An assay method for bacterial collagenase using sterile collagen-agarose plates was developed to isolate collagenolytic bacteria. In order to avoid heat-denaturation of collagen, the plates were made at 37 °C mixing microwave-sterilized collagen with low melting point-agarose. Sensitivities to various proteases were tested on the plates; it was shown that whereas the collagen in these plates was digested only by the collagenases, the collagen could also be hydrolyzed by other proteolytic enzymes if it had been either sterilized by ethylene oxide or solidified with conventional agar at a higher temperature. Colonies of Clostridium histolyticum cells grown on the plates had clear zones around them and their collagenase activities could be readily detected.
The enamel organ of the growing rat incisor was perfusion-fixed with a mixture of formaldehyde and glutaraldehyde and processed for indirect immunogold labeling of calmodulin on post-embedded ultrathin sections. Throughout the zones of presecretion, secretion, and maturation of enamel, specific protein A-immunogold labeling was localized on polyribosomes and those attached to endoplasmic reticulum, mitochondria, nuclear chromatin, phagolysosomes, and cytoplasm adjacent to the plasma membrane, and tonofilaments associated with desmosomes of ameloblasts and cells of outer layer of enamel organ. Golgi membranes, condensing vacuoles, secretion granules, primary lysosomes, and micropinocytotic coated vesicles were hardly labeled. In the presecretion zone, the basal lamina of the preameloblasts and the matrix vesicles and collagen fibrils of the predentin matrix were not immunoreactive. Tomes' process of secretory ameloblast and adjacent enamel crystals were labeled. In addition to the above immunoreactive structures, some phagolysosomes, ferritin granules, and the cytoplasm of the ruffled border zone of maturation ameloblast contained immunogold particles. In control sections incubated with either protein A-gold complex alone, or antiserum preabsorbed with an excess of calmodulin and protein A-gold complex, only a few gold particles were observed to be randomly associated with the tissues. These results indicate that calmodulin is present in the cells of the enamel organ through all stages of amelogenesis. Its wide distribution is consistent with its involvement in various cytoplasmic functions.
The present study was to identify the physiological properties of the afferent fibers from the masseter muscle to the caudal part of the spinal trigeminal nucleus of rat using a microelectrode technique. When electrical stimulation was applied to the masseter muscle, the evoked potentials were recorded with a shorter latency (the S-response) or a longer latency (the L-response). The threshold current intensity of the S-response was lower than that of the L-response. There were statistical significant differences between the S-response and the L-response in latency and in threshold current intensity. Both the S-and L-responses could follow by stimulation of low frequencies (10 to 30 Hz), indicating that the evoked responses were the component of the secondary neuron activities. On the anatomical and physiological assumption, the conduction velocities of the S-and the L-responses were calculated and they were in the range of that of A-delta fibers. We also observed that both the 5-and the L-responses could not follow high frequency stimulation of the masseter muscle. This effect may reflect the phenomenon of fatigue in the finely myelinated fibers or polymodal nociceptors. Thus, the present study suggested the involvement of the afferents from the masseter muscle to the spinal trigeminal nucleus in the transmission and the relay of the masticatory muscle pain.
Our previous study reported that the afferents to the spinal trigeminal nucleus from the masseter muscle were predominantly of A-delta range fibers having fast conducting-low threshold and slow conducting-high threshold. In the present study, further experiment was undertaken to obtain more definite information on the central transmission of muscle pain. When the electrical stimulation was delivered to the masseter muscle of rat, the evoked potentials with a shorter latency (the S-response) or longer latency (the L-response) were recorded from the caudal part of the spinal trigeminal nucleus. In both the S-and the L-responses, the relationship between the amplitudes of the responses and the stimulus intensity almost followed the power function with the exponent of about 3.4 and 4.0, respectively. The stimulus intensity range of the S-response, in which the power function was formed, was wider than that of the L-response. The most of the S-response were recorded from the trigeminal subnucleus caudalis and adjacent reticular formation, whereas the recording sites of the L-response were located mainly in the trigeminal subnucleus caudalis. These results suggest that the trigeminal subnucleus caudalis and adjacent reticular formation are an important portion of the afferent projection areas and of the modulator system of pain sensation from the masseter muscle.