Stichoposide C and Rhizochalin as Potential Aquaglyceroporin Modulators.

Aquaporins (AQPs) are a family of integral membrane proteins that selectively transport water and glycerol across the cell membrane. Because AQPs are involved in a wide range of physiological functions and pathophysiological conditions, AQP-based therapeutics may have the broad potential for clinical utility, including for disorders of water and energy balance. However, AQP modulators have not yet been developed as suitable candidates for clinical applications. In this study, to identify potential modulators of AQPs, we screened 31 natural products by measuring the water and glycerol permeability of mouse erythrocyte membranes using a stopped-flow light scattering method. None of the tested natural compounds substantially affected the osmotic water permeability. However, several compounds considerably affected the glycerol permeability. Stichoposide C increased the glycerol permeability of mouse erythrocyte membranes, whereas rhizochalin decreased it at nanomolar concentrations. Immunohistochemistry revealed that AQP7 was the main aquaglyceroporin in mouse erythrocyte membranes. We further verified the effects of stichoposide C and rhizochalin on aquaglyceroporins using human AQP3-expressing keratinocyte cells. Stichoposide C, but not stichoposide D, increased AQP3-mediated transepithelial glycerol transport, whereas the peracetyl aglycon of rhizochalin was the most potent inhibitor of glycerol transport among the tested rhizochalin derivatives. Collectively, stichoposide C and the peracetyl aglycon of rhizochalin might function as modulators of AQP3 and AQP7, and suggests the possibility of these natural products as potential drug candidates for aquaglyceroporin modulators.

Differential Expressions of Aquaporin Subtypes in the Adult Mouse Testis.

Many efforts have been made to study the expression of aquaporins (AQP) in the mammalian reproductive system, but there are not enough data available regarding their localized expression to fully understand their specific roles in male reproduction. The present study investigated the expression and localization patterns of different AQP subtypes in the adult mouse testes and testicular spermatozoa using an immunofluorescence assay. All the studied AQPs were expressed in the testes and revealed subtype-specific patterns in the intensity and localization depending on the cell types of the testes. AQP7 was the most abundant and intensive AQP subtype in the seminiferous tubules, expressing in Leydig cells and Sertoli cells as well as all stages of germ cells, especially the spermatids and testicular spermatozoa. The expression pattern of AQP3 was similar to that of AQP7, but with higher expression in the basal and lower adluminal compartments rather than the upper adluminalcompartment. AQP8 expression was limited to the spermatogonia and Leydig cells whereas AQP9 expression was exclusive to tails of the testicular spermatozoa and elongated spermatids. Taken together, the abundance and distribution of the AQPs across the different cell types in the testes indicating to their relavance in spermatogenesis, as well as in sperm maturation, transition, and function.

Constitutive expression of cytochrome P450 1B1 endows testicular Leydig cells with susceptibility to 7,12-dimethylbenzanthracene-induced cell death.

Testicular Leydig cells produce testosterone through the participation of steroidogenic proteins. The CYP1B1 enzyme has been shown to catalyze 7,12-dimethylbenzanthracene (DMBA), a representative polycyclic aromatic hydrocarbon. We hypothesized that exposure to DMBA causes Leydig cell cytotoxicity through activation of CYP1B1. Leydig cells were exposed to various concentrations of DMBA for the induction of CYP1B1 expression and activity. The status of CYP1B1 function was monitored by evaluation of cytotoxicity-mediated cell death. Our data show that exposure to DMBA causes cytotoxicity in Leydig cells by CYP1B1 activation. DMBA evoked a significant increase in the generation of reactive oxygen species (ROS) by which the depolarization of mitochondrial membrane potential (MMP) is initiated and caspase-3 activation is augmented. The knockdown of CYP1B1 expression resulted in the suppression of DMBA-induced apoptosis via reduced p53 activation and caspase-3 activation, suggesting that a final metabolite of DMBA (i.e., DMBA-DE) bioactivated by CYP1B1 induces p53 activation by binding to DNA and subsequently causing apoptosis via caspase-3 activation. This finding provides evidence for constitutive expression of CYP1B1 in Leydig cells, which is a trait that only requires an initiating signal for its activity. Further research on CYP1B1 activation-provoked steroid metabolism in Leydig cells may provide decisive clues for elucidating its innate function.

