Efficient and multiplexable genome editing using Platinum TALENs in oleaginous microalga, Nannochloropsis oceanica NIES-2145.

Algae accumulate large amounts of lipids produced by photosynthesis, and these lipids are expected to be utilized as feedstocks for sustainable new energies, known as biodiesels. Nannochloropsis species are eukaryotic microalgae that produce high levels of lipids. However, since the production costs of algal biodiesels are higher than those of fossil fuels, the improved productivity of algal lipids by molecular breeding of algae is required for practical use. In the present study, we developed a highly efficient genome-editing system involving Platinum transcription activator-like effector nucleases (TALENs) in Nannochloropsis oceanica. Platinum TALENs codon-optimized for N. oceanica were synthesized, and their DNA-binding activity was confirmed by single-strand annealing assays in human HEK293T cells. All-in-one expression vectors for Platinum TALEN targeting the nitrate reductase gene, NoNR, and acyltransferase gene, LPAT1, were transfected into Nannochloropsis species. The introduction of each Platinum TALEN revealed high genome-editing efficiency with no detectable off-target mutations at the candidate sites in N. oceanica. By simultaneously introducing TALENs targeting two genes, we obtained double mutant strains. The loss-of-function phenotype of NoNR was also confirmed. These findings will provide an essential technology for molecular breeding in Nannochloropsis species.

Genome editing with removable TALEN vectors harboring a yeast centromere and autonomous replication sequence in oleaginous microalga.

Algal lipids are expected to become a basis for sustainable fuels because of the highly efficient lipid production by photosynthesis accompanied by carbon dioxide assimilation. Molecular breeding of microalgae has been studied to improve algal lipid production, but the resultant gene-modified algae containing transgenes are rarely used for outdoor culture because the use of genetically modified organisms (GMOs) is strictly restricted under biocontainment regulations. Recently, it was reported that plasmids containing yeast centromere and autonomous replication sequence (CEN/ARS) behaved as episomes in Nannochloropsis species. We previously reported that the Platinum TALEN (PtTALEN) system exhibited high activity in Nannochloropsis oceanica. Therefore, we attempted to develop a genome editing system in which the expression vectors for PtTALEN can be removed from host cells after introduction of mutations. Using all-in-one PtTALEN plasmids containing CEN/ARS, targeted mutations and removal of all-in-one vectors were observed in N. oceanica, suggesting that our all-in-one PtTALEN vectors enable the construction of mutated N. oceanica without any transgenes. This system will be a feasible method for constructing non-GMO high-performance algae.

Neuroendovascular Training Using Multisource Video-Recording System in a Hybrid Operating Room.

BACKGROUND: Increasing restrictions over trainees' working hours and the recent coronavirus disease 2019 pandemic warrant new educational methods of surgical skills. We assessed a novel video-recording system for neuroendovascular skill education, developed with the installation of a hybrid operating room (OR) at our institution. METHODS: A single-plane angiography unit with a large flat display (FlexVision XL; Philips Medical Systems) was installed in our OR. All media sources in the OR, including live fluoroscopy and ceiling-mounted camcorders, were connected to a video switcher. This video switcher laid up to 8 video images into one big image, which was transferred to the large display and the professional-use Blu-ray recorder. The recording was performed continuously during the procedure. This recording system was evaluated retrospectively with a questionnaire administered to the 5 trainees. RESULTS: Using this system, 68 interventional procedures were recorded. Among the potential merits, the trainees assigned the greatest value to the simultaneous recording of the operator's hand motions and the fluoroscopy images. Among the potential limitations of the system, the prolonged time and the increased volume of the video data bothered the trainees the most. The recorded video looked like a live demonstration. CONCLUSIONS: Our "selfie" video recording system was useful for skill training of neuroendovascular interventions.

A homolog of Arabidopsis SDP1 lipase in Nannochloropsis is involved in degradation of de novo-synthesized triacylglycerols in the endoplasmic reticulum.

