP (20.7+/-1.2 nmol Pi liberated mg protein(-1) min(-1)). In line with the presence of this hydrolytic activity, liver samples separated by non-denaturing gel electrophoresis and subsequently exposed to either ATP, ADP or UTP yielded a single band with enzyme activity and similar electrophoretic mobility. Subsequent SDS-PAGE electrophoresis of the active bands resulted in the appearance of two protein bands with molecular masses of 70 and 64 kDa. Immunoblotting of soluble extracts and microsomes obtained from goldfish liver, using a monoclonal antibody against CD39 (a well-known E-NTPDase), detected a single 97-kDa protein. The enzyme activity measured in solution and in native gels, together with structural information from denaturing gels plus immunoblots, points to the existence, in goldfish liver, of at least two different E-NTPDases.

Hormone uptake and morphological changes in cultured pituicytes exposed to oxytocin and vasopressin.

Enzymatically dispersed cells, isolated from adult female rat neural lobes, were cultured for 7 days. Routine cultures showed pituicytes with compact, sometimes ovoid, cell bodies. The cytoplasmic processes of these cells exhibited several varicosities and made contact with neighboring cells forming networks. The cultured pituicytes were immunocytochemically characterized using antisera to glial fibrillary acidic protein and to S-100. Most pituicytes, when exposed during culture to oxytocin (OXY) and vasopressin (VP; 1 microM each), were devoid of their characteristic processes. Immunocytochemical staining for OXY or VP revealed that the pituicytes were capable of incorporating these hormones during culture. In cultures without added hormones, no significant staining reaction for OXY or VP could be detected. The lack of projections in pituicytes exposed to the hormones during culture is in agreement with the morphological changes observed by other authors in situ after acute hormone release. The uptake of OXY and VP may be indicative for a regulatory mechanism, by which the pituicytes control the amount of hormones present in the intercellular space.

Knockdown of hepatocyte aquaporin-8 by RNA interference induces defective bile canalicular water transport.

Aquaporin-8 (AQP8) water channels, which are expressed in rat hepatocyte bile canalicular membranes, are involved in water transport during bile formation. Nevertheless, there is no conclusive evidence that AQP8 mediates water secretion into the bile canaliculus. In this study, we directly evaluated whether AQP8 gene silencing by RNA interference inhibits canalicular water secretion in the human hepatocyte-derived cell line, HepG2. By RT-PCR and immunoblotting we found that HepG2 cells express AQP8 and by confocal immunofluorescence microscopy that it is localized intracellularly and on the canalicular membrane, as described in rat hepatocytes. We also verified the expression of AQP8 in normal human liver. Forty-eight hours after transfection of HepG2 cells with RNA duplexes targeting two different regions of human AQP8 molecule, the levels of AQP8 protein specifically decreased by 60-70%. We found that AQP8 knockdown cells showed a significant decline in the canalicular volume of approximately 70% (P < 0.01), suggesting an impairment in the basal (nonstimulated) canalicular water movement. We also found that the decreased AQP8 expression inhibited the canalicular water transport in response either to an inward osmotic gradient (-65%, P < 0.05) or to the bile secretory agonist dibutyryl cAMP (-80%, P < 0.05). Our data suggest that AQP8 plays a major role in water transport across canalicular membrane of HepG2 cells and support the notion that defective expression of AQP8 causes bile secretory dysfunction in human hepatocytes.

Potassium transmembrane fluxes in anoxic hepatocytes from goldfish (Carassius auratus L.).

