Gene expression and immunohistochemical localization of megalin in the anterior pituitary gland of helmeted guinea fowl (Numida meleagris).

Megalin/the low density lipoprotein receptor-related protein-2 (LRP-2) is expressed in a variety of epithelia and mediates endocytosis of numerous substances. Megalin is also shown to bind clusterin with high affinity. In the pituitary gland, clusterin is localized in endocrine cells, folliculostellate (FS) cells and colloids. The present study examines the expression pattern of megalin within the gland and assesses its cellular localization to that of clusterin so as to deduce their functional implications in colloidal accumulation as relevant in vivo. Quantity of megalin mRNA expression in pituitary and other endocrine tissues was quantified by real-time PCR using SYBR-green I detective system. High levels were detected in kidneys and pituitary. In situ hybridization showed megalin mRNA in FS cells. Megalin protein detected by immunohistochemistry was also observed in FS cells. Immunoelectron microscopy clearly showed the localization of megalin in peripheral region of colloid-containing follicles and on vesicular structures in FS cells. Immunolabeling was also found to be associated with membranes of vacuoles in apoptotic endocrine cells and cell remnants engulfed by FS cells. Double immunofluorescence labeling was performed to determine whether megalin and clusterin in the anterior pituitary were present within the same cell. Simultaneous localization was detected in almost all FS cells surrounding colloids and in several foci of FS cells surrounding endocrine cells. These findings suggest that megalin may drive ingestion of clusterin complexes with products of digested apoptotic endocrine cells in FS cells, and thereby providing a potential mechanism for a receptor mediated uptake of degenerating endocrine cells and secretion of colloid.

Characterization and distribution of an arginine vasotocin receptor in mouse.

A cDNA, which has a high homology with teleost Platichthys flesus [Arg8] vasotocin (AVT) receptor (GenBank: AK033957), was found in mouse genome database. Analyses of the deduced amino acid sequence revealed that a cDNA has several features of AVT receptor. We tentatively named it as a mouse vasotocin receptor (MVTR). A two-electrodes voltage clamp technique was applied to characterize the MVTR expressed in Xenopus laevis oocytes. AVT induced Ca2+-dependent Cl- currents in Xenopus oocytes injected with MVTR cRNA. On the other hand, [Arg8] vasopressin, oxytocin and isotocin did not induce such currents. RT-PCR showed that MVTR mRNA was specifically expressed in the brain. In situ hybridization analysis demonstrated significant expression of MVTR mRNA in suprachiasmatic nucleus, arcuate nucleus and medial habenular nucleus of mouse brain. These results suggest that MVTR may mediate a variety of physiological functions in mouse.

Fibrin stimulates the proliferation of human keratinocytes through the autocrine mechanism of transforming growth factor-alpha and epidermal growth factor receptor.

In the field of dermatology and plastic and reconstructive surgery, fibrin gel is regarded as a material that promotes wound healing. To test the hypothesis that fibrin may promote the growth of the epidermis, we examined its effects on the proliferation of cultured keratinocytes. Human keratinocytes were cultivated in fibrin-coated wells, and the cell numbers and transforming growth factor (TGF)-alpha, secreted into the cultured medium, were measured. We also assessed the capacity of epidermal growth factor receptor (EGF-R) that is responsible for all known actions of TGF-alpha and epidermal growth factor. The keratinocytes increased dramatically in their number, and the TGF-alpha secretion and the binding capacity of EGF-R were also increased dramatically in the presence of fibrin. These findings suggest that fibrin supports the proliferation of keratinocytes in an autocrine fashion via EGF-R; namely, fibrin stimulates keratinocytes to secrete TGF-alpha, which in turn increases cell proliferation and EGF-R capacity. We propose that fibrin can support the wound healing process of the epidermis via the TGF-alpha/EGF-R pathway.

Distribution, histochemical and enzyme histochemical characterization of mast cells in dogs.