Organisms of the microalgal genus Nannochloropsis produce high levels of triacylglycerols (TAGs), an efficient raw material for biofuels. A complete understanding of the TAG-breakdown pathway is critical for improving the productivity of TAGs to meet future needs. Among a number of lipases annotated as TAG lipase in the genomes of every organism, Arabidopsis SUGAR-DEPENDENT 1 (AtSDP1) lipases are characterized as a type of crucial TAG lipase in plants, similar to ScTgl3-5 in Saccharomyces cerevisiae. Homologs of the AtSDP1 TAG lipases are universally found in the genomes of plants, fungi, and algae. Here we identified two homologs of AtSDP1 TAG lipases in the oleaginous microalga species Nannochloropsis oceanica, NoTGL1 and NoTGL2. We generated single- and double-knockout strains for these lipases by homologous recombination. Whereas overall TAG content in the NoTGL2 single-knockout mutant was identical to that of wild type, the NoTGL1 knockout showed a two-fold increase in TAG content per cell in early log phase under nutrient-sufficient conditions without affecting growth. Homologs of AtSDP1 in S. cerevisiae are localized to the surface of lipid droplets, and AtSDP1 is transported from peroxisomes to the surface of lipid droplets. In contrast, NoTGL1 localized to the endoplasmic reticulum in both Nannochloropsis and yeast. We suggest that homologs of AtSDP1 lipases in Nannochloropsis modulate de novo TAG biosynthesis in the endoplasmic reticulum, unlike the roles of these lipases in other organisms. These results provide important insights into the mechanisms of TAG metabolism catalyzed by homologs of AtSDP1 lipase, which are highly conserved across species.

Angiotensin II mimics the hypoxic effect on regulating trophoblast proliferation and differentiation in human placental explant cultures.

Regulation of cytotrophoblast differentiation toward extravillous trophoblasts (EVTs) is critical for establishing successful pregnancy. Previous studies have focused primarily on the factors promoting the differentiation, while inhibitory regulators except hypoxia have been less documented. In this study, to test our hypothesis that angiotensin II (Ang II) would inhibit EVT differentiation, we investigated the effects of Ang II on trophoblast outgrowth and the expression of molecules associated with the proliferation and invasion of trophoblasts using human first trimester villous explant cultures. Ang II increased EVT outgrowth and the number of cells in cell columns. Moreover, Ang II-treated explants exhibited increased Ki67 and integrin alpha5 immunoreactivity in EVTs as well as matrix metalloproteinase-2 activity in the conditioned media, and decreased alpha1 integrin immunoreactivity, which are compatible with the features of the proliferative phenotype EVTs. These effects of Ang II were similar to those of hypoxia (3% O(2)). Ang II stimulated the expression of hypoxia inducible factor-1alpha at both mRNA and protein levels, and also enhanced the expression of plasminogen activator inhibitor-1 (PAI-1). Data presented herein suggest a possible role for Ang II in impairing trophoblast differentiation toward an invasive phenotype, which might be associated with shallow invasion in preeclamptic placentas.

Microsurgery Simulator of Cerebral Aneurysm Clipping with Interactive Cerebral Deformation Featuring a Virtual Arachnoid.

BACKGROUND: A virtual reality simulator for aneurysmal clipping surgery is an attractive research target for neurosurgeons. Brain deformation is one of the most important functionalities necessary for an accurate clipping simulator and is vastly affected by the status of the supporting tissue, such as the arachnoid membrane. However, no virtual reality simulator implementing the supporting tissue of the brain has yet been developed. OBJECTIVE: To develop a virtual reality clipping simulator possessing interactive brain deforming capability closely dependent on arachnoid dissection and apply it to clinical cases. METHODS: Three-dimensional computer graphics models of cerebral tissue and surrounding structures were extracted from medical images. We developed a new method for modifiable cerebral tissue complex deformation by incorporating a nonmedical image-derived virtual arachnoid/trabecula in a process called multitissue integrated interactive deformation (MTIID). MTIID made it possible for cerebral tissue complexes to selectively deform at the site of dissection. Simulations for 8 cases of actual clipping surgery were performed before surgery and evaluated for their usefulness in surgical approach planning. RESULTS: Preoperatively, each operative field was precisely reproduced and visualized with the virtual brain retraction defined by users. The clear visualization of the optimal approach to treating the aneurysm via an appropriate arachnoid incision was possible with MTIID. CONCLUSION: A virtual clipping simulator mainly focusing on supporting tissues and less on physical properties seemed to be useful in the surgical simulation of cerebral aneurysm clipping. To our knowledge, this article is the first to report brain deformation based on supporting tissues.

