Calbindin D28k-like immunoreactivity in the developing and regenerating circumvallate papilla of the rat

The distribution of calbindin D28k (CB)-like immunoreactivity (-LI) in the circumvallate papilla (CVP) was examined during development and regeneration following bilateral crush injury to the glossopharyngeal nerve in the rat. In the adult CVP, CB-like immunoreactive (-IR) nerve fibers were observed in the subgemmal region and some penetrated into the taste buds. CB-LI was also detected in the cytoplasm of the spindle-shaped gustatory cells in the lower half of the trench epithelium, which contained numerous synaptic vesicles and bundles of intermediate filaments. These CB-IR gustatory cells made synapse-like contacts with CB-IR nerve terminals. Some CB-IR nerve terminals made contacts with the gustatory cells negative for CB-LI. At least three developmental stages were defined with regard to the developmental changes in the distribution of CB-LI: (1) Stage I (embryonic day (E) 18–postnatal day (P)5): CB-IR nerve fibers appeared in the lamina propria just beneath the newly-formed CVP at E18, but the gustatory epithelium of the CVP contained no CB-IR structures. Taste buds with taste pores appeared at P1. (2) Stage II (P5–10): thin CB-IR nerve fibers began entering the trench epithelium, but no CB-IR cells were observed. (3) Stage III (P10–adult): in addition to the intragemmal and perigemmal CB-IR nerve fibers, very few CB-IR cells appeared in the taste buds around P10, and their numbers increased progressively. The changes in the distribution of taste buds and CB-LI following glossopharyngeal nerve injury were similar to those observed during development. On post-operative day (PO) 4, the taste buds and CB-IR cells decreased markedly in number. These CB-IR cells became round in shape, and the number of CB-IR nerve fibers decreased markedly. On PO8, both taste buds and CB-IR cells disappeared completely. The regenerated taste buds were first observed on PO12, increased rapidly in number by PO20, and increased slowly thereafter. CB-IR nerve fibers accumulated at the subgemmal region and began penetrating into the trench wall epithelium around PO16. CB-IR cells appeared between PO20 and PO24, and their numbers increased progressively and reached the normal level on PO40. The topographical localizations of the taste buds and CB-IR cells during development and regeneration were comparable to those of normal animals. The delay of the time courses for appearance of CB-IR nerve fibers and CB-IR cells compared to the appearance of taste buds during development and regeneration suggests that CB in the gustatory epithelium may participate in the survival of the taste bud cells rather than in the induction of the taste buds.     

Cryopreservation of developing teeth

By using a slow rate of cooling, successful storage of developing teeth has been performed in liquid nitrogen. Dye exclusion tests indicate that 78 ± 8% of cells remain viable and transplantation to isologous hosts demonstrates that 86% of the stored developing teeth commence to grow and produce normal dental hard and soft tissue elements. The length of storage at −196 °C, for periods up to 3 months, appears to have no detrimental effect upon subsequent survival and development of tooth germs.     

Phosphoglucose isomerase in developing teeth

Summary The distribution of phosphoglucose isomerase in developing teeth of rats, using an indirect tetrazolium technique, is described. The demonstration of this enzyme, which occupies a position of considerable importance in carbohydrate metabolism, provides additional information regarding the biochemistry of odontogenic cells.     

Glucagon stimulation of ruthenium red-insensitive calcium ion transport in developing rat liver.

The maturation of glucagon-stimulated Ruthenium Red-insensitive Ca2+-transport activity was determined in livers of rats ranging in age from 5 days preterm to 10 weeks of adult life. Previous indications are that this activity is confined to vesicles derived mainly from the endoplasmic reticulum. Perinatal-rat liver contains near-adult values of Ruthenium Red-insensitive Ca2+-transport activity, and exhibits large transient increases in the rate of this activity at two stages of development, immediately after birth, and at 2-5 days after birth. The administration of glucagon to foetal rats, at developmental stages after 19.5 days of gestation (2.5 days before birth), results in a large stable increase (greater than 100%) of Ca2+-transport activity in a subsequently isolated 'heavy' microsomal fraction. That this fraction was enriched in vesicles derived from the rough endoplasmic reticulum was indicated by both an electron-microscopic examination and a marker-enzyme analysis of the subcellular fractions. The administration of glucagon into newborn animals only hours old does not enhance further the initial rate of Ca2+-transport activity, and from day 1 to 10 weeks after birth the administration of the hormone results in the moderate enhancement of Ca2+ transport. Experiments with cyclic AMP and inhibitors of phosphodiesterase activity suggest that cyclic AMP plays a key role in the enhancement by glucagon of Ruthenium Red-insensitive Ca2+ transport, and arguments are presented that this transport system has an important metabolic role in the redistribution of intracellular Ca2+ in liver tissue.     

