L-Alanyl-L-alanyl-L-phenylalanine 4-methylcoumaryl-7-amide (AAF-MCA) is one of the classic substrates for use with tripeptidyl peptidases (TPP-I and TPP-II). We have previously clarified the tissue distribution of TPP-I in detail and noted that the protein expression of TPP-I is often incompatible with its enzyme activity. Herein, we describe the unknown peptidase, which could effectively hydrolyze AAF-MCA, in the rat kidney. The peptidase was purified after four chromatography steps, and its enzyme characteristics were elucidated. The peptidase activity was inhibited by amastatin, bestatin, and o-phenanthroline and was also inhibited by zinc and copper ions. The substrate specificity for several monoamino acidic-MCAs revealed that the peptidase had an affinity for alanyl-MCA. The amino terminal amino acid sequence of the peptidase was x-Ala-Pro-x-Leu-Pro-Gly-Ser-Thr-Ser-Ala-Thr-x-x-Ser, where x indicates undetectable amino acid residues, and the antiserum against the peptidase was immunopositive for the brush border of a renal proximal tubule and the small intestine, and the surface membrane of bile canaliculi. These results indicate that the unknown peptidase that hydrolyzed AAF-MCA is the soluble form of aminopeptidase N/CD13, and caution is required when using AAF-MCA as a substrate for tripeptidyl peptidase assays.
Although salivary glands are one of the target organs in graft-versus host disease (GVHD), mechanism of disease-related tissue injury has been poorly understood. To investigate it, we employed rat GVHD model including irradiation and donor cell transfer. Immunostaining revealed that submandibular and parotid, but not sublingual glands showed acinar reduction whereas ducts were almost intact. Ductal area of sublingual glands was significantly smaller than those of submandibular and parotid glands. Ductal epithelial cells upregulated expression of class II major histocompatibility complex antigens overtime. This expression preceded donor cell infiltration. Donor cell infiltration was preferentially found in ductal area at the early phase of the disease. While ductal epithelial cells exhibited upregulated mRNA expression of Th1-type chemokines, infiltrating donor cells exhibited their receptors CXCR3 and CCR5. These results suggest ductal epithelial cells have a pivotal role in the progression of GVHD.
The pancreas comprises exocrine and endocrine portions, the latter of which is a glucose-responsive tissue that secretes hormones in response to serum glucose levels. One pathway implicated in the regulatory mechanism of this gland is the phosphoinositide (PI) cycle, which generates second messengers. Diacylglycerol (DG), the major second messenger in the PI signaling cascade, is catalyzed by the diacylglycerol kinase (DGK) family. We previously described characteristic expression and localization patterns of DGKs in various organs under pathophysiological conditions. Nevertheless, little is known about the characteristics and morphological aspects of this enzyme family in the pancreas. This study was conducted to investigate the pancreas, specifically the expression and localization of the DGK family. RTPCR analysis reveals that DGKζ is the major isozyme in the pancreas. Additionally, we show that DGKζ is expressed in pancreatic islet cells, but not in the exocrine cells. It localizes predominantly to the nuclei of α-, β-, and δ-cells. We found further that DGKζ translocates from the nucleus to the cytoplasm in β-cells in response to a β-cell-selective toxin streptozotocin (STZ) and that it disappears over time. These findings will substantiate and extend our understanding of the functional roles of DGKζ in pancreatic islet cells.
The microvascularization of the pancreas of larval and adult South African Clawed Toads Xenopus laevis, was studied by scanning electron microscopy of vascular corrosion casts and light microscopy of paraplast embedded Goldner-stained serial tissue sections. We showed that branches of left and right gastric artery, hepatic artery and anterior intestinal artery, namely anterior pancreatic, anterior middle pancreatic, posterior middle pancreatic and caudal pancreatic arteries supply and pancreatic veins drain the adult pancreas into hepatic portal vein, anterior and middle gastric vein, gastroduodenal vein, and anterior duodenal vein. In premetamorphosis the pancreas showed a dense but immature vascular bed with signs of ongoing sprouting and non-sprouting angiogenesis (=intussusceptive microvascular growth; IMG). During metamorphic climax the pancreas shrinked dramatically paralleled by vascular regression.The larval pancreas had an underdeveloped ductal system which in the course of pancreas remodeling during metamorphic climax developed into a complex ductal system. In adult Xenopus laevis the pancreas showed intralobular islets of Langerhans only and an insuloacinar portal vessel system as described sofar in reptiles, birds and mammals. Islets in Xenopus located superficially and within deeper regions and emitted both insulo-acinar portal vessels and insulo-venous efferent vessels. Intrainsular microvascular patterns found suggest that in Xenopus islets blood flows first to β-cells and subsequently to the other endocrine cells present in the endocrine pancreas. Key words: Xenopus, pancreas, microvascularization, vascular casts, histomorphology
ATP-sensitive K+ (KATP) channel subunits Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B in the rat pituitary gland were first investigated by RT-PCR assay and immunohistochemical staining. The results of RTPCR analysis showed that the rat pituitary gland expressed the five KATP channel subunits mentioned above. Immunohistochemical staining showed that these KATP channel subunits were widely localized in the anterior lobe, intermediate lobe, and posterior lobe at different intensities. Immunofluorescence double and triple staining showed that these KATP channel subunits colocalized with cells containing adrenocorticotropic hormone (ACTH) in the anterior lobe of the pituitary gland. Interestingly, neither Kir6.1 nor Kir6.2 colocalized with cells containing prolactin (PRL), follicular stimulating hormone (FSH), and growth hormone (GH). These results suggest that ACTH cells contain four types of KATP channels: Kir6.1/SUR2A, Kir6.2/SUR2A, Kir6.1/SUR2B, and Kir6.2/SUR2B. KATP channels may play some important roles in ACTH cells in the pituitary gland. The different compositions of KATP channel subunits in corticotrophs but not in the PRL, FSH, and GH cells might be due to the different metabolic situations of these cells. Key words: ATP-sensitive K+ channel; Kir6.x; SUR2; immunohistochemistry; pituitary gland; rat