Radiosurgery-Induced Anterior Inferior Cerebellar Artery Pseudoaneurysm Treated with Trapping and Bypass.

BACKGROUND: Stereotactic radiosurgery (SRS) is an established modality for treatment of vestibular schwannomas (VSs). However, its long-term vascular complications have not been well studied. Among 360 patients who underwent SRS for VS in our institution and lived for >5 years thereafter, we identified only 1 patient who exhibited a complication secondary to a late-onset aneurysm for an estimated incidence of 0.3%. CASE DESCRIPTION: A 78-year-old man who had undergone SRS 19 years previously for a right VS presented with right peripheral facial palsy. Radiographic examinations revealed a distal anterior inferior cerebellar artery (AICA) fusiform aneurysm that was embedded in the tumor and progressively enlarged over 17 months. Although the right AICA perfused a large area of the cerebellum, the aneurysm was successfully treated with AICA trapping in conjunction with an occipital artery-AICA bypass. CONCLUSIONS: Distal AICA pseudoaneurysm formation is a rare but potentially severe late complication after SRS for VS. This pathology was successfully treated with AICA trapping with occipital artery-AICA bypass.

P-LAP/IRAP-induced cell proliferation and glucose uptake in endometrial carcinoma cells via insulin receptor signaling.

BACKGROUND: Hyperglycemia or hyperinsulinemia contributes to poorer endometrial cancer survival. It was shown that P-LAP/IRAP translocates to the plasma membrane in response to insulin stimulation. Recently, we demonstrated that P-LAP/IRAP is associated with a poor prognosis in endometrial adenocarcinoma patients. The aim of this study was to examine whether the malignant potential of endometrial cancer enhanced by P-LAP/IRAP is due to increased glucose uptake via the P-LAP/IRAP-mediated activation of insulin signaling. METHODS: We transfected P-LAP/IRAP cDNA into A-MEC cells (endometrial adenocarcinoma cell line), and A-MEC-LAP cells expressed a remarkably high level of GLUT4 proteins. RESULTS: 3H-2-deoxyglucose uptake which responds to insulin in A-MEC-LAP cells was significantly higher than that of A-MEC-pc cells. A-MEC-LAP cells exhibited a significant growth-stimulatory effect compared to A-MEC-pc cells. A-MEC-LAP cells expressed a remarkably high level of p85PI3K protein compared to A-MEC-pc cells, and showed a higher degree of AKT phosphorylation by insulin stimulation. CONCLUSION: In summary, P-LAP/IRAP was involved in the increasing malignant potential of endometrial cancer mediated by insulin. P-LAP/IRAP was suggested to be a potential new target of molecular-targeted therapy for endometrial cancer.

Neurosurgical Virtual Reality Simulation for Brain Tumor Using High-definition Computer Graphics: A Review of the Literature.

Simulation and planning of surgery using a virtual reality model is becoming common with advances in computer technology. In this study, we conducted a literature search to find trends in virtual simulation of surgery for brain tumors. A MEDLINE search for "neurosurgery AND (simulation OR virtual reality)" retrieved a total of 1,298 articles published in the past 10 years. After eliminating studies designed solely for education and training purposes, 28 articles about the clinical application remained. The finding that the vast majority of the articles were about education and training rather than clinical applications suggests that several issues need be addressed for clinical application of surgical simulation. In addition, 10 of the 28 articles were from Japanese groups. In general, the 28 articles demonstrated clinical benefits of virtual surgical simulation. Simulation was particularly useful in better understanding complicated spatial relations of anatomical landmarks and in examining surgical approaches. In some studies, Virtual reality models were used on either surgical navigation system or augmented reality technology, which projects virtual reality images onto the operating field. Reported problems were difficulties in standardized, objective evaluation of surgical simulation systems; inability to respond to tissue deformation caused by surgical maneuvers; absence of the system functionality to reflect features of tissue (e.g., hardness and adhesion); and many problems with image processing. The amount of description about image processing tended to be insufficient, indicating that the level of evidence, risk of bias, precision, and reproducibility need to be addressed for further advances and ultimately for full clinical application.