Effects of osmolality and solutes on the morphology of red blood cells according to three-dimensional refractive index tomography.

Phosphate-buffered saline (PBS) and Alsever's solution (AS) are frequently used as media in blood-related studies, while 0.9% normal saline (NS) is frequently used in transfusion medicine. Despite the frequent use, the effects of these solutions on the shape and volume of red blood cells (RBCs) have not been reported. We collected blood samples from five healthy adults and used three-dimensional refractive index tomography to investigate the changes in the morphology of RBCs caused by changes in osmolality and solutes at the single-cell level. After diluting 2 µL of RBCs 200-fold with each solution (PBS, AS, and 0.9% NS), 40 randomly selected RBCs were microscopically observed. RBC shape was measured considering sphericity, which is a dimensionless quantity ranging from 0 (flat) to 1 (spherical). RBCs in plasma or AS showed a biconcave shape with a small sphericity, whereas those in 0.9% NS or PBS showed a spherical shape with a large sphericity. Moreover, we confirmed that sodium chloride alone could not elicit the biconcave shape of RBCs, which could be maintained only in the presence of an osmotic pressure-maintaining substance, such as glucose or mannitol. Although 0.9% NS solution is one of the most commonly used fluids in hematology and transfusion medicine, RBCs in 0.9% NS or PBS are not biconcave. Therefore, as the debate on the use of NS continues, future clinical studies or applications should consider the effect of glucose or mannitol on the shape of RBCs.

Differential Expressions of Aquaporin Subtypes in Female Reproductive Tract of Mice.

Although many aquaporin (AQP) transcripts have been demonstrated to express in the female reproductive tract, the defined localizations and functions of AQP subtype proteins remain unclear. In this study, we investigated the expression of AQP1, AQP3, AQP5, AQP6, and AQP9 proteins in female reproductive tract of mouse and characterized their precise localizations at the cellular and subcellular levels. Immunofluorescence analyses for AQP1, AQP3, AQP6, and AQP9 showed that these proteins were abundantly expressed in female reproductive tract and that intense immunoreactivities were observed in mucosa epithelial cells with a subtype-specific pattern. The most abundant aquaporin in both vagina and uterine cervix was AQP3. Each of AQP1, AQP3, AQP6, and AQP9 exhibited its distinct distribution in stratified squamous or columnar epithelial cells. AQP9 expression was predominant in oviduct and ovary. AQP1, AQP3, AQP6, and AQP9 proteins were mostly seen in apical membrane of ciliated epithelial cells of the oviduct as well as in both granulosa and theca cells of ovarian follicles. Most of AQP subtypes were also expressed in surface epithelial cells and glandular cells of endometrium in the uterus, but their expression levels were relatively lower than those observed in the vagina, uterine cervix, oviduct and ovary. This is the first study to investigate the expression and localization of 5 AQP subtype proteins simultaneously in female reproductive tract of mouse. Our results suggest that AQP subtypes work together to transport water and glycerol efficiently across the mucosa epithelia for lubrication, proliferation, energy metabolism and pH regulation in female reproductive tract.

Loss of Aquaporin-3 in Placenta and Fetal Membranes Induces Growth Restriction in Mice.

