ABSTRACT
Pasteurella multocida toxin (PMT) activates the G-proteins Gαi(1â3), Gα(q), Gα11, Gα12 and Gα13 by deamidation of specific glutamine residues. A number of these alpha subunits have signalling roles in neurones. Hence we studied the action of this toxin on rat superior cervical ganglion (SCG) neurones and NG108-15 neuronal cells. Both Gα(q) and Gα11 could be identified in SCGs with immunocytochemistry. PMT had no direct action on Kv7 or Cav2 channels in SCGs. However PMT treatment enhanced muscarinic receptor mediated inhibition of M-current (Kv7.2 + 7. 3) as measured by a 19-fold leftward shift in the oxotremorine-M concentration-inhibition curve. Agonists of other receptors, such as bradykinin or angiotensin, that inhibit M-current did not produce this effect. However the amount of PIP2 hydrolysis could be enhanced by PMT for all three agonists. In a transduction system in SCGs that is unlikely to be affected by PMT, Go mediated inhibition of calcium current, PMT was ineffective whereas the response was blocked by pertussis toxin as expected. M1 muscarinic receptor evoked calcium mobilisation in transformed NG108-15 cells was enhanced by PMT. The calcium rises evoked by uridine triphosphate acting on endogenous P2Y2 receptors in NG108-15 cells were enhanced by PMT. The time and concentration dependence of the PMT effect was different for the resting calcium compared to the calcium rise produced by activation of P2Y2 receptors. PMT's action on these neuronal cells would suggest that if it got into the brain, symptoms of a hyperexcitable nature would be seen, such as seizures.
Subject(s)
Bacterial Proteins/pharmacology , Bacterial Toxins/pharmacology , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Neurons/drug effects , Animals , Calcium/metabolism , Cells, Cultured , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effectsABSTRACT
Two new prototype assembly methods have been evaluated for biosensors that combine an integrated circuit (IC) sensor with a culture chamber. The first method uses a poly-ethylene glycol (PEG) mould to mask the IC sensor during application of a room temperature vulcanising (RTV) silicone elastomer used to insulate the bondpads and bondwires. The second method utilises the 'partial encapsulation' service offered by Quik-Pak, USA. Both methods were shown to provide good electrical insulation and demonstrated biocompatibility with the NG108-15 cell line. These methods are particularly useful for the assembly of low-cost ICs with a small (< 4 mm²) sensor area.
