Expression of hepatic calcium-binding protein regucalcin mRNA is elevated by refeeding of fasted rats: Involvement of glucose,insulin and calcium as stimulating factors

The effect of refeeding on the expression of Ca²⁺-binding protein regucalcin mRNA in the liver of fasted rats was investigated. When rats were fasted overnight, the hepatic regucalcin mRNA level was reduced about 70% of that in feeding rats. Refeeding produced a remarkable elevation of hepatic regucalcin mRNA level (about 150–170% of fasted rats). Liver regucalcin concentration was appreciably increased by refeeding, although it was not altered by fasting. The oral administration of glucose (2 g/kg body weight) to fasted rats caused a significant increase in hepatic regucalcin mRNA level. Moreover, hepatic regucalcin mRNA level was clearly elevated by a single subcutaneous administration of insulin (10 and 100 U/kg) to fasted rats. The hormonal effect was not further enhanced by the simultaneous administration of calcium chloride (250 mg Ca/kg) to fasted rats, although calcium administration stimulated regucalcin mRNA expression in the liver. The present study suggests that the expression of hepatic regucalcin mRNA stimulated by refeeding is significantly involved in the action of insulin and/or calcium as stimulating factors.     

Hamster pulmonary endocrine cells with positive immunostaining for calbindin-D28K

 We have examined the distribution of calcium-binding proteins (CaBPs) in adult and fetal lungs of Syrian golden hamsters (Mesocricetus auratus) using immunostaining with confocal laser microscopy and electron microscopy. Single and grouped (neuroepithelial body; NEB) endocrine cells were distributed from bronchi to alveolar ducts in the adult lung. Serial frozen sections immunostained for CaBPs in combination with immunostaining for endocrine markers such as calcitonin gene-related peptide, serotonin, PGP9.5, and synaptophysin revealed that positive immunostaining for calbindin-D_28K (CB-D28K) was seen in single endocrine cells and NEBs. However, other so-called EF-hand family CaBPs, parvalbumin and calretinin, were not detected. Electron microscopically, positive immunoreaction for CB-D28K was mainly in the organelle-free cytoplasmic matrix of endocrine cells, and partly in nuclei and associated with secretory granules and endoplasmic reticulum. In fetal developing lungs, endocrine cells appeared first on gestational day 13, and they were positive for all the endocrine markers used. However, pulmonary endocrine cells were positively immunostained for CB-D28K from gestational days 15 and 16 onward. In summary, our observations suggest that CB-D28K is a useful marker for endocrine cells of the lung, and CB-D28K could function as a mediator of endocrine stimulation or calcium homeostasis in pulmonary endocrine cells. Accepted: 17 June 1997     

Changes in cytokeratin expression in epidermal keratinocytes during wound healing

In order to investigate the re-epithelialization process during wound healing, the hair on the back of guinea pigs was shaved and then excisional wounds were made through the entire thickness of the skin. Histological changes were observed and changes in the expression of different cytokeratin polypeptides were examined using an immunohistochemical technique. Immunohisto chemical study revealed that the proliferating and migrating keratinocytes expressed the same cytokeratins as the basal cells of normal epidermis. In addition, the entire epidermis of fairly remote areas from the edges of the wound where no thickening was observed showed a temporarily abnormal staining pattern. The suprabasal cells in the regenerating epidermis temporarily expressed cytokeratins not only specific for suprabasal cells but also specific for basal cells. The cytokeratins expressed in normal basal keratinocytes were also present in the thickened granular layers. These data indicate that the expression of cytokeratins in the epidermal keratinocytes (even in fairly remote areas from the wound edges) changes during wound healing, that the origin of the migrating keratinocytes from the remaining epidermis seems to be the basal cells in the epidermis, and that the appearance of keratohyalin granules is not related to changes in cytokeratin expression.     