Usefulness of high-resolution 3D multifusion medical imaging for preoperative planning in patients with posterior fossa hemangioblastoma: technical note.

Successful resection of hemangioblastoma depends on preoperative assessment of the precise locations of feeding arteries and draining veins. Simultaneous 3D visualization of feeding arteries, draining veins, and surrounding structures is needed. The present study evaluated the usefulness of high-resolution 3D multifusion medical imaging (hr-3DMMI) for preoperative planning of hemangioblastoma. The hr-3DMMI combined MRI, MR angiography, thin-slice CT, and 3D rotated angiography. Surface rendering was mainly used for the creation of hr-3DMMI using multiple thresholds to create 3D models, and processing took approximately 3-5 hours. This hr-3DMMI technique was used in 5 patients for preoperative planning and the imaging findings were compared with the operative findings. Hr-3DMMI could simulate the whole 3D tumor as a unique sphere and show the precise penetration points of both feeding arteries and draining veins with the same spatial relationships as the original tumor. All feeding arteries and draining veins were found intraoperatively at the same position as estimated preoperatively, and were occluded as planned preoperatively. This hr-3DMMI technique could demonstrate the precise locations of feeding arteries and draining veins preoperatively and estimate the appropriate route for resection of the tumor. Hr-3DMMI is expected to be a very useful support tool for surgery of hemangioblastoma.

Dipeptidyl peptidase IV in tumor progression.

Dipeptidyl peptidase IV (DPPIV) is a 110-kDa glycoprotein with ubiquitous expression. Several recent studies have shown that DPPIV affects tumor progression in several human malignancies. We found that ovarian carcinoma cell lines with higher DPPIV expression showed less invasive potential. Furthermore, introduction of DPPIV cDNA into SKOV3 cells (SKDPIV), derived from serous cystadenocarcinoma showing little DPPIV expression, caused a significant decrease in both migration and invasive potential. In addition, nude mice inoculated with SKDPIV cells showed significantly less peritoneal dissemination and longer survival time than those inoculated with parental or vector-transfected cells. We further examined the mechanisms of anti-invasive ability of DPPIV. The expression of E-cadherin was positively correlated with DPPIV expression among five independent ovarian carcinoma cell lines. The SKDPIV cells showed enhanced expression of E-cadherin with a cellular morphological change from a fibroblastic and motile phenotype to an epithelial phenotype compared to parental and MOCK cells. In addition, matrix metalloproteinase 2 (MMP-2) and membrane type 1 matrix metalloprotease (MT1-MMP), which are important markers associated with invasive and metastatic potential, were remarkably reduced in SKDPIV cells. In contrast, tissue inhibitors of matrix metalloproteinases (TIMPs) were enhanced by DPPIV transfection. These findings imply that DPPIV may functionally suppress peritoneal dissemination and progression of ovarian carcinoma by regulating the expression levels of several molecules associated with carcinoma cell invasion and progression.

Gene regulation and physiological function of placental leucine aminopeptidase/oxytocinase during pregnancy.

Human pregnancy serum and placenta have the ability to degrade uterotonic peptide oxytocin (OT). Placental leucine aminopeptidase (P-LAP), which is also called cystine aminopeptidase, is the only membrane aminopeptidase known to functionally degrade OT as oxytocinase (OTase). P-LAP/OTase hydrolyzes several peptides other than OT including vasopressin and angiotensin III. P-LAP/OTase predicted from cDNA sequence is a type II integral membrane protein, which is converted to a soluble form existing in maternal serum by metalloproteases, possibly ADAM (a disintegrin and metalloproteinase) members. P-LAP/OTase activity increases with normal gestation, while decreases in the patients with preterm delivery and severe preeclampsia. In placenta, P-LAP/OTase is predominantly expressed in differentiated trophoblasts, syncytiotrophoblasts. Activator protein-2 (AP-2) and Ikaros transcription factors play significant roles in exerting high promoter activity of P-LAP/OTase in the trophoblastic cells. Moreover, P-LAP/OTase is transcriptionally regulated in a trophoblast-differentiation-dependent fashion via up-regulation of AP-2, putatively AP-2alpha. P-LAP/OTase may be involved in maintaining pregnancy homeostasis via metabolizing peptides such as OT and vasopressin.