Aquaporin (AQP) 3, a facilitated transporter of water and glycerol, expresses in placenta and fetal membranes, but the detailed localization and function of AQP3 in placenta remain unclear. To elucidate a role of AQP3 in placenta, we defined the expression and cellular localization of AQP3 in placenta and fetal membranes, and investigated the structural and functional differences between wild-type and AQP3 null mice. Gestational sacs were removed during mid-gestational period and amniotic fluid was aspirated for measurements of volume and composition. Fetuses with attached placenta and fetal membranes were weighed and processed for histological assessment. AQP3 strongly expressed in basolateral membrane of visceral yolk sac cells of fetal membrane, the syncytiotrophoblasts of the labyrinthine placenta and fetal nucleated red blood cell membrane. Mice lacking AQP3 did not exhibit a significant defect in differentiation of trophoblast stem cells and normal placentation. However, AQP3 null fetuses were smaller than their control litter mates in spite of a decrease in litter size. The total amniotic fluid volume per gestational sac was reduced, but the amniotic fluid-to-fetal weight ratio was increased in AQP3 null mice compared with wild-type mice. Glycerol, free fatty acid and triglyceride levels in amniotic fluid of AQP3 null mice were significantly reduced, whereas lactate level increased when compared to those of wild-type mice. These results suggest a role for AQP3 in supplying nutrients from yolk sac and maternal blood to developing fetus by facilitating transport of glycerol in addition to water, and its implication for the fetal growth in utero.

The effect of AQP3 deficiency on fuel selection during a single bout of exhausting exercise.

Aquaporin-3 (AQP3) is an integral membrane protein that facilitates the transport of water and glycerol across cell membranes. However, the precise localization and function of AQP3 in skeletal muscles is currently unknown. In this study, we investigated the capacity of AQP3 knockout mice to perform a single bout of exhausting exercise and analyzed the parameters related to skeletal muscle energy metabolism during exhausting exercise. Mice were exposed to a single bout of treadmill running at a speed of 12 m/min with 10° inclination until exhaustion, and sacrificed immediately, 24 h and 48 h after exercise. Both immunohistochemistry and double immunofluorescence staining revealed that AQP3 is expressed at the cell surface with no evidence of colocalization with either AQP1 or AQP4 in hamstring skeletal muscles. When exposed to a single bout of exhaustive exercise, AQP3 knockout mice fatigued more easily with the average time to exhaustion shorter than the wild-type mice. After exhausting exercise, plasma glucose, muscle glycogen, muscle triglyceride, and muscle free fatty acid levels decreased compared with the values before exercise in both AQP3 knockout and wild-type mice. However, muscle glycerol concentration after exercise decreased in the wild-type mice, but rather increased in AQP3 knockout mice. These findings suggest that decreased glycerol efflux from the skeletal muscles in AQP3 knockout mice may result in low exercise capacity, presumably due to the limitations in the constant energy supply through hepatic gluconeogenesis from glycerol during the prolonged endurance exercise.

Three-dimensional culture and interaction of cancer cells and dendritic cells in an electrospun nano-submicron hybrid fibrous scaffold.

An artificial three-dimensional (3D) culture system that mimics the tumor microenvironment in vitro is an essential tool for investigating the cross-talk between immune and cancer cells in tumors. In this study, we developed a 3D culture system using an electrospun poly(ε-caprolactone) (PCL) nanofibrous scaffold (NFS). A hybrid NFS containing an uninterrupted network of nano- and submicron-scale fibers (400 nm to 2 µm) was generated by deposition onto a stainless steel mesh instead of an aluminum plate. The hybrid NFS contained multiplanar pores in a 3D structure. Surface-seeded mouse CT26 colon cancer cells and bone marrow-derived dendritic cells (BM-DCs) were able to infiltrate the hybrid NFS within several hours. BM-DCs cultured on PCL nanofibers showed a baseline inactive form, and lipopolysaccharide (LPS)-activated BM-DCs showed increased expression of CD86 and major histocompatibility complex Class II. Actin and phosphorylated FAK were enriched where unstimulated and LPS-stimulated BM-DCs contacted the fibers in the 3D hybrid NFS. When BM-DCs were cocultured with mitoxantrone-treated CT26 cells in a 3D hybrid NFS, BM-DCs sprouted cytoplasm to, migrated to, synapsed with, and engulfed mitoxantrone-treated CT26 cancer cells, which were similar to the naturally occurring cross-talk between these two types of cells. The 3D hybrid NFS developed here provides a 3D structure for coculture of cancer and immune cells.

