Prevalence and pathogenicity of binary toxin-positive Clostridium difficile strains that do not produce toxins A and B.

Clostridium difficile causes antibiotic-associated diarrhoea and pseudomembranous colitis. The main virulence factors of C. difficile are the toxins A (TcdA) and B (TcdB). A third toxin, called binary toxin (CDT), can be detected in 17% to 23% of strains, but its role in human disease has not been clearly defined. We report six independent cases of patients with diarrhoea suspected of having C. difficile infection due to strains from toxinotype XI/PCR ribotype 033 or 033-like, an unusual toxinotype/PCR ribotype positive for CDT but negative for TcdA and TcdB. Four patients were considered truly infected by clinicians and were specifically treated with oral metronidazole. One of the cases was identified during a prevalence study of A(-)B(-)CDT(+) strains. In this study, we screened a French collection of 220 nontoxigenic strains and found only one (0.5%) toxinotype XI/PCR ribotype 033 or 033-like strain. The description of such strains raises the question of the role of binary toxin as a virulence factor and could have implications for laboratory diagnostics that currently rarely include testing for binary toxin.

Alpha2-adrenoceptor activation increases a cationic conductance and spontaneous GABAergic synaptic activity in dopaminergic neurones of the rat substantia nigra.

Noradrenaline (NA) plays an important role in compensating for the loss in dopaminergic (DA) function following lesions of the DA neurones of the substantia nigra (SN). Alpha2-adrenoceptors are largely expressed in these neurones, but the cellular response to their activation is unknown. Whole-cell patch-clamp recordings were made from DA neurones of rat SN. At a holding potential of -60 mV, bath application of NA (50 microM) induced an inward current (-20.3+/-10.0 pA) in 50% of the recorded neurones. This effect was mimicked by UK-14304 (50 microM), a specific alpha2-adrenoceptor agonist, whereas alpha1-adrenoceptor and beta-adrenoceptor agonists failed to induce a response. Surprisingly, alpha2-adrenoceptor antagonists (idazoxan, RX-811059, SKF-86466 and yohimbine) also induced an inward current that could occlude the one induced by UK-14304, suggesting that they may act as alpha2-adrenoceptor agonists. The inward current results from an increase in cationic conductance identical to the one previously described in these neurones, as neurotensin (1 microM), known to activate it, occluded the inward current induced by UK-14304. In addition, GABAergic miniature inhibitory postsynaptic current frequency was increased by activation of presynaptic alpha2-adrenoceptors. We conclude that the effects of NA on alpha2-adrenoceptors can contribute to the previously described composite action of NA on DA neurone firing and can be pharmacologically differentiated from the effect of NA on DA and neighbouring neurones known to be mediated through alpha1-adrenoceptors.

Neurotensin inhibition of the hyperpolarization-activated cation current (Ih) in the rat substantia nigra pars compacta implicates the protein kinase C pathway.

1. Whole-cell patch-clamp recording was performed from principal neurones of the substantia nigra pars compacta (SNc). In 66% of these neurones, neurotensin (NT) induced, at -60 mV, an inward current associated with an increase in conductance. 2. Principal neurones displayed, in response to hyperpolarizing voltage steps, the voltage-dependent inward cationic current, Ih. This current activated at potentials more negative than -65 mV and reached a maximum at -106 +/- 4 mV, with a half-activation potential of -86 +/- 3 mV. Its estimated reversal potential was -43 +/- 7 mV and its activation curve was fitted with two exponentials. 3. In 41% of neurones showing the inward current, NT (0.5 microM) also reversibly reduced the amplitude of Ih. The diminution was 48.5 +/- 12% when voltage steps were made from -60 to -95 mV. The decrease in Ih resulted from a reduction in the maximal current with no change in the voltage dependence of activation. 4. Forskolin (10 microM), an activator of adenylate cyclase, increased Ih by shifting its activation range to more positive potentials, but it did not alter the NT inhibition of Ih. 5. The effect of NT was blocked by staurosporine (0.5 microM) and by PKC-(19-31) (0.5 microM), a specific protein kinase C (PKC) inhibitor, but was unaffected by Walsh's peptide (100 microM), a specific inhibitor of protein kinase A. The reduction of Ih was mimicked by 1-oleoyl-2-acetyl-sn-glycerol (0.5-10 microM), an analogue of diacylglycerol, an endogenous PKC activator. 6. These results suggest that the inhibition of Ih by NT involves a phosphorylation mechanism that implies activation of PKC.

Hepatocytic ultrastructure in splenectomized rats treated with pregnenolone-16alpha-carbonitrile, a microsomal enzyme inducer.

Pregnenolone-16alpha-carbonitrile (PCN), given orally at the dose of 6.8 mg/100 g body wt, twice daily for 3 or 6 days, increased liver weight in intact rats, and reduced zoxazolamine paralysis in both unoperated and splenectomized animals. The steroid induced smooth endoplasmic reticulum proliferation in the hepatocytes of intact and splenectomized rats, while splenectomy alone caused rough endoplasmic reticulum fragmentation and vesiculation. It appears that the spleen does not influence the hepatic action of PCN.