This study describes the distribution, proteoglycan properties and protease activity of mast cells from 15 different dog organs. In beagles and mixed breed dogs, staining with Alcian Blue-Safranin O revealed mast cells in all the organs examined. However, their numbers varied and they demonstrated unique localization patterns within some of these organs. Berberine sulphate fluorescence-positive mast cells were observed in the submucosa, muscularis and serosa of the intestines, as well as the tongue and liver (within the connective tissue). Mast cells within the intestinal mucosa were negative for, or demonstrated weak, berberine sulphate staining. Heterogeneity of mast cells in terms of the proteoglycans contained within their granules was further confirmed by determination of critical electrolyte concentrations (CECs). The CECs of mast cells within the connective tissue of several organs, including the intestines (submucosal and muscularis-serosal layers) were all greater than 1.0 M. The results from CEC experiments together with berberine staining indicate that heparin was contained within their granules. Relative to the CECs of mast cells in other organs, mast cells in the intestinal mucosa exhibited lower CECs, suggesting that the proteoglycans within their granules were of lower charge density and/or molecular weight. Although mast cells were classified into two groups by proteoglycans within the granules, enzyme histochemical analysis in beagles revealed three subtypes of mast cells: chymase (MC(C)), tryptase (MC(T)) and dual positive (MC(TC)) cells. There was no correlation between the proteoglycan content and enzyme properties of the mast cell granules.

Standardized data and relationship between bone growth and bone metabolism in female Göttingen minipigs.

Minipigs have been studied as a model of osteoporosis. However, little information is available regarding their bone physiology. We established standardized bone data and investigated the relationship between bone growth and bone metabolism in female minipigs. Blood and urine samples were obtained from 53 female Göttingen minipigs, 3-76 months of age, for measurement of bone biomarkers (i.e., BAP, OC, NTX, and DPD). The lumbar vertebra and femur were excised to determine the growth plate condition, bone length, bone mineral content (BMC), and bone mineral density (BMD). High levels of bone biomarkers were observed during the initial period after birth, decreasing thereafter with age. Bone biomarkers were confirmed to be highly correlated with age (R(2) > 0.7). The growth plates of the lumbar vertebra and the femur began to close at 21 and 25 months of age, respectively, and closed completely at 42 months of age. Bone length increased rapidly before growth plate closure, and reached a peak at 21 and 28 months of age in the lumbar vertebra and the femur, respectively. The levels of BMC and BMD increased rapidly before growth plate closure, and continued to increase slowly until 76 months of age. A high negative correlation (-0.855 < r < -0.711, p<0.001) was confirmed between the bone biomarkers and the bone measurement data. These results indicate that the bone turnover velocity is consistent with the bone growth velocity in female Göttingen minipigs.

Age-related changes of bone mineral density and microarchitecture in miniature pigs.

Bone mineral density (BMD), distribution of its density and bone histomorphometric parameters were evaluated in lumbar vertebra of normally growing miniature pigs. The fourth lumbar vertebra (L4) of the Göttingen miniature pig were used in this cross-sectional study in vitro. The BMD of the miniature pig was similar to that of humans in tendency of gender differences and some growth patterns during puberty. In these regards this animal appears useful as a model for human bone study. However, the trabecular and cortical BMDs of lumbar spine were extremely high value (399.43 +/- 26.36 mg/cm(3) in female trabeculae; 973.06 +/- 69.55 mg/cm(3) in female cortical bone; 419.04 +/- 34.84 mg/cm(3) in male trabeculae; 1038.81 +/- 125.72 mg/cm(3) in male cortical bone in pigs 30 months or more). Furthermore, histomorphometric analysis yielded values that were remarkably different from those found in humans. From these results, it was revealed that miniature pig had a higher bone mass and denser trabecular network than human, indicating that its bone is probably stronger. Therefore, care should be taken in choosing the miniature pig as a bone study model.