Relationships between chemical structures and functions of triterpene glycosides isolated from sea cucumbers.

Many marine triterpene glycosides have in vitro and in vivo activities with very low toxicity, suggesting that they are suitable agents for the prevention and treatment of different diseases, particularly cancer. However, the molecular mechanisms of action of natural marine compounds in cancer, immune, and other various cells are not fully known. This review focuses on the structural characteristics of marine triterpene glycosides and how these affect their biological activities and molecular mechanisms. In particular, the membranotropic and membranolytic activities of frondoside A and cucumariosides from sea cucumbers and their ability to induce cytotoxicity and apoptosis have been discussed, with a focus on structure-activity relationships. In addition, the structural characteristics and antitumor effects of stichoposide C and stichoposide D have been reviewed along with underlying their molecular mechanisms.

Time-dependent expression patterns of cardiac aquaporins following myocardial infarction.

Aquaporins (AQPs) are expressed in myocardium and the implication of AQPs in myocardial water balance has been suggested. We investigated the expression patterns of AQP subtypes in normal myocardium and their changes in the process of edema formation and cardiac dysfunction following myocardial infarction (MI). Immunostaining demonstrated abundant expression of AQP1, AQP4, and AQP6 in normal mouse heart; AQP1 in blood vessels and cardiac myocytes, AQP4 exclusively on the intercalated discs between cardiac myocytes and AQP6 inside the myocytes. However, neither AQP7 nor AQP9 proteins were expressed in CD1 mouse myocardium. Echocardiography revealed that cardiac function was reduced at 1 week and recovered at 4 weeks after MI, whereas myocardial water content determined by wet-to-dry weight ratio increased at 1 week and rather reduced below the normal at 4 weeks. The expression of cardiac AQPs was up-regulated in MI-induced groups compared with sham-operated control group, but their time-dependent patterns were different. The time course of AQP4 expression coincided with that of myocardial edema and cardiac dysfunction following MI. However, expression of both AQP1 and AQP6 increased persistently up to 4 weeks. Our findings suggest a different role for cardiac AQPs in the formation and reabsorption of myocardial edema after MI.

Omega-3 fatty acid supplementation increases 1,25-dihydroxyvitamin D and fetuin-A levels in dialysis patients.

Vitamin D deficiency, low levels of fetuin-A, and fibroblast growth factor 23 (FGF-23) are related to vascular calcification, which is associated with cardiovascular disease. We hypothesized that omega-3 fatty acid (FA), which has cardioprotective properties, modifies vitamin D status, fetuin-A, and FGF-23 levels in dialysis patients. In a randomized, open-label, controlled study, a total of 47 patients treated with dialysis for at least 1 year were randomized to treatment for 6 months with omega-3 FAs (Omacor, 3 g/d; Pronova, Sandefjord, Norway) or a control group. Levels of fetuin-A and FGF-23 were measured by enzyme-linked immunoassay, 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were measured by radioimmunoassay. The mean age of the enrolled patients was 57.4 ± 10.4 years, and mean dialysis duration was 46.5 ± 28.1 months. Twenty-seven hemodialysis patients and 16 peritoneal dialysis patients finished this trial. After 6 months, the levels of 1,25-dihydroxyvitamin D and fetuin-A were significantly increased in the group taking the omega-3 FA supplement compared with baseline. Levels of calcium, phosphorous, parathyroid hormone, 25-hydroxyvitamin D, FGF-23, and lipid profiles were not significantly changed in the omega-3 FA-supplemented group after 6 months compared with baseline. The erythrocyte membrane contents of eicosapentaenoic acid and docosahexaenoic acid were significantly increased, and oleic acid content was significantly decreased in the omega-3 FA-supplemented group after 6 months compared with baseline. Regarding vascular calcification and cardiovascular disease, omega-3 FA supplementation may have a clinical benefit caused by activating vitamin D, increasing fetuin-A levels, and modifying erythrocyte membrane FA contents in dialysis patients.

