Biomedical subjects
Publications and source records attributed to S Wakisaka.
Vasoactive intestinal polypeptide (VIP)- and substance P (SP)-like immunoreactivities were examined in the tongue of the guinea pig by using the double immunofluorescence method. Coexistence of VIP- and SP-like immunoreactivities was suggested in many nerve fibers innervating the lingual salivary glands, as well as in a few intralingual ganglionic cells.
The incorporation of 3H-proline into the secretory ameloblasts of rat molar tooth germs cultured with or without colchicine was studied by light and electron microscope radioautography to determine the function of microtubules in the transport of precursor enamel protein from the rough-surfaced endoplasmic reticulum (rER) to the Golgi cisternae. The grain counts over the transitional vesicles, which accumulated in various cellular regions with colchicine treatment, continued to increase with chase time, unlike in controls. At 30 and 90 min chase, these counts were significantly higher than in controls. Moreover, the total grain count over the organelles (rER, pale granules, and transitional vesicles), which are positioned before the Golgi cisternae in the synthetic pathway, maintained a significantly higher level at 90 min chase in colchicine-treated tooth germs than in controls. The transport of synthesized protein to the Golgi cisternae via transitional vesicles was suppressed in colchicine-treated tooth germs. Some grains appeared with time over pale granular materials that appeared in the intercellular spaces of secretory ameloblasts with colchicine treatment. However, at each chase period, the grain count over pale granular materials was not so high as the count over the enamel in control. The present results indicate that colchicine affects the transport of newly synthesized protein from the rER to the Golgi cisterna via transitional vesicles, probably by interfering with the oriented transport related to microtubular function. It is suggested that the microtubular system may be concerned with the movement of the transitional vesicles.
The budding of small vesicles from the rough-surfaced endoplasmic reticulum (rER) was examined in the secretory ameloblast of rat molar tooth germs by ordinary fixation or prolonged osmium fixation. The budding of small vesicles from the rER was observed not only at the special region (transitional region) of the rER system, which abutted on the cis-face of the Golgi apparatus, but also at other regions of the rER in the secretory ameloblast. Small vesicles (presumed to be transitional vesicles) were adjacent to the rER, which also showed budding of vesicles. After prolonged osmium treatment, osmium deposits appeared in small vesicles, as well as in the cisternae of the cis saccule of the Golgi apparatus. Small vesicles containing osmium deposits were located at various regions of the cell, including the cis-face of the Golgi apparatus. These findings indicate that the budding of small vesicles from the rER is not restricted to the transitional region of the rER system of the secretory ameloblast, but is found at various regions of the cell. This indicates that newly synthesized proteins may be transferred from the rER cisternae to the transitional vesicles not only in the transitional region of the rER system adjacent to the Golgi apparatus, but also in other regions of the secretory ameloblast.
The distribution and origin of neurokinin A (NKA)-like immunoreactivity were investigated in feline dental pulp by an indirect immunofluorescence method. NKA-containing nerve fibres with varicosities, which entered the dental pulp via apical foramen, were distributed throughout this tissue. Many NKA-containing nerve fibres were localized around blood vessels, but some were observed apart therefrom. At the odontoblastic layer, thin NKA-containing nerve fibres were observed running straight toward the pulp-predentinal border between odontoblasts. After inferior alveolar nerve section, all NKA-containing nerve fibres disappeared in the dental pulp, while the removal of the superior cervical ganglion resulted in no change in the distribution of these fibres. The correlation of NKA-like immunoreactivity and substance P (SP)-like immunoreactivity was also investigated by double-immunofluorescence technique. The distribution of NKA-containing nerve fibres was very similar to that of SP-containing nerve fibres; it appeared that all NKA-containing nerve fibres contained SP.
Staining patterns of the surface and interior of maturing enamel of these tooth germs were examined using the glyoxal bis(2-hydroxyanil) (GBHA) solution which chelates with calcium loosely bound to hydroxyapatite to form insoluble red precipitates. An intense red, band-like pattern of GBHA staining, 1-2 mm wide, first appeared on the incisal portion of lingual enamel surface as complete loops, concentrically arranged; these gradually increased in number. Most of the later-formed bands encircled the entire periphery of the maturing enamel surface. GBHA also revealed reactive areas on the labio-lingual, cut and ground surface of maturing enamel, corresponding exactly to the surface GBHA bands. GBHA did not stain EDTA-etched surface enamel, but did reveal regular staining patterns on the ground surface, disclosed after EDTA treatment. These observations suggest an intimate correlation between the properties of interior and surface enamel, at least with regard to the state of local calcium. The maturation of bovine incisor enamel may start at the lingual aspect of the tooth crown.
