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BACKGROUND: Standard treatment for diffuse peritonitis due to colorectal perforation may be insufficient to suppress inflammatory reaction in sepsis. Thus, developing new treatments is important. This study aimed to examine whether intraperitoneal irradiation by artificial sunlight suppresses inflammatory reaction in a lipopolysaccharide (LPS)-induced peritonitis model after surgical treatments. MATERIALS AND METHODS: Mice were divided into naive, nontreatment (NT), and phototherapy (PT) groups. In the latter two groups, LPS was intraperitoneally administered to induce peritonitis and removed by intraperitoneal lavage after laparotomy. The PT group was irradiated with artificial sunlight intraperitoneally. We evaluated the local and systemic inflammatory reactions. Murine macrophages were irradiated with artificial sunlight after stimulation by LPS, and cell viability and expression of tumor necrotizing factor-α (TNF-α) were evaluated. RESULTS: As a local inflammatory reaction, the whole cell count, the expression of interleukin-6 and TNF-α in the intra-abdominal fluid, and the peritoneal thickness were significantly lower in the PT group than in the NT group. As a systematic inflammatory reaction, the expression of serum TNF-α, granulocyte macrophage colony-stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1α, and MIP-1ß were significantly lower in the PT group than in the NT group. Irradiation by artificial sunlight suppressed the expression of TNF-α in murine macrophages without affecting cell viability. CONCLUSIONS: Intraperitoneal irradiation by artificial sunlight could suppress local and systemic inflammatory reactions in the LPS-induced peritonitis murine model. These effects may be associated with macrophage immune responses.
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Perfuração Intestinal/complicações , Peritônio/efeitos da radiação , Peritonite/terapia , Fototerapia/métodos , Luz Solar , Animais , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Perfuração Intestinal/imunologia , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/efeitos da radiação , Masculino , Camundongos , Peritônio/imunologia , Peritonite/imunologia , Células RAW 264.7RESUMO
BACKGROUND: Iatrogenic inferior vena cava (IVC) injury is a rare but potentially life-threatening complication during laparoscopic surgery. This experimental study aimed to assess the hemostatic ability of a new device, double balloon-equipped central venous (DB-CV) catheter, for IVC injury. METHODS: The DB-CV catheter comprises a triple-lumen sphincterotome combined with two dilating balloons having a diameter of 25 mm. The experimental procedures were performed in five pigs. The DB-CV catheter was inserted via the right femoral vein. For the IVC occlusion test, correct placement of the balloons was confirmed by indocyanine green fluorescence imaging, and hemodynamic data were recorded. For the IVC injury test, a 3- to 4-mm circumferential incision was created in IVC, and hemostasis was initiated using balloon inflation 5 s after the injury. RESULTS: Hemodynamic changes were minimal, with a 20 mmHg reduction in the mean arterial pressure because of IVC occlusion. All bleeding from IVC injuries was successfully temporarily stopped by direct balloon compression, with a mean time to hemostasis of 69 s and mean blood loss of 32 ml. Subsequently, the positioning of IVC injuries between two balloons made it possible to suture the injured IVC. CONCLUSIONS: Balloon occlusion using the DB-CV catheter provides a rapid temporal hemostatic effect and can overcome the serious condition of massive hemorrhage from IVC injuries.
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Oclusão com Balão/instrumentação , Cateteres Venosos Centrais , Doença Iatrogênica , Laparoscopia/efeitos adversos , Lesões do Sistema Vascular/terapia , Veia Cava Inferior/lesões , Animais , Modelos Animais , SuínosRESUMO
OBJECTIVES: After gastrectomy with extended lymph node (LN) dissection, the damage of celiac plexus seems to cause of disorder of eating habits. To clarify the indication of gastrectomy with limited LN dissection for elderly patients, the pathological characteristics of advanced gastric cancer in elderly patients were examined in this study. METHODS: Forty-seven patients aged ≥80 years with advanced gastric cancer (deeper than pT2) who underwent curative gastrectomy from 1998 to 2015 were enrolled. Patients were classified into two groups by extent of LN metastasis: Group A, with N0 or only perigastric LN metastasis (n = 33); Group B, LN metastasis beyond the perigastric area (n = 14). Pathological factors were then evaluated. RESULTS: No significant differences were observed in age, sex, body mass index, American Society of Anesthesiologists physical status classification, serum level of carcinoembryonic antigen, surgical procedure, extent of LN dissection, and number of dissected LNs. Pathological findings showed no significant differences in tumor location, macroscopic type, histologic type, and lymphovascular invasion. However, significant differences were observed in tumor maximum diameter at the cut-off level of 40 mm (Group A: ≤40 mm, n = 10 and >40 mm, n = 23; Group B: ≤40 mm, n = 0 and >40 mm, n = 14; P = 0.02). CONCLUSION: In the elderly patients, LN metastasis in advanced gastric cancer of ≤40 mm in diameter was limited to be within the perigastric area. Gastrectomy with only perigastric LN dissection may be adopted in these patients.