Role of aquaporin 3 in development, subtypes and activation of dendritic cells.

Dendritic cells (DCs) uptake soluble antigens and large volumes of fluid through macropinocytosis and migrate for antigen presentation. Aquaporin 3 (AQP3), a water and glycerol transporting protein, is highly expressed in immature DCs. To elucidate the role of AQP3 in DC function, we investigated subtype and activation of DCs in AQP3 knock-out (AQP3(-/-)) mice. Depletion of AQP3 did not affect the development of bone marrow-derived DCs (BM-DCs) by GM-CSF or the Flt3 ligand and the level of expression of CD86 on unstimulated and LPS-stimulated BM-DCs. In addition, the percentage of CD86(+) cells among splenic cDCs after LPS treatment in both in vitro and in vivo conditions was similar in wild type and AQP3(-/-) mice. However, the frequency of CD4(+) cDCs in the spleen of AQP3(-/-) mice was significantly lower than that of wild type mice. There was higher expression of CD103 in the CD8(+) subpopulation of splenic cDCs from AQP3(-/-) mice than wild type mice. In the dermis, more CD103-expressing cells were detected in AQP3(-/-) mice than in wild type mice and the LPS-induced decrease of CD103(+) dermal DCs was impaired in AQP3(-/-) mice. AQP3 depletion did not affect the uptake of either albumin or dextran by CD11c(+) splenic DCs. However, HgCl(2), which is an AQP inhibitor, significantly inhibited the uptake of albumin but not dextran by CD11c(+) splenic DCs. These results suggest that AQP3 may play a role in modulating DC population and migration.

GD3 accumulation in cell surface lipid rafts prior to mitochondrial targeting contributes to amyloid-ß-induced apoptosis.

Neuronal apoptosis induced by amyloid ß-peptide (Aß) plays an important role in the pathophysiology of Alzheimer's disease (AD). However, the molecular mechanism underlying Aß-induced apoptosis remains undetermined. The disialoganglioside GD3 involves ceramide-, Fas- and TNF-α-mediated apoptosis in lymphoid cells and hepatocytes. Although the implication of GD3 has been suggested, the precise role of GD3 in Aß-induced apoptosis is still unclear. Here, we investigated the changes of GD3 metabolism and characterized the distribution and trafficking of GD3 during Aß-induced apoptosis using human brain-derived TE671 cells. Extracellular Aß-induced apoptosis in a mitochondrial-dependent manner. GD3 level was negligible in the basal condition. However, in response to extracellular Aß, both the expression of GD3 synthase mRNA and the intracellular GD3 level were dramatically increased. Neosynthesized GD3 rapidly accumulated in cell surface lipid microdomains, and was then translocated to mitochondria to execute the apoptosis. Disruption of membrane lipid microdomains with methyl-ß-cyclodextrin significantly prevented both GD3 accumulation in cell surface and Aß-induced apoptosis. Our data suggest that rapidly accumulated GD3 in plasma membrane lipid microdomains prior to mitochondrial translocation is one of the key events in Aß-induced apoptosis.

Vascular calcification score on plain radiographs of the feet as a predictor of peripheral arterial disease in patients with chronic kidney disease.