The Golgi apparatus and Golgi-associated endoplasmic reticulum lysosome (GERL) were examined in the ameloblasts with a cytochemical marker, osmium impregnation, and two enzyme markers, thiamine pyrophosphatase (TPPase) and acid phosphatase (ACPase). In control cultured germs, osmium deposit appeared in one to two immature side cisternae of Golgi stacks; TPPase activity was restricted in a few mature side cisternae and condensing vacuoles. ACPase activity existed in the GERL and, sometimes, in the mature side-cisternae and condensing vacuoles. These findings show that Golgi stacks of ameloblasts consist of several distinct compartments. In colchicine-treated tooth germs, there were morphological and cytochemical changes in both Golgi stacks and GERL. The Golgi apparatus was fragmented and its stacks were scattered throughout the supranuclear region. In some stacks, the number of osmium-positive cisternae was greater than normal; in others they were absent. TPPase and ACPase activity was absent or diminished. These findings suggest the importance of microtubules in the organization of Golgi complex and GERL in the secretory ameloblast.
Substance P-like immunoreactive (SPLI) nerve fibers were demonstrated in the Krause corpuscles of the dog's tongue using the indirect immunofluorescence method and cholinesterase histochemistry. SPLI nerve fibers were often in contact with Krause end bulbs and occasionally entered them. From this result it was suggested that substance P might be involved in sensory mechanism of the Krause apparatus.
The occurrence of peptidergic cellular elements in the feline vagus nerve was examined by an immunohistochemical method. Some methionine-enkephalin-like immunoreactive cells were present around a few capillaries within the vagus nerve, whereas substance-P- and vasoactive-intestinal-polypeptide-like immunoreactive cells were not observed. The methionine-enkephalin-like immunoreactive cells had an oval shape and their diameter was 7-10 micron. These cells appeared to be in contact with the pericyte of the capillaries. Though the physiological function of these enkephalin-positive cells is unclear, our findings suggest that they can act as endocrine cells and release methionine-enkephalin into the capillaries.
The location of calcium in a rapid-frozen and freeze-substituted maturation stage enamel organ of the rat incisors was demonstrated by means of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, which formed insoluble red precipitates of calcium-GBHA complex. In the ameloblast layer, highly GBHA-reactive tubulo-vesicular structures corresponding to mitochondria and some other membrane-bound structures were localized in both ruffle-ended and smooth-ended ameloblasts, although no significant GBHA reaction was localized in the nucleus, Golgi region, nor along the plasma membrane of these cells. In addition, numerous granular GBHA reactions appeared exclusively in association with the ruffled border of ruffle-ended ameloblasts. GBHA reactions were positive, but were considerably weaker in papillary cells than in the ameloblast. These observations provide a first published histochemical mapping of calcium in the maturation stage enamel organ, and suggest the active participation of mitochondria in maturation stage ameloblasts in calcium regulation.
A method of combination therapy was proposed for the treatment of malignant gliomas, and the clinical results were reported. This combination consisted of ACNU (nimustine), UFT (tegafur + uracil), Radiation, vitamin A and PSK (krestin), and was named AUFRAP therapy. Intracarotid infusion of ACNU (100 or 150 mg/body) was done after the administration of vitamin A (100,000 units), in the first and last week of radiation therapy with a total dose of 60-70 Gy. UFT (400-600 mg/day) and PSK (3g/day) were also given orally. After the induction of remission, patients were treated in the outpatient clinic under a similar maintaining protocol. Two patients who received 150 mg of ACNU and daily 600 mg of UFT in combination with radiation therapy showed severe myelosuppression in the early stage of treatment, and the combination therapy was aborted. Two of four patients who received 100mg of ACNU and daily 400mg of UFT did not show such severe side effects and two showed transient, moderate myelosuppression and vomiting. The serum phenitoin concentration doubled and was thought to be a side effect of tegafur. All four patients completed the induction protocol and follow-up CT scans disclosed shrinkage of the remained tumors, decreased contrast enhancement or no evidence of recurrence. Interactions of these drugs and radiation were discussed and the literature was reviewed.