Alkaline phosphatase reactivity in rabbit airway epithelium: a potentially useful marker for airway basal cells

The purpose of this study was to investigate alkaline phosphatase (ALPase) reactivity in rabbit airway epithelial cells. Acetone-fixed, methyl benzoate and xylene-cleared (AMeX-treated) paraffin sections of trachea, bronchus, and lung tissue were stained by an azo dye coupling method for ALPase and examined by light microscopy. Electron histochemical staining was also performed in order to study the sensitivity and specificity of reactivity in each cell type. ALPase reactivity at the light microscopic level was observed exclusively in trachco-bronchial basal cells, and not in bronchiolar basal cells. By electron microscopy, ALPase reactivity was noted in 97.9% of basal cells in the trachea, 97.0% of basal cells in the bronchus, and 94.5% of basal cells and 15.4% of Clara cells in the bronchiole. This was also true for dispersed tracheal epithelial cells. Reactivity was rarely observed in ciliated cells, non-goblet-type secretory cells, and undetermined cells. The reactivity was heatlabile, levamisole-sensitive, and of a non-specific type. These findings indicate that basal cells of rabbit trachea and bonchus have fairly high specificity for ALPase of a non-specific isozyme (92.2% and 95.6%, respectively). Therefore, ALPase is considered to be a useful marker for these cells.     

Photochemical reaction of 9-cis-retro-γ-rhodopsin at low temperatures

9-cis-Retro-γ;rhodopsin (λ_max = 420 nm) was prepared from 9-cis-retro-γ-retinal and cattle opsin. After cooling to liquid nitrogen temperature (77 K), the pigment was irradiated with light at 380 nm. The spectrum shifted to the longer wavelengths, owing to formation of a batho product. This fact indicates that the conjugated double bond system from C-5 to C-8 of the chromophoric retinal in rhodopsin was not necessary for formation of bathorhodopsin. Reirradiation of the batho product with light at wavelengths longer than 520 nm yielded a mixture composed of presumably 9- or 11-cis forms of retro-γ-rhodopsin. These three isomers are interconvertible by light at liquid nitrogen temperature. Thus the retro-γ-rhodopsin system is similar in photochemical reaction at 77 K to cattle rhodopsin system. Each system has its own batho product. Based on these results, it was infered that the formation of bathorhodopsin is due to photoisomerization of the chromophoric retinal of rhodopsin and is not due to translocation of a proton on the ring or on the side chain from C-6 to C-8 of the chromophoric retinal to the Schiff-base nitrogen.     

Concentration-dependent differntial effects of cortisol on synthesis of α-lactalbumin and of casein in cultured mouse mammary gland explants: Importance of prolactin concentration

Summary Cortisol was previously shown to elicit a concentration-dependent inhibition of α-lactalbumin accumulation in midpregnant mouse mammary gland cultured in medium containing optimal concentrations of 5 μg/ml prolactin and insulin. In contrast, casein accumulation under these conditions was progressively stimulated by addition of increasing amounts of cortisol (Ono, M.; Oka, T. Cell 19: 473–480; 1980). In the present study we found that in the presence of a suboptimal concentration of 0.5 μg/ml prolactin, 2.8×10⁻⁹ M to 2.8×10⁻⁷ M cortisol stimulated α-lactalbumin accumulation. Furthermore, higher concentrations of cortisol produced a smaller inhibition of α-lactalbumin accumulation as compared to that obtained in cultures containing 5 μg/ml prolactin. The maximal increase in α-lactalbumin accumulation attained in the presence of 1.4×10⁻⁸ M cortisol, 0.5 μg/ml prolactin, and insulin was comparable to that observed in culture containing 5 μg/ml prolactin and insulin. Similar results were obtained in a cortisol concentration-response study of α-lactalbumin accumulation in cultures containing a suboptimal concentration of 0.5 μg/ml human placental lactogen. Measurement of the rate of α-lactalbumin synthesis in cultured tissue indicated that the opposing effects of low and high concentrations of cortisol on α-lactalbumin accumulation involved an alteration in the rate of synthesis of the milk protein. In contrast to α-lactalbumin, the synthesis of casein was stimulated in a concentration-dependent manner by addition of cortisol that acted synergistically with either 0.5 μg/ml or 5 μg/ml prolactin. The maximal increases were obtained in the presence of 2.8×10⁻⁶ M cortisol. These results indicated that the action of cortisol on α-lactalbumin accumulation can be modulated by the concentration, of prolactin and suggest that the interplay between cortisol and prolactin in regulation of α-lactalbumin synthesis may be different from that involved in casein synthesis.     