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Excisão de Linfonodo/métodos , Metástase Linfática/patologia , Neoplasias Gástricas/patologia , Fatores Etários , Idoso de 80 Anos ou mais , Feminino , Gastrectomia , Humanos , Masculino , Invasividade Neoplásica , Estadiamento de Neoplasias , Estudos Retrospectivos , Neoplasias Gástricas/cirurgiaRESUMO
The permeability of cells is important for cryopreservation. Previously, we showed in mice that the permeability to water and cryoprotectants of oocytes and embryos at early cleavage stages (early embryos) is low because these molecules move across the plasma membrane predominantly by simple diffusion through the lipid bilayer, whereas permeability of morulae and blastocysts is high because of a water channel, aquaporin 3 (AQP3). In this study, we examined the pathways for the movement of water and cryoprotectants in bovine oocytes/embryos and the role of AQP3 in the movement by determining permeability, first in intact bovine oocytes/embryos, then in bovine morulae with suppressed AQP3 expression, and finally in mouse oocytes expressing bovine AQP3. Results suggest that water moves through bovine oocytes and early embryos slowly by simple diffusion, as is the case in mice, although channel processes are also involved in the movement. On the other hand, water appears to move through morulae and blastocysts predominantly by facilitated diffusion via channels, as in mice. Like water, cryoprotectants appear to move through bovine oocytes/early embryos mostly by simple diffusion, but channel processes could also be involved in the movement of glycerol and ethylene glycol, unlike that in mice. In bovine morulae, although glycerol and ethylene glycol would move predominantly by facilitated diffusion, mostly through AQP3, as in mice, dimethylsulfoxide appears to move predominantly by simple diffusion, unlike in mice. These results indicate that permeability-related properties of bovine oocytes/embryos are similar to those of mouse oocytes/embryos, but species-specific differences do exist.
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Aquaporina 3/metabolismo , Blastocisto , Tamanho Celular/efeitos dos fármacos , Criopreservação , Crioprotetores/farmacologia , Deslocamentos de Líquidos Corporais/efeitos dos fármacos , Oócitos , Animais , Aquaporina 3/genética , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Bovinos , Crioprotetores/metabolismo , Crioprotetores/farmacocinética , Difusão/efeitos dos fármacos , Ectogênese , Difusão Facilitada/efeitos dos fármacos , Fertilização in vitro , Inativação Gênica , Soluções Hipertônicas , Técnicas de Maturação in Vitro de Oócitos , Camundongos , Camundongos Endogâmicos ICR , Mórula/efeitos dos fármacos , Mórula/metabolismo , Mórula/patologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/patologia , Permeabilidade/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Especificidade da EspécieRESUMO
[This corrects the article DOI: 10.1155/2019/2710587.].
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AIM: We investigated the clinical impact of D3 lymph node dissection preserving left colic artery (LCA) compared to D3 without LCA preservation using data from JCOG0404. LCA preservation is expected to maintain adequate blood supply, which is effective in preventing anastomotic leakage, intestinal paralysis, and bowel obstruction. Whether D3 with LCA preservation (Group A) improves clinical outcomes following resection of sigmoid colon cancer compared to D3 without LCA preservation (Group B) is unclear. METHODS: Procedure type was identified from photographs of the surgical field collected for central surgical review in JCOG0404. Clinical outcomes were compared between each procedure. RESULTS: Among the 1057 randomized patients in JCOG0404, 631 patients receiving sigmoid colectomy or anterior resection were included in the subgroup analysis. Group A comprised of 135 patients and Group B of 496 patients. Patient backgrounds did not differ between groups. Median operative time, blood loss, anastomotic leakage, and intestinal paralysis were not remarkably different (Group A vs Group B: 185 vs 186 minutes, 60 vs 50 mL, 3.0% vs 5.0%, and 2.2% vs 3.8%). More overall postoperative complications occurred in Group B than Group A (21.6% vs 9.6%, P = .022). Five-year relapse-free survival (RFS) and overall survival (OS) tended to be better in Group A than Group B (RFS: 83.7% and 80.5%, HR 0.80 [95% CI 0.51-1.26], OS: 96.3% and 91.1%, HR 0.41 [95% CI 0.19-0.89]). CONCLUSIONS: Short- and long-term outcomes tend to be better in Group A than Group B, indicating that preservation of LCA could be an alternative treatment.