Monoclonal antibodies directed against the calcium binding protein Calbindin D-28k

We have produced 25 clones secreting antibodies directed against chicken Calbindin D-28k. Two of them, 300 and 318, recognize determinants conserved in fish, chicken, mouse, rat, rabbit, monkey and human Calbindin D-28k. We demonstrate their use in the immunohistochemical localization of Calbindin D-28k, and in the detection of Calbindin D-28k on immunoblots.     

Histochemical studies of dehydrogenases in the developing teeth

Summary Histochemical observations were made of oxidative and reducing enzymes in relation to several metabolic pathways in the developing tooth. The localization and activity of dehydrogenases and oxidases could be divided roughly into two types.Type one refers to enzymatic activities of lactic, succinic, malic and glucose-6-phosphate dehydrogenase and aconitase and cytochrome oxidase, which were low in undifferentiated dental epithelium and increased in proportion to cell differentiation. Comparatively high enzymatic activities were reflected in high cell function.Type two did not increase in the enzymatic activity during differentiation. Glyceraldehyde-3-phosphate, TPN isocitric, 6-phosphogluconate, glutamic, -glycerophosphate and -hydroxybutyric dehydrogenase and monoamine oxidase belong to this type.With 12 Figures in the TextPresented in Parts at the 3rd Annual Meeting in September, 1962 of the Japanese Histochemical Association.     

The "independence principle" in the processes of water transport.

The processes of membrane transport exhibiting permeability coefficients depending on the species activities do not obey the "independence principle" and are assumed to take place by a mechanism of discrete nature, analyzable by a kinetic formalism. In this article, we study the dependence of the osmotic permeability coefficient on the water activities, from the steady-state analysis of a kinetic model of single-file water transport that simultaneously incorporates the vacancy-mediated and "knock-on" mechanisms into the state diagram. In particular, we study the relation between the near-equilibrium osmotic permeability (Pe) and the equilibrium water activity of the compartments (w). The analysis and numerical calculations performed for a simple case of the model show that, for values of the parameters consistent with experimental data, Pe exhibits only a small variation with w within the physiological range in the majority of the situations considered here. It is not possible to predict, from the study of these simple models, whether more complicated kinetic diagrams of water transport may be characterized by permeability coefficients with a more evident dependence on the water activities. Nevertheless, the results obtained here suggest that, for the case of physiological water pores, the analysis of the kinetic dependence of the permeability coefficients on the water activities may not yield evidence pointing to a discrete nature for the transport process.     

Immunofluorescent localization of amelogenins in developing bovine teeth.

Antiserum was prepared to the proteins (amelogenins) isolated from fetal bovine enamel matrix. This antiserum was used to localize the amelogenins in the developing bovine molar by immunofluorescent microscopy. Amelogenins could be identified in the preameloblasts before enamel matrix deposition had begun as well as in the secretory ameloblasts. The closely adherent layer of stratum intermedium cells also contained some immunoreactive material, suggesting that they may contribute protein to the enamel matrix. The newly deposited enamel matrix consisted of brightly fluorescent particles. Mature enamel matrix did not contain the immunoreactive protein except in a thin layer along the dentino-enamel junction and adjacent to the ameloblasts. No other portion of the tooth bud or other tissues reacted with the specific antiserum.     

Characterization of the calcium biomineral in the radular teeth of Chiton pelliserpentis

The radula in a group of molluscan invertebrates, the chitons (Polyplacophora), is a ribbon-like apparatus used for feeding and which bears a series of distinctive mineralized teeth called the major lateral teeth. While some chiton species deposit only iron biominerals in these teeth, many others deposit both iron and calcium. In this study, the calcium biomineral in the teeth of one of the latter types of species, the Australian east-coast chiton, Chiton pelliserpentis, has been isolated and examined for the first time. Spectroscopic and crystallographic techniques have identified the biomineral as a carbonate-substituted apatite with significant fluoride substitution also likely. Fourier-transform infrared and laser Raman spectroscopy indicated that the carbonate content was less than that of either bovine tibia cortical bone or human tooth enamel. X-ray diffraction analysis showed the biomineral to be poorly crystalline due to small crystal size and appreciable anionic substitution. The lattice parameters were calculated to be a=9.382?Å and c=6.883?Å, which are suggestive of a fluorapatite material. It is postulated that structural and biochemical differences in the tooth organic matrix of different chiton species will ultimately determine if the teeth become partly calcified or iron mineralized only.     

Dietary-induced modification of calcium transport in mitochondria isolated from flight-muscle of developing sheep blowfly Lucilia cuprina

The activity of mitochondrial calcium transport in flight-muscle of the sheep blowfly $\text{Lucilia cuprina}$ is shown to depend on the nature of the diet upon which the adult fly is maintained. The finding raises the possibility that calcium transport in mitochondria from other tissues and species also might be influenced by the dietary intake.     

