Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles.

Extracellular vesicles (EVs), which are found in almost all cells and human body fluids, are currently being studied as a source of pathophysiological information. Previously, we demonstrated that at least two types of EVs can be isolated from human whole saliva (WS) using enzymatic activity of dipeptidyl peptidase IV (DPP IV) as a marker for differentiating the EV subsets. In the present study, EV fractions, termed EV-I 20 k-ppt and EV-II 100 k-ppt, were prepared by a combination of size-exclusion chromatography of improved condition and sequential centrifugation. The EV-I 20 k-ppt fraction contained medium/large EVs with a diameter of 100-1,000 nm, including aminopeptidase N (APN), mucin 1, ezrin, and Annexin A1. EV-II 100 k-ppt contained small EVs with a diameter of 20-70 nm, with DPP IV and CD9, programmed cell death 6-interacting protein, and tumor susceptibility gene 101 as characteristic proteins. Proteomic analyses also revealed distinctive repertoires of constituent proteins. Immunoprecipitation of several membrane proteins of the EVs with respective antibodies suggested their differential local membrane environment between the two types of salivary vesicles. Thus, we identified two distinctive types of EVs, one is APN/MUC1- rich EVs (EV-I, large/medium EVs) and the other is DPP IV/CD9-rich EVs (EV-II, small EVs). Furthermore, analysis of the binding of the EVs to coronavirus spike proteins showed that EV-II 100 k-ppt, but not EV-I 20 k-ppt, significantly bound to the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we developed a simple method to prepare two distinctive EVs from only 1 mL of human WS using sequential immunoprecipitation. Elucidating the features and functions of these two types of salivary EVs may help us understand their pathophysiological roles in the oral cavity and gastrointestinal tract.

Stability of human salivary extracellular vesicles containing dipeptidyl peptidase IV under simulated gastrointestinal tract conditions.

BACKGROUND: Extracellular vesicles (EVs) have been isolated from various sources, including primary and cultured cell lines and body fluids. Previous studies, including those conducted in our laboratory, have reported the stability of EVs under various storage conditions. METHODS: EVs from human whole saliva were separated via size-exclusion chromatography. To simulate the effects of gastric or intestinal fluids on the stability of EVs, pepsin or pancreatin was added to the samples. Additionally, to determine the effect of bile acids, sodium cholate was added. The samples were then subjected to western blotting, dynamic light scattering, and transmission electron microscopy analyses. In addition, the activity of dipeptidyl peptidase (DPP) IV retained in the samples was examined to monitor the stability of EVs. RESULTS: Under acidic conditions, with pepsin mimicking the milieu of the stomach, the EVs remained stable. However, they partially lost their membrane integrity in the presence of pancreatin and sodium cholate, indicating that they may be destabilized after passing through the duodenum. Although several associated proteins, such as mucin 5B and CD9 were degraded, DPP IV was stable, and its activity was retained under the simulated gastrointestinal conditions. CONCLUSION: Our data indicate that although EVs can pass through the stomach without undergoing significant damage, they may be disrupted in the intestine to release their contents. The consistent delivery of active components such as DPP IV from EVs into the intestine might play a role in the efficient modulation of homeostasis of the signal transduction pathways occurring in the gastrointestinal tract.

Right testicular necrosis and left vanishing testis in a neonate.

We describe a rare case of right testicular necrosis and left vanishing testis in a neonate. The patient presented with a right swollen testis and left non-palpable testis at birth. Exploratory laparotomy was performed at 23 days after birth, and the right testis was extirpated because of extensive necrosis due to torsion of the spermatic cord. Histopathological examination revealed a massively necrotic testicular structure with hypertrophy of Leydig cells. Hypergonadotropinemia was also recognized before operation. On laparoscopy at the age of 5.3 years, a normal left vas deferens and spermatic vessels exiting through the internal inguinal ring were confirmed, although the patient's left testis was not found in the intraperitoneal cavity. An hCG test demonstrated no response of serum testosterone. These findings suggested a diagnosis of left vanishing testis due to testicular regression. We therefore speculated that bilateral asynchronous testicular torsion had occurred prenatally in this patient.

Regulation of SR-BI protein levels by phosphorylation of its associated protein, PDZK1.

Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein (HDL) receptor that mediates the selective uptake of HDL cholesterol and cholesterol secretion into bile in the liver. Previously, we identified an SR-BI-associated protein, termed PDZK1, from rat liver membrane extracts. PDZK1 contains four PSD-95/Dlg/ZO-1 (PDZ) domains, the first of which in the N-terminal region is responsible for the association with SR-BI. PDZK1 controls hepatic SR-BI expression in a posttranscriptional fashion both in cell culture and in vivo. In this study, we demonstrated that the C-terminal region of PDZK1 is crucial for up-regulating SR-BI protein expression. Metabolic labeling experiments and phosphoamino acid analysis revealed that PDZK1 is phosphorylated at Ser residues within this region. Point-mutation analysis demonstrated that PDZK1 is phosphorylated at Ser-509. Interestingly, a mutant PDZK1, in which Ser-509 was replaced with Ala, lost the ability to up-regulate SR-BI protein. We identified Ser-509 of PDZK1 as the residue that is phosphorylated by the cAMP-dependent PKA in vitro as well as in cell culture. Ser-509 of PDZK1 in rat liver was also phosphorylated, as shown by an Ab that specifically detects phosphorylated Ser-509. Administration of glucagon to Wistar rats increased PDZK1 phosphorylation as well as hepatic SR-BI and PDZK1 expression while it decreased plasma HDL levels, indicating that PDZK1 phosphorylation is hormonally regulated. These findings suggest that phosphorylation of PDZK1 has an important role in the regulation of hepatic SR-BI expression and, thus, influences plasma HDL levels.

Enhanced antitumor activity of xanthohumol, a diacylglycerol acyltransferase inhibitor, under hypoxia.

Cancer chemotherapy for hypoxic tumor cells is thought to be an important issue, since hypoxia is related to tumor growth, apoptosis, angiogenesis and metastasis. Here, the bioactivities of xanthohumol (XN), a diacylglycerol acyltransferase inhibitor, against hypoxic cells were investigated. At first, the inhibitory effects of XN on the formation of lipid droplets in the cytoplasm were evaluated in hypoxia. Hypoxia upregulated the synthesis of triglyceride and promoted the formation of lipid droplets in the cytoplasm, however, the treatment of XN downregulated the triglyceride synthesis and completely canceled the appearance of lipid droplets. Second, the effects of XN on the proliferation and the motility of HT-1080 human fibrosarcoma were investigated. The proliferation of HT-1080 was significantly suppressed in the presence of XN only in hypoxic condition but not in normoxic condition. XN also suppressed the motility of HT-1080 that was enhanced by hypoxia. Since, most cells in solid tumor were thought to be in hypoxic condition and acquired malignancy in response to hypoxia, these data suggest that XN may have potent and specific activities against cancerous cells. Furthermore, these data suggested that lipid metabolism may play an important role for hypoxic tumor cells and proposed a new therapeutic target for cancer chemotherapy.

Damaged interlobular bile ducts in primary biliary cirrhosis show reduced expression of glutathione-S-transferase-pi and aberrant expression of 4-hydroxynonenal.

BACKGROUND/AIMS: Chronic inflammation induces oxidative stress by producing reactive oxygen species. We investigated how the oxidative stress associated with chronic cholangitis induce bile duct damages in primary biliary cirrhosis. METHODS: The intracellular status of lipid peroxidation due to oxidative stress and that of glutathione, an endogenous cytoprotective molecule, were examined in primary biliary cirrhosis and controls by immunostaining of 4-hydroxynonenal and glutathione-S-transferase-pi. The former is a by-product of lipid peroxidation, and the latter is involved in the formation of intracellular glutathione. RESULTS: In the damaged bile ducts of primary biliary cirrhosis, glutathione-S-transferase-pi expression was markedly reduced, reflecting reduction of intracellular glutathione, and perinuclear expression of 4-hydroxynonenal was frequent, reflecting active lipid peroxidation associated with biliary epithelial damages. There was diffuse/luminal expression of 4-hydroxynonenal in the bile ducts frequent in primary biliary cirrhosis and controls, likely reflecting absorption of 4-hydroxynonenal, also a component of oxidized low-density lipoprotein, from bile via scavenger receptor class B type 1 on biliary epithelium. CONCLUSIONS: The data suggest that lipid peroxidation in the bile ducts with reduced expression of glutathione-S-transferase-pi, may be an important pathologic process leading to the bile duct damage of primary biliary cirrhosis.

CD36, serves as a receptor for advanced glycation endproducts (AGE).

Interaction of advanced glycation endproducts (AGE) with AGE receptors induces several cellular phenomena relating potentially to diabetic complications. Five AGE receptors identified so far are receptor for AGE (RAGE), 80 K-H, OST-48, galectin-3, and macrophage scavenger receptor, types I and II (SR-A) [Eur. J. Biochem. 230 (1995) 408; Nature 386 (1997) 292.]. Since SR-A is known to belong to the class A scavenger receptor family and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36 belonging to class B scavenger receptor family (SR-B) can recognize AGE proteins as a ligand. This was tested in the present study at the cellular level by using Chinese hamster ovary (CHO) cells overexpressing human CD36 (CHO-CD36 cells). 125I-AGE-bovine serum albumin (BSA) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-CD36, but not wild-type CHO cells. Endocytic uptake of 125I-AGE-BSA by these cells was inhibited 50% by oxidized low-density lipoprotein (Ox-LDL) and 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of Ox-LDL. Our results indicate that CD36 expressed by these cells mediates endocytic uptake and subsequent intracellular degradation of AGE proteins. Since CD36 is one of the major Ox-LDL receptors and is up-regulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, the present results suggest that, like Ox-LDL, AGE proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.