Endoplasmic reticulum aminopeptidase-1 mediates leukemia inhibitory factor-induced cell surface human leukocyte antigen-G expression in JEG-3 choriocarcinoma cells.

Maternal immune tolerance is required for extravillous trophoblasts (EVTs) to invade the decidua without rejection. Endoplasmic reticulum aminopeptidase-1 (ERAP1) generates human leukocyte antigen (HLA) class I-adapted antigenic peptides, but its function in trophoblasts lacking classical HLA class I molecules remains undetermined. Leukemia inhibitory factor (LIF) is produced from decidua during the implantation period and plays a necessary role in establishing pregnancy. This study is intended to investigate the location and the function of ERAP1 in trophoblastic cells, focusing on LIF. Immunohistochemistry showed strong ERAP1 expression in cultured EVTs. In choriocarcinoma cell lines used as a model for trophoblasts, ERAP1 was expressed more intensively in JEG-3 than BeWo cells. Immunoblot analysis and immunocytochemistry localized ERAP1 to the endoplasmic reticulum (ER) in JEG-3 cells. Flow cytometry with HLA-G antibody to monitor the supply of antigenic peptides presenting to HLA-G in the ER showed that reducing ERAP1 transcripts by RNA interference did not affect cell surface expression of membrane HLA-G1 (mHLA-G1) in JEG-3 cells under basal conditions. In LIF-treated JEG-3 cells, cell surface mHLA-G1 expression was increased along with ERAP1 protein and promoter activities. In contrast to nonstimulated cells, eliminating ERAP1 from LIF-treated JEG-3 cells reduced the cell surface mHLA-G1 expression and soluble HLA-G1 secretion. This study provides the first evidence showing that ERAP1 is localized in the ER of trophoblasts and is involved in regulating cell surface HLA-G expression in the presence of LIF. Consequently, ERAP1 would function to present antigenic peptides to HLA-G in trophoblasts.

Twist expression predicts poor clinical outcome of patients with clear cell carcinoma of the ovary.

OBJECTIVES: Twist is a highly conserved basic helix-loop-helix transcription factor that regulates the expression of E-cadherin and promotes the epithelial-mesenchymal transition, which is critical for tumor infiltration. We examined the distribution and expression of this molecule in clear cell carcinoma of the ovary (CCC) to elucidate their clinical significance. METHODS: Paraffin sections from CCC tissues (n = 27) were immunostained with Twist antibody and staining intensities were evaluated. Stratified with various clinicopathological factors, overall survival (OS) and progression-free survival (PFS) were evaluated. RESULTS: In the 27 carcinomas, negative Twist immunoexpression was observed in 14 cases (51.9%), and positive immunoexpression in 13 (48.1%). Twist, when categorized into negative versus positive expression, was associated with FIGO stage and peritoneal cytology. In addition, positive Twist expression significantly predicted a poorer OS and PFS compared with negative expression (p < 0.0001). Furthermore, the multivariate analyses revealed that positive Twist expression was an independent prognostic factor for OS and PFS of patients with CCC in this study (p = 0.0077 and 0.0033, respectively). CONCLUSION: The current findings suggest that the assessment of Twist immunoreactivity may be a useful prognostic indicator and that Twist may play a critical role in the progression of CCC.

Advances in treatment of epithelial ovarian cancer.