Somatostatin and intestinal calcium transport in the rat

In intact rats we studied the influence of low doses of intravenously (i.v.) administered somatostatin (SRIF) on the net absorption and the bidirectional fluxes (lumen-to-plasma, LP; plasma-to-lumen, PL) of calcium in the duodenum, jejunum, ileum and caecum. In the duodenum SRIF inhibited the LP-flux and the net absorption of Ca significantly at infusion rates of 0.75 and 1.0 microgram SRIF . kg-1 . h-1. The PL-flux was not altered by any of the SRIF doses administered. In the other gut segments studied (jejunum, ileum, caecum) neither the net absorption nor the bidirectional Ca fluxes were changed by i.v. SRIF. It is concluded that SRIF in the plasma levels achieved in this study has an influence on the duodenal calcium absorption (CaA) of the rat; questions regarding the mechanisms of this action as well as the physiological significance of our findings are as yet unresolved.     

Expression patterns of neurotrophic factor mRNAs in developing human teeth

Neurotrophic factors regulate survival, differentiation, growth and plasticity in the nervous system. In addition, based on their specific and shifting temporospatial expression patterns, neurotrophic factors have been implicated in morphogenetic events during tooth development in rodents. To determine whether these findings in rodents could be related to humans, we have now studied nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell-line derived neurotrophic factor (GDNF), and neurturin (NTN) mRNA expression patterns in developing human teeth during gestational weeks 6.5-11. Using in situ hybridization histochemistry, we found distinct and specific patterns of neurotrophin and GDNF mRNA expression in the developing human teeth. NGF mRNA labeling was weak and confined predominantly to the dental papilla. BDNF mRNA labeling was stronger than NGF mRNA and was seen in the mesenchyme located lateral to the dental organ, as well as in epithelial structures (inner dental epithelium and enamel knot). NT-3 mRNA was observed in the dental papilla and in the area of the cervical loop. NT-4 mRNA was expressed in both oral and dental epithelia in all stages studied. GDNF mRNA was found in the dental follicle and at different sites in the inner dental epithelium. Weak NTN mRNA labeling was also found in the developing teeth. Based on these findings, we suggest that neurotrophins, GDNF and NTN might be involved in morphogenetic events during early stages of tooth development in humans. Protein gene product (PGP) 9.5-immunoreactive nerve fibers were observed in the dental follicle by 11 weeks coinciding with the labeling for neurotrophic factor mRNAs in this structure. This suggests that these neurotrophic factors might be involved in the innervation of dental structures. The rich expression of neurotrophic factors in developing dental tissues suggests that developing, or possibly adult, dental tissue might be used as an allograft source of trophic support for diseases of the nervous system.     

Nutrient transport and the blood-brain barrier in developing animals

Structural alterations in the development of the blood-brain barrier (BBB) can be seen in capillary profiles from the rat cortex. The neonatal luminal membrane is amplified with irregular folds, a possible adaptation to reduced cerebral blood flow rates. By 21 days the capillaries have resolved to a smooth-surfaced, adult-like appearance. Developmental alterations in the basement membrane, tight junctions, capillary seams, Golgi, pinocytotic vesicles, and cytoplasmic thickness are observed. Two studies have addressed developmental modulations in BBB polarity; both indicate that brain-to-blood transport mechanisms that were inoperative in the early neonatal rat become functional in weanlings. Six of the seven major independent BBB nutrient transport systems that regulate plasma-to-brain uptake have been kinetically characterized in the newborn rabbit, and comparisons have been made in the weanling (28-day-old) rabbit. All of these saturable transport systems are operative at birth, which suggests that the immature rabbit has a mature BBB with respect to regulation of nutrients. Purine base permeability, affinity, and uptake velocities are virtually unchanged during postnatal development. Subtle alterations in amino acid and amine transport were suggested by the lower-affinity (high-capacity) transport mechanisms characterized in the newborn as compared to the 28-day-old BBB. Under conditions of elevated plasma levels (typical of the neonate), these higher-capacity mechanisms would facilitate a relative increase in metabolite influx to the developing brain. Significant differences in kinetics were also observed for the monocarboxylic acid and hexose transport systems in the absence of developmental changes in permeability times surface area products. A low-affinity, high-capacity monocarboxylic acid transport system operates at birth. It supplies the developing brain with increased quantities of ketone bodies, but is seen as a high-affinity, low-capacity mechanism in the 28-day-old rabbit. Concomitantly, the higher-affinity glucose carrier defined in newborn rabbits modulates, and by 28 days becomes a lower-affinity, high-capacity mechanism capable of delivering about 2 mumol X min-1 X g-1 of glucose to the (anesthetized) brain.