This report defines the identity of a calcium-regulated membrane guanylate cyclase transduction system in the cilia of olfactory sensory neurons, which is the site of odorant transduction. The membrane fraction of the neuroepithelial layer of the rat exhibited Ca(2+)-dependent guanylate cyclase activity, which was eliminated by the addition of EGTA. This indicated that the cyclase did not represent a rod outer segment guanylate cyclase (ROS-GC), which is inhibited by free Ca(2+). This interpretation was supported by studies with the Ca(2+) binding proteins, GCAPs (guanylate cyclase activating proteins), which stimulate photoreceptor ROS-GC in the absence of Ca(2+). They did not stimulate the olfactory neuroepithelial membrane guanylate cyclase. The olfactory neuroepithelium contained a Ca(2+) binding protein, neurocalcin, which stimulated the cyclase in a Ca(2+)-dependent fashion. The cyclase was cloned from the neuroepithelium and was found to be identical in structure to that of the previously cloned cyclase termed GC-D. The cyclase was expressed in a heterologous cell system, and was reconstituted with its Ca(2+)-dependent activity in the presence of recombinant neurocalcin. The reconstituted cyclase mimicked the native enzyme. Immunocytochemical studies showed that the guanylate cyclase coexists with neurocalcin in the apical region of the cilia. Deletion analysis showed that the neurocalcin-regulated domain resides at the C-terminal region of the cyclase. The findings establish the biochemical, molecular, and functional identity of a novel Ca(2+)-dependent membrane guanylate cyclase transduction system in the cilia of the olfactory epithelium, suggesting a mechanism of the olfactory neuroepithelial guanylate cyclase regulation fundamentally distinct from the phototransduction-linked ROS-GC.
The mechanism by which the individual odor signals are translated into the perception of smell in the brain is unknown. The signal processing occurs in the olfactory system which has three major components: olfactory neuroepithelium, olfactory bulb, and olfactory cortex. The neuroepithelial layer is composed of ciliated sensory neurons interspersed among supportive cells. The sensory neurons are the sites of odor transduction, a process that converts the odor signal into an electrical signal. The electrical signal is subsequently received by the neurons of the olfactory bulb, which process the signal and then relay it to the olfactory cortex in the brain. Apart from information about certain biochemical steps of odor transduction, there is almost no knowledge about the means by which the olfactory bulb and cortical neurons process this information. Through biochemical, functional, and immunohistochemical approaches, this study shows the presence of a Ca(2+)-modulated membrane guanylate cyclase (mGC) transduction system in the bulb portion of the olfactory system. The mGC is ROS-GC1. This is coexpressed with its specific modulator, guanylate cyclase activating protein type 1 (GCAP1), in the mitral cells. Thus, a new facet of the Ca(2+)-modulated GCAP1--ROS-GC1 signaling system, which, until now, was believed to be unique to phototransduction, has been revealed. The findings suggest a novel role for this system in the polarization and depolarization phenomena of mitral cells and also contradict the existing belief that no mGC besides GC-D exists in the olfactory neurons.
Aim:The present study was conducted to detect and identify the virulence genes in Pasteurella multocida isolates of porcine origin from Assam.Materials and Methods:A total of 21 porcine P. multocida isolates were subjected to capsular typing and detection of virulence-associated genes (pfhA, tbpA, hgbB, toxA, oma87, ompH, and nanB) using various polymerase chain reaction (PCR) methods reported elsewhere. Further, pathogenicity of the porcine isolates of P. multocida was studied in mice. For each strain of P. multocida selected for pathogenicity trial, the group of mice was injected intraperitoneally (i/p) with 0.1 ml of the inoculum prepared from respective field isolates, containing 109 organisms per ml.Results:Capsular typing of the isolates by multiplex PCR showed two capsular types, type A (66.66%) and type D (33.33%). All the isolates were positive for outer membrane protein genes, oma87 and ompH genes. Iron acquisition genes, tbpA and hgbB, were detected in 14.28% and 19.04% of the isolates. The dermonecrotoxin encoding gene, toxA, was present in 23.80% of the isolates. Filamentous hemagglutinin encoding gene, pfhA, was detected in 28.57%. The virulence gene distribution pattern of the isolates indicates the important role of the genes in disease pathogenesis.Conclusion:From the present study, it can be concluded that toxA gene is an important marker gene for defining the pathogenic potential of P. multocida strains in swine.
An investigation was carried out to compare the effect of growth of P. multocida type A of pig origin in iron restricted and in iron sufficient media on the basis of their outer membrane protein extract. Pasteurella multocida serotype A was cultured in two ways. In one process only BHI medium was used and on the other process same medium was used which was supplemented with 2, 2'-Dipyridyl (ironrestricted medium). Both these cultures were used to obtain Outer Membrane Proteins (OMPs) by extraction of bacterial cells with 1% Sarcosyl. Separation of the OMPs in SDS-PAGE showed that OMPs were mixture of protein fraction with molecular weight ranging from 110 to 22.6 kDa in case of ironrestricted OMP and 47.3 to 29.9 kDa in case of iron sufficient OMP. The OMP with molecular masses 29.9 kDa grew in both iron-restricted as well as in iron sufficient medium.
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