Despite the fact that anoxic goldfish hepatocytes can maintain the transmembrane gradients of Na(+), H(+) and Ca(2+), cyanide (CN) intoxication leads to a rapid breakdown of K(+) homeostasis. In this study, [(86)Rb(+)] K(+) fluxes across the plasma membrane of goldfish hepatocytes were studied in order to identify the possible causes of this imbalance. Four minutes of cyanide incubation induced an acute and stable 61% decrease of K(+) influx (mostly driven by Na,K-ATPase activity), whereas K(+) efflux increased by 24.3%, this imbalance yielding a net K(+) efflux of 0.279+/-0.024 nmol 10(-6) cells(-1) min(-1). This uncoupling was not observed when glycolytic ATP production was inhibited with iodoacetic acid. Although the CN-induced decrease of K(+) influx was fully reversible upon washout of the inhibitor, it could not be prevented by any of the following treatments: (1) addition of 2% bovine serum albumin, which binds extracellular fatty acids known to activate specific K(+) channels; (2) addition of ascorbate, which acts as a radical scavenger; (3) inclusion of 5 mM glucose as an extracellular carbon source; and (4) removal of medium oxygen (obtained by nitrogen bubbling). Regarding the elevation of K(+) efflux in the presence of CN, neither ATP-dependent K(+) channels nor the KCl cotransporter appeared to be activated, whereas BaCl(2), an inhibitor of voltage-gated K(+) channels, decreased K(+) efflux of CN-intoxicated cells to control levels. In summary, these results indicate that, in goldfish hepatocytes, the CN-induced K(+) imbalance results from acute Na,K-ATPase inhibition together with the activation of voltage-dependent K(+) channels, the latter probably resulting from transient membrane depolarization.

Volumetric and ionic responses of goldfish hepatocytes to anisotonic exposure and energetic limitation.

The relationship between cell volume and K(+) transmembrane fluxes of goldfish (Carassius auratus) hepatocytes exposed to anisotonic conditions or energetic limitation was studied and compared with the response of hepatocytes from trout (Oncorhynchus mykiss) and rat (Rattus rattus). Cell volume was studied by video- and fluorescence microscopy, while K(+) fluxes were assessed by measuring unidirectional (86)Rb(+) fluxes. In trout and rat hepatocytes, hyposmotic (180 mosmoll(-1)) exposure at pH 7.45 caused cell swelling followed by a regulatory volume decrease (RVD), a response reported to be mediated by net efflux of KCl and osmotically obliged water. By contrast, goldfish hepatocytes swelled but showed no RVD under these conditions. Although in goldfish hepatocytes a net ((86)Rb(+))K(+) efflux could be activated by N-ethylmaleimide, this flux was not, or only partially, activated by hyposmotic swelling (120-180 mosmoll(-1)). Blockage of glycolysis by iodoacetic acid (IAA) did not alter cell volume in goldfish hepatocytes, whereas in the presence of cyanide (CN(-)), an inhibitor of oxidative phosphorylation, or CN(-) plus IAA (CN(-)+IAA), cell volume decreased by 3-7%. Although in goldfish hepatocytes, energetic limitation had no effect on ((86)Rb(+))K(+) efflux, ((86)Rb(+))K(+) influx decreased by 57-66% in the presence of CN(-) and CN(-)+IAA but was not significantly altered by IAA alone. Intracellular K(+) loss after 20 min of exposure to CN(-) and CN(-)+IAA amounted to only 3% of the total intracellular K(+). Collectively, these observations suggest that goldfish hepatocytes, unlike hepatocytes of anoxia-intolerant species, avoid a decoupling of transmembrane K(+) fluxes in response to an osmotic challenge. This may underlie both the inability of swollen cells to undergo RVD but also the capability of anoxic cells to maintain intracellular K(+) concentrations that are almost unaltered, thereby prolonging cell survival.

Hormone uptake and morphological changes in cultured pituicytes exposed to oxytocin and vasopressin

Enzymatically dispersed cells, isolated from adult female rat neural lobes, were cultured for 7 days. Routine cultures showed pituicytes with compact, sometimes ovoid, cell bodies. The cytoplasmic processes of these cells exhibited several varicosities and made contact with neighboring cells forming networks. The cultured pituicytes were immunocytochemically characterized using antisera to glial fibrillary acidic protein and to S-100. Most pituicytes, when exposed during culture to oxytocin (OXY) and vasopressin (VP; 1 microM each), were devoid of their characteristic processes. Immunocytochemical staining for OXY or VP revealed that the pituicytes were capable of incorporating these hormones during culture. In cultures without added hormones, no significant staining reaction for OXY or VP could be detected. The lack of projections in pituicytes exposed to the hormones during culture is in agreement with the morphological changes observed by other authors in situ after acute hormone release. The uptake of OXY and VP may be indicative for a regulatory mechanism, by which the pituicytes control the amount of hormones present in the intercellular space