BACKGROUND: The incidence of lower extremity amputation is increasing in patients with chronic kidney disease (CKD), but symptoms of peripheral arterial disease (PAD) in patients with CKD are uncommon. The aim of the current study was to assess the value of the vascular calcification (VC) score on plain radiographs of the feet for the prediction of PAD in patients with CKD. METHODS: We recruited 102 patients with CKD (24 pre-dialysis, 58 hemodialysis [HD], and 20 peritoneal dialysis [PD]). We defined the VC score on the plain radiographs of the feet as follows: score 0, no calcification; score 1, the length of the VC is <5 cm in a single foot or <2 cm in both feet; and score 2, the length of the VC is >5 cm in a single foot or >2 cm in both feet. We evaluated the atherosclerotic calcified plaques of the femoral or popliteal artery with Doppler ultrasonography and compared it with the VC score. RESULTS: Patients with high VC scores showed more atherosclerotic calcified plaques in the femoral or popliteal artery (P < 0.01). The prevalence of PAD in patients with CKD was closely related with the VC score (P < 0.01). PAD was associated with diabetes, a higher hsCRP, and a lower total cholesterol level (P < 0.01). The VC score was a significant predictor of the presence of PAD (odds ratio: 6.66, P < 0.001). CONCLUSIONS: Asymptomatic CKD patients, including those on HD and PD, may have PAD and routine testing by plain radiographs of the feet followed by Doppler ultrasonography are of pivotal significance, especially if the patients are diabetic.

P2 Receptor-mediated Inhibition of Vasopressin-stimulated Fluid Transport and cAMP Responses in AQP2-transfected MDCK Cells.

We cultured canine kidney (MDCK) cells stably expressing aquaporin-2 (AQP2) on collagen-coated permeable membrane filters and examined the effect of extracellular ATP on arginine vasopressin (AVP)-stimulated fluid transport and cAMP production. Exposure of cell monolayers to basolateral AVP resulted in stimulation of apical to basolateral net fluid transport driven by osmotic gradient which was formed by addition of 500 mM mannitol to basolateral bathing solution. Pre-exposure of the basolateral surface of cell monolayers to ATP (100 microM) for 30 min significantly inhibited the AVP-stimulated net fluid transport. In these cells, AVP-stimulated cAMP production was suppressed as well. Profile of the effects of different nucleotides suggested that the P2Y(2) receptor is involved in the action of ATP. ATP inhibited the effect of isoproterenol as well, but not that of forskolin to stimulate cAMP production. The inhibitory effect of ATP on AVP-stimulated fluid movement was attenuated by a protein kinase C inhibitor, calphostin C or pertussis toxin. These results suggest that prolonged activation of the P2 receptors inhibits AVP-stimulated fluid transport and cAMP responses in AQP2 transfected MDCK cells. Depressed responsiveness of the adenylyl cyclase by PKC-mediated modification of the pertussis-toxin sensitive G(i) protein seems to be the underlyihng mechanism.

Associations between oxidized LDL to LDL ratio, HDL and vascular calcification in the feet of hemodialysis patients.

Cardiovascular mortality is associated with vascular calcification (VC) in hemodialysis (HD) patients. The present study was designed to find factors related with medial artery calcification on the plain radiography of feet by comparing C-reactive protein (CRP), plasminogen activator inhibitor type 1 (PAI-1) and lipid profile including oxidized low density lipoprotein (ox-LDL) and to elucidate associations among these factors in HD patients. Forty-eight HD patients were recruited for this study. VC in the feet was detected in 18 patients (37.5%) among total patients and 12 patients (85.7%) among diabetic patients. Diabetes, cardiovascular disease (CVD), pulse pressure, ox-LDL/LDL were higher and high density lipoprotein (HDL) was lower in patients with VC than in patients without VC. Negative associations were found between HDL and CRP, PAI-1. PAI-1 had positive association with ox-LDL/LDL. History of CVD was the only determinant of vascular calcification on the plain radiography of feet. Ox-LDL/LDL, HDL, CRP, and PAI-1 were closely related with one another in HD patients. History of CVD is the most important factor associated with the presence of VC and low HDL and relatively high oxidized LDL/LDL ratio may affect VC formation on the plain radiography in the feet of HD patients.

Interactions of acetylcholinesterase with caveolin-1 and subsequently with cytochrome c are required for apoptosome formation.