Levels of endogenous ethylene, carbon dioxide, and soluble pectin, and activities of pectin methylesterase and polygalacturonase in ripening tomato fruits

In 4 cultivars of tomato (Lycopersicon esculentum Mill.), theearly detachment of fruits advanced ripening and considerablyreduced the threshold value of endogenous C₂H₄. This indicatesa supply from the vegetative parts of (a) labile ripening-inhibitingsubstance(s) antagonizing the action of C₂H₄. The endogenous level of CO₂ increased shortly after the risein C₂H₄, and maximum levels of C₂H₄ and CO₂ occurred almostsimultaneously. The activity of PE showed no connection with ripening, but PGactivity did not occur until the onset of ripening. However,this activity increased at considerably higher C₂H₄ concentrationsthan the rise in WSP, and was independent of the possible presenceof ripening inhibitor(s). Hence PG is considered not to be involvedin the primary events leading to fruit ripening. Exposure of fruits to different C₂H₄ concentrations in the ambientatmosphere also showed PG activity to increase only after therise in WSP had started. Other pectin degrading or synthesizingenzymes may be involved. In the non-ripening Rin mutant of cv. Rutgers, no rise occurredin C₂H₄, CO₂, WSP, and PG activity. ¹ Present address: Department of Agricultural Chemistry, Facultyof Agriculture, Kochi University, Otsu 200 Monobe, Nangoku City,Kochi Prefecture 783, Japan. (Received February 16, 1978; )     

A ring of uncharged polar amino acids as a component of channel constriction in the nicotinic acetylcholine receptor.

The channel pore of the nicotinic acetylcholine receptor (AChR) has been investigated by analysing single-channel conductances of systematically mutated Torpedo receptors expressed in Xenopus oocytes. The mutations mainly alter the size and polarity of uncharged polar amino acid residues of the acetylcholine receptor subunits positioned between the cytoplasmic ring and the extracellular ring. From the results obtained, we conclude that a ring of uncharged polar residues comprising threonine 244 of the alpha-subunit (alpha T244), beta S250, gamma T253 and delta S258 (referred to as the central ring) and the anionic intermediate ring, which are adjacent to each other in the assumed alpha-helical configuration of the M2-containing transmembrane segment, together form a narrow channel constriction of short length, located close to the cytoplasmic side of the membrane. Our results also suggest that individual subunits, particularly the gamma-subunit, are asymmetrically positioned at the channel constriction.     

Adductor muscles of the thigh of crab-eating monkeys (Macaca fascicularis)

On the basis of 44 hindlimbs of 14 male and 14 female crab-eating monkeys (Macaca fascicularis), the morphology of the adductor muscles of the thigh was described and some functional indices were calculated. The results obtained from this study agreed generally with those of otherMacaca species reported by various authors. For the classification and nomenclature of the adductors, the criteria proposed byUhlmann (1967, 1968) was well adapted to the crab-eating monkey. The adductors comprise the m. gracilis, m. pectineus, m. adductor longus, pars longa and pars brevis of m. adductor brevis, pars lata and pars minima of m. adductor magnus and m. obturatorius externus. In males, the adductors are generally inserted further down the femur, and the insertions of pars brevis of the m. adductor brevis and pars minima of the m. adductor magnus have longer attachments to the femur than in females. The arrangement of each adductor muscle and of each fasciculus of a thigh muscle may invoke a principle of organization.