A rapid enzymatic assay method for ammonia was developed by using glutamine synthetase from glutamate-producing bacteria together with pyruvate kinase, lactate dehydrogenase, and NADH. The time required for determination of 25 nmol of ammonia was 5 min with 1 unit of glutamine synthetase, as opposed to 14-30 min with 1 unit of glutamate dehydrogenases from various sources. The present method was used to determine ammonia in serum, microbiol-culture broth, and waste water. The method can be modified for spectrophotometry in the visible region by substituting pyruvate oxidase, peroxidase, and appropriate chromogens for lactate dehydrogenase and NADH. With 4-aminoantipyrine (4AA) and phenol, and with 4AA and N-ethyl-N-2-hydroxyethyl-m-toluidine as chromogens, the sensitivity of ammonia determination was 0.65 and 1.7 times that with glutamate dehydrogenase, respectively. The present method was also applicable to the continuous detection of the activity of some ammonia-forming enzymes such as guanase, adenosine deaminase, and urease and to the determination of 0.5-30 microM ATP-ADP after some modification of the mixture.
The origin and distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity in feline dental pulp were studied using indirect immunofluorescence. Nerve fibres with varicosities exhibiting CGRP-like immunoreactivity were observed to enter the pulp with blood vessels. Many CGRP-containing nerve fibres were found to extend along blood vessels in the central pulp, and some of these fibres exhibited a network arrangement in the walls of dental pulp blood vessels. However, some of fibres were apparently not associated with blood vessels. Some thin, CGRP-containing nerve fibres formed a part of the nerve plexus in the subodontoblastic area and penetrated into the odontoblastic layer. In animals that had undergone transection of the inferior alveolar nerve, no CGRP-containing nerve fibres were observed. Application of a double-immunofluorescence staining technique also revealed that the distribution of CGRP-containing nerve fibres is very similar to that of substance P-containing nerve fibres.
The correlation between substance P (SP) and vasoactive intestinal polypeptide (VIP)-containing nerve fibres in the pulp was examined by double immunofluorescence. Both SP- and VIP-containing fibres entered the pulp in bundles with the blood vessels and spread throughout the pulp. In the coronal pulp, both SP- and VIP-containing nerve fibres formed networks on the walls of blood vessels, but both peptide-containing nerve fibres were not observed together in relation to vessels. There were more SP-containing fibres than VIP-containing ones. In the odontoblast layer, there were many SP-containing fibres but few VIP-containing ones.
Using an indirect immunofluorescence method, after 14 days, regenerated SP-containing nerve fibres first appeared around the blood vessels in apical and coronal regions of the pulp and apical and middle regions of the periodontal ligament. Penetration of regenerated SP-positive fibres into the dentine and predentine was observed 25 days after operation. The number of regenerated SP-containing fibres recovered to nearly to the same level as in intact controls by 35 days. They had a similar distribution in the tissues.
The influence of vanadate, a potent inhibitor of Ca2+-ATPase and Na+-K+-ATPase, on 45Ca uptake in maturing enamel of the rat incisor was investigated by a vascular perfusion method combined with 45Ca autoradiography. The morphological integrity of the maturation-stage enamel organ was well-retained during vascular perfusion under all the experimental conditions. Distinct patterns of 45Ca labeling, comparable with those found in previous in vivo 45Ca autoradiographic studies, appeared in the maturing enamel after vascular perfusion with a standard perfusate. One mmol/L vanadate added to the standard perfusate caused a drastic decrease in 45Ca uptake in the maturing enamel, corresponding to the ruffle-ended ameloblasts, leaving narrow peaks of moderate intensity corresponding to the bands of the overlying smooth-ended ameloblasts. The in vitro labeling of exposed enamel surfaces with 45Ca revealed blackening of autoradiographic emulsion in wide bands separated by unlabeled or slightly labeled narrow ones resembling the distribution of smooth-ended ameloblasts in both control and vanadate-treated incisors. Our observations indicate that the ruffle-ended ameloblasts of the rat incisor serve as an efficient diffusion barrier to calcium ions and regulate transcellular calcium transport to the maturing enamel, at least in part, by a vanadate-sensitive mechanism.
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