Since most cases of epithelial ovarian cancer are advanced at diagnosis, this disease is one of the most lethal malignancies of the female genital tract. In recent years, aggressive cytoreductive surgery and chemotherapy have been employed in an attempt to improve the survival rate in patients with epithelial ovarian cancer. Introduction of platinum anticancer drugs increased survival rate, and several randomized studies have been tried to establish the better combination of anticancer drugs. As a result, the combination of paclitaxel and carboplatin was considered as standard regimen for the first-line treatment of patients with advanced ovarian cancer. Since International Federation of Gynecology and Obstetrics (FIGO) accepted a postoperative staging system in 1988, staging laparotomy needs hysterectomy, bilateral adnexectomy, omentectomy, and pelvic and para-aorta lymphadenectomy. However, the influence of lymphadenectomy on survival still remains controversial. Complete resection of the tumor is often difficult since the disease has spread to the abdominal cavity. In such cases, interval debulking surgery is performed after chemotherapy to remove tumors completely. The effectiveness of neoadjuvant chemotherapy and interval debulking surgery still remains unclear. This review will describe the advances in surgical procedures and chemotherapy in treatment of ovarian cancer patients.

Successful surgical strategy for a cervical hemangioblastoma: Case report.

BACKGROUND: Hemangioblastomas are hypervascular lesions and hence their surgical management is challenging. In particular, if complete resection is to be attained, all feeding and draining vessels must be occluded. Although most intramedullary spinal cord tumors are treated utilizing a posterior approach, we describe an anterior surgical strategy for resection of an intramedullary cervical hemangioblastoma. CASE DESCRIPTION: A 36-year-old female with a spinal hemangioblastoma located in the anterior cervical spinal cord presented with a long-standing history of motor weakness of the right upper extremity. Magnetic resonance imaging revealed a large multilevel extensive syrinx and a focal intramedullary enhanced tumor at the C6 level. Angiography showed that the main feeder to the tumor was the left radicular artery (C8), which originated from the thyrocervical trunk, penetrated the dura mater, and branched both rostrally and caudally into the anterior spinal artery (ASA). Three-dimensional computer graphic images showed the tumor was located in the anterior part of the spinal cord, adjacent to and supplied by the ASA. The planned anterior surgical approach involved a total corpectomy of C6 and partial corpectomies of C5 and C7. The tumor was entirely removed despite multiple adhesions, and was successfully freed from the ASA. Patency of the ASA was confirmed utilizing intraoperative indocyanine green videoangiography. Intraoperatively, no monitoring changes were encountered. The pathological diagnosis was of a hemangioblastoma. No postoperative deficit occurred. CONCLUSIONS: An anterior approach for the resection of an anteriorly located intramedullary spinal hemangioblastomas was successfully accomplished in this case.

Wall-to-lumen ratio of intracranial arteries measured by indocyanine green angiography.

BACKGROUND: The wall-to-lumen ratio (WLR) is an important parameter in vascular medicine because it indicates the character of vascular wall as well as the degree of stenosis. Despite the advances in medical imaging technologies, it is still difficult to measure the thin-walled normal intracranial arteries, and the reports on the WLR of normal intracranial artery are limited. It might be possible to calculate the WLR using the indocyanine green (ICG) angiography, which is used to observe intracranial vessels during microsurgery. PURPOSE: To evaluate the WLR of normal intracranial arteries using ICG angiography. MATERIALS AND METHODS: From the three cases in which ICG angiography was recorded with a ruler during microsurgery, 20 measurement points were chosen for the analysis. The ICG was injected intravenously with a dose of 0.2 mg/kg, and the vessels were inspected at high magnification using an operating microscope equipped with near-infrared illumination system. The vessel outer diameter and the luminal diameter were measured using the images before and after the ICG arrival based on the pixel ratio method using a ruler as reference, respectively. The WLR was calculated as 0.5 × (vessel outer diameter - vessel luminal diameter). RESULTS: The WLR (mean ± standard deviation) of normal intracranial arteries was 0.086 ± 0.022. The WLR tended to be high in small arteries. CONCLUSION: The WLR of normal intracranial arteries calculated using ICG angiography was consistent with the WLR reported in the previous reports based on human autopsy.

Suppression of invasion and peritoneal carcinomatosis of ovarian cancer cells by overexpression of AP-2alpha.