Effect of catecholamines on the hyperpolarization-activated cationic Ih and the inwardly rectifying potassium I(Kir) currents in the rat substantia nigra pars compacta.

Whole-cell ruptured-patch and perforated-patch recordings were used in principal neurons of the rat substantia nigra pars compacta (SNc) to study the effect of catecholamines both on the hyperpolarization-activated cationic (Ih) and the inwardly rectifying potassium (I(Kir)) currents. In internal potassium, a 2 min bath application of noradrenaline (NA; 50 microM) or dopamine (DA; 50 microM) both inhibited Ih and induced an outward current associated with an increase in I(Kir) conductance. These two effects recovered poorly after wash-out. Protein kinase A (PKA), protein kinase C (PKC) and phosphatases 1 and 2A inhibitors did not modify the NA and DA effects on the amplitude of Ih and I(Kir) currents. They also had no effect on the recovery of the catecholamine responses. In perforated-patch experiments, NA and DA also induced an inhibition of Ih and revealed an outward current associated with an increase in conductance. However, both effects recovered in less than 5 min following the wash-out. These results indicate that neither PKA, PKC, nor phosphatases 1 or 2A were required in the NA and DA modulation of these two currents and that an intracellular factor, that could be either washed-out or inversely up-regulated in the ruptured-patch configuration, was implicated in the recovery of both effects. In the presence of external barium (300 microM) or internal caesium which both blocked the outward current and the increase in conductance, neither NA nor DA affected Ih, suggesting that the effect on Ih observed is secondary to the activation of the I(Kir) channels. Increasing chloride conductance of the cell by activation of GABA(A) receptors also induced an inhibition of Ih. All together these results suggest that the NA or DA induced inhibition of Ih could result from an occlusion of Ih by a space-clamp effect.

Modulation of glycine responses by dihydropyridines and verapamil in rat spinal neurons.

Although glycine receptors (GlyRs) are responsible for the main spinal inhibitory responses in adult vertebrates, in the embryo they have been reported to mediate depolarizing responses, which can sometimes activate dihydropyridine-sensitive L-type calcium channels. However, these channels are not the only targets of dihydropyridines (DHPs), and we questioned whether GlyRs might be directly modulated by DHPs. By whole-cell recording of cultured spinal neurons, we investigated modulation of glycine responses by the calcium channel antagonists, nifedipine, nitrendipine, nicardipine and (R)-Bay K 8644, and by the calcium channel, agonist (S)-Bay K 8644. At concentrations between 1 and 10 microM, all these DHPs could block glycine responses, even in the absence of extracellular Ca2+. The block was stronger at higher glycine concentrations, and increased with time during each glycine application. Nicardipine blocked GABAA responses from the same neurons in a similar manner. In addition to their blocking effects, nitrendipine and nicardipine potentiated the peak responses to low glycine concentrations. Both effects of extracellular nitrendipine on glycine responses persisted when the drug was present in the intracellular solution. Thus, these modulations are related neither to calcium channel modulation nor to possible intracellular effects of DHPs. Another type of calcium antagonist, verapamil (10-50 microM), also blocked glycine responses. Our results suggest that some of the effects of calcium antagonists, including the neuroprotective and anticonvulsant effects of DHPs, might result partly from their interactions with ligand-gated chloride channels.

Developmental profile of the changing properties of NMDA receptors at cerebellar mossy fiber-granule cell synapses.

During cerebellar development, granule cells display well characterized changes in the expression of NMDA receptor (NMDAR) NR2 subunits, switching from NR2B to NR2A and NR2C in mature cells. Although various studies, including experiments on mutant mice with one or more NR2 subunit types deleted, suggest that NR2A, NR2B, and NR2C subunits contribute to synaptic NMDARs, changes in the properties of the mossy fiber EPSC during development have not been fully evaluated. In particular, information on NMDAR EPSCs in mature animals is lacking. We have examined pharmacological and kinetic properties of NMDARs at mossy fiber-granule cell synapses from their formation to maturity [postnatal day 7 (P7)-P40 rats]. Significant changes were seen in the relative amplitudes of the non-NMDAR- and NMDAR-mediated components of the evoked EPSC and in the decay kinetics of the latter. The NMDA/non-NMDA ratio was similar at P7, P21, and P40, but showed a clear peak at P12. This change coincided with a speeding of the NMDAR EPSC decay, accompanied by a decrease in sensitivity to ifenprodil (selective NR2B-antagonist). By P21, sensitivity of the NMDAR EPSC to Mg(2+) was approximately threefold less than that at P12 (IC(50), 76 vs 28 microm), suggesting incorporation of the NR2C subunit. However, the predicted slowing of decay kinetics to a value more characteristic of NR2C deactivation, was not seen until P40. Our data are consistent with the known switch from NR2B to NR2A subunits during the first two postnatal weeks, but suggest a gradual incorporation of the NR2C subunit that modifies Mg(2+) sensitivity and only later influences EPSC kinetics.