Acetylcholinesterase (AChE) is emerging as an important component in leading to apoptosis. Our previous study demonstrated that silencing of the AChE gene blocked the interaction between cytochrome c and apoptotic protease-activating factor-1 (Apaf-1) in etoposide-induced apoptosis of HT-29 cells. We undertook this study to further dissect the molecular role of AChE in apoptosome formation. The present study elicited that small interfering RNA (siRNA) to cytochrome c gene blocked the interaction of AChE with Apaf-1, whereas siRNA to Apaf-1 gene did not block the interaction of AChE with cytochrome c, indicating that the interaction of AChE with cytochrome c is required for the interaction between cytochrome c and protease-activating factor-1. We further observed that AChE is localized to caveolae via interacting with caveolin-1 during apoptosis and that the disruption of caveolae prevented apoptosome formation. These data indicate that the interactions of AChE with caveolin-1 and subsequently with cytochrome c appear to be indispensable for apoptosome formation.

Netrin induces down-regulation of its receptor, Deleted in Colorectal Cancer, through the ubiquitin-proteasome pathway in the embryonic cortical neuron.

The proper regulation of temporal and spatial expression of the axon guidance cues and their receptors is critical for the normal wiring of nervous system during development. Netrins, a family of secreted guidance cues, are involved in the midline crossing of spinal commissural axons and in the guidance of cortical efferents. Axons normally lose the responsiveness to their attractants when they arrive at their targets, where the attractant is produced. However the molecular mechanism is still unknown. We investigated the molecular mechanism of down-regulation of netrin-1 signaling in the embryonic cortical neurons. Netrin-1 induced the ubiquitination and proteolytic cleavage of Deleted in Colorectal Cancer (DCC), a transmembrane receptor for netrin, in dissociated cortical neurons. A dramatic decrease of DCC level particularly on the cell surface was also observed after netrin-1 stimulation. Specific ubiquitin-proteasome inhibitors prevented the netrin-induced DCC cleavage and decrease of cell surface DCC. We suggest that the ligand-mediated down-regulation of DCC might participate in the loss of netrin-responsiveness in the developing nervous system.

Cloning, characterization and neuronal expression profiles of tumor endothelial marker 7 in the rat brain.

Tumor endothelial marker7 (TEM7) is a putative transmembrane protein that is highly expressed in the tumor endothelium. In the present study, the expression profile of TEM7 was investigated in TEM7-transfected human embryonic kidney (HEK) 293 cells and the rat brain. The extracellular secretion of the recombinant N-terminal ectodomain of TEM7, not full-length TEM7, was observed in the transiently transfected HEK 293 cells. The full-length TEM7 was found inside and membrane part of cells as demonstrated by confocal microscopy. In situ hybridization study revealed that TEM7 mRNA expressions were localized to specific neuronal areas, such as cerebellar Purkinje cells, the layer IV and V of cerebral cortex, hippocampal pyramidal cells and hypothalamic magnocellular nuclei. Immunohistochemical investigation of TEM expression with specific antibodies against TEM7 further supported the spatial expression patterns of TEM7 mRNA. The temporal expression of TEM7 mRNA in the cerebellar Purkinje cells demonstrated a postnatal developmental regulation of TEM7 expression. Our results indicate that TEM7 plays a role as a transmembrane receptor in some neuronal populations of the vertebrate brains.

Identification of vasopressin-induced genes in AQP2-transfected MDCK cells by suppression subtractive hybridization.

Arginine vasopressin (AVP) plays a major role in the modulation of water reabsorption in mammalian kidney. In addition to short-term regulation of aquaporin 2 (AQP2) trafficking, AVP also has long-term effects to regulate the expression of AQP2 in renal collecting duct. However, the detailed mechanism of the long-term effects of AVP in kidney remains to be elucidated. We have searched for genes induced by AVP using the polymerase chain reaction-based suppression subtractive hybridization technique in AVP-responsive AQP2-transfected MDCK cells. We found that the expression of the genes such as VIP17/MAL, annexin II, stimulatory GTP binding protein, tubulin, and mitochondrial ATP synthase was induced by AVP treatment for 4h. These results suggest that AVP might induce the expression of several genes related to the apical targeting of newly synthesized AQP2 as well as that of AQP2 for the long-term modification of water permeability in renal collecting duct.