A previous report demonstrated that AP-2alpha favors the survival of ovarian cancer patients by clinical findings. However, the functional roles of AP-2alpha in human ovarian cancers have not been determined. To clarify the roles, we overexpressed AP-2alpha in SKOV3 human ovarian cancer cells, which originally possess little AP-2alpha. AP-2alpha overexpression changed cell morphology from spindle to epithelioid type and suppressed cell proliferation and invasion, which would be partially correlated with decreased phosphorylation levels of the erbB2, Akt and ERK pathways, increased E-cadherin and reduced pro-matrix metalloproteinase-2 levels. Moreover, nude mice intraperitoneally injected with AP-2alpha-overexpressing cells survived longer than those with neo-transfected cells. The present data represent the first direct evidence that AP-2alpha plays a tumor suppressive role in ovarian cancer.

Possible involvement of aminopeptidase A in hypertension and renal damage in Dahl salt-sensitive rats.

BACKGROUND: Although aminopeptidase A (APA), which is abundant in the kidneys, is responsible for metabolizing angiotensin II (Ang II), its association with salt sensitivity remains uncertain. We aimed to clarify the involvement of APA in salt-induced hypertension and renal damage. METHODS: Male Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats were fed low-salt (0.3%) or high-salt diet (8%) from 6 weeks of age for 12 weeks. Tail-cuff-measured blood pressure (BP), renal APA activity, renal Ang II levels, histologic renal damage, and APA immunoreactivity were periodically examined. RESULTS: Systolic BP progressively increased only in DS rats given the high-salt diet (DS-8% rats). The DR-8% rats had approximately 3-fold higher renal APA activity than the rats given the low-salt diet (DR-0.3% rats) during the maintenance on the high-salt diet. However, although DS-8% rats also had 2.5-fold higher renal APA activity than DS-0.3% rats at 10 weeks, continuing the high-salt diet afterward suppressed the activity in DS-8% rats below the levels observed in DS-0.3% rats. High-salt diet reduced renal Ang II levels by 30% in DR rats, whereas it showed a small and nonsignificant decrease in DS rats. The number of injured glomeruli was markedly elevated in DS-8% rats after 10 weeks. The APA immunostaining in DS-8% rats was enhanced in glomeruli displaying mild damage, diminished in the severely injured glomeruli, and absent in lesions with hyalinization. CONCLUSIONS: High-salt diet in DS rats increased renal APA activity, although renal injury remained mild, but then reduced it along with the progression of glomerulosclerosis, suggesting that reduced APA activity may be involved in the deterioration of salt-induced hypertension and renal injury.

Possible role of placental leucine aminopeptidase in the antiproliferative effect of oxytocin in human endometrial adenocarcinoma.

PURPOSE: Oxytocin (OT) was reported to inhibit the proliferation of various neoplastic tissues and cells, however, the regulation system remains unclear. This study examined the role of OT and its regulatory ability in endometrial adenocarcinoma. EXPERIMENTAL DESIGN: To investigate the possible function of placental leucine aminopeptidase (P-LAP) in endometrial adenocarcinoma, we transfected P-LAP cDNA into A-MEC cells, showing the lowest enzyme activity of P-LAP. Also we examined P-LAP protein expression in human endometrial adenocarcinoma. RESULTS: We demonstrated the presence of P-LAP, which is identical to cystine aminopeptidase as oxytocinase, in human endometrial adenocarcinoma tissues and found that the expression of P-LAP increase with advances in the grade. Exposure of endometrial adenocarcinoma cell lines to OT caused dose- and time-dependent inhibition of growth. Treatment with 10(-7) M OT for 72 h reduced cell growth by 62, 25, and 30% in A-MEC, HEC1A, and Ishikawa cells, respectively. P-LAP-transfectant cells not only partially recovered from OT-induced growth inhibition but also showed a higher growth rate than parental cells under condition without OT. An OT receptor antagonist and a protein kinase A inhibitor blocked OT-induced growth inhibition in A-MEC and A-MEC-pc cells but not in A-MEC-LAP cells. CONCLUSIONS: These findings suggested that P-LAP might be functionally positive on carcinoma cell growth by degrading suppressive peptides such as OT.