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BACKGROUND: An alkalescent (pH 8.3) mineral water (AMW) of Hita basin, located in the northwestern part of Kyushu island in Japan, has been recognized for the unique quality of ingredients including highly concentrated silicic acid, sodium, potassium, and hydrogen carbonate. The biological effects of AMW intake were evaluated with a particular focus on its "antiobesity" properties through its modulation of the gut microbiota population. METHODS: Two groups of C57BL6/J mice (8-week-old male) were maintained with a standard diet and tap water (control: TWC group) or AMW (AMW group) for 6 months and the following outputs were quantitated: (1) food and water intake, (2) body weight (weekly), (3) body fat measurements by CT scan (monthly), (4) sera biochemical values (TG, ALT, AST, and ALP), and (5) UCP-1 mRNA in fat tissues (terminal point). Two groups of ICR mice (7-week-old male) were maintained with the same method and their feces were collected at the 0, 1st, 3rd, and 6th month at which time the population rates of gut microbiota were quantitated using metagenomic sequencing analysis of 16S-rRNA. RESULTS: Among all antiobesity testing items, even though a weekly dietary consumption was increased (p=0.012), both ratios of weight gain (p=1.21E - 10) and visceral fat accumulation (p=0.029) were significantly reduced in the AMW group. Other criteria including water intake (p=0.727), the amounts of total (p=0.1602), and subcutaneous fat accumulation (p=0.052) were within the margin of error and UCP-1 gene expression level (p=0.171) in the AMW group was 3.89-fold higher than that of TWC. Among 8 major gut bacteria families, Lactobacillaceae (increased, p=0.029) and Clostridiaceae (decreased, p=0.029) showed significant shift in the whole population. CONCLUSION: We observed significantly reduced (1) weight gaining ratio (average -1.86%, up to -3.3%), (2) visceral fat accumulation ratio (average -4.30%, up to -9.1%), and (3) changes in gut microbiota population. All these consequences could support the "health benefit" functionality of AMW.
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In zebrafish oocytes, it has been reported that a 60 or 75% Leibovitz L-15 medium or simple balanced saline solution containing 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP) is effective for nuclear maturation. However, most of the oocytes that matured under these conditions were not fertilized and did not hatch. Thus, these in vitro maturation methods could not support the cytoplasmic maturation of zebrafish oocytes. Therefore, we tried to develop a reliable in vitro maturation method for zebrafish oocytes, which supports their ability to be fertilized and to develop till hatching. When zebrafish oocytes at stage III were cultured in 50-100% Leibovitz L-15 medium supplemented with DHP, the highest rates of cleavage (24%) and hatching (12%) were obtained from oocytes matured in 90% Leibovitz L-15 medium. When we examined the suitable pH (7.5-9.5) of the 90% medium, higher rates of cleavage (45%) and hatching (33%) were obtained in oocytes matured at pH 9.0 than at pH 7.5, 8.5, or 9.5 (cleavage rate, 16-29%; hatching rate, 8-21%). In oocytes matured in 90% Leibovitz L-15 medium at pH 9.0, high rates of cleavage (70%) and hatching (63%) were obtained when oocytes were cultured for 270 min with 0.5 mg/ml BSA. Thus, 90% Leibovitz L-15 medium at pH 9.0 containing 0.5 mg/ml BSA was effective for normal maturation of zebrafish oocytes. This method will become a powerful tool for understanding the mechanism of in vitro maturation in zebrafish oocytes and for the practical use of immature oocytes.
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Oócitos/fisiologia , Oogênese/fisiologia , Peixe-Zebra/fisiologia , Animais , Meios de Cultura , Citoplasma/fisiologia , Feminino , Fertilização in vitro/métodos , Concentração de Íons de Hidrogênio , Hidroxiprogesteronas , Compostos Orgânicos , Concentração Osmolar , Soroalbumina Bovina , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
OBJECTIVES: This study aimed to evaluate the long-term outcomes of neoadjuvant chemoradiotherapy with S-1 in patients with locally advanced rectal cancer. METHODS: A multi-institutional, prospective, phase II trial was conducted between April 2009 and August 2011. The study enrolled 37 patients with histologically proven rectal carcinoma (T3-4 N0-3 M0) who underwent neoadjuvant chemoradiotherapy with S-1. Total mesorectal excision with D3 lymphadenectomy was performed 4-8 weeks after completion of neoadjuvant chemoradiotherapy with S-1 in 36 patients. We then analyzed late adverse events, overall survival, and disease-free survival. RESULTS: The median patient age was 59 years (range: 32-79 years); there were 24 men and 13 women. Ten patients had Stage II disease, and 27 had Stage III disease. Severe late adverse events occurred in 7 patients (18.9%). The 5-year disease-free survival was 66.7%, and the 5-year overall survival was 74.7%. The median follow-up period was 57 months. Local recurrences developed in 5 patients (13.5%), and distant metastases developed in 8 (21.6%). CONCLUSION: Neoadjuvant-synchronous chemoradiotherapy with S-1 for locally advanced rectal cancer is feasible in terms of adverse events and long-term outcomes. (UMIN Clinical Trial Registry: UMIN000003396).
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BACKGROUND: Endoscopic resection is accepted as the standard treatment for early mucosal gastric cancer, and its indications have recently been expanded while its long-term outcomes are still unclear. Herein, we present a didactic case of undifferentiated-type mucosal gastric cancer fulfilling the expanded indication and curative criteria for endoscopic submucosal dissection (ESD), having synchronous multiple lymph node metastases. CASE PRESENTATION: A 40-year-old woman was found to have a Helicobacter pylori infection at a standard health check with no abdominal symptoms. She received an upper gastrointestinal endoscopy and found to have an undifferentiated-type mucosal gastric cancer with the size of 15 mm in diameter without ulceration, which fulfilled the expanded indication for ESD. According to patient's preference, we performed laparoscopy-assisted distal gastrectomy with D1+ lymph node dissection, and routine pathological analysis revealed a predominantly signet ring cell carcinoma limited to the mucosa without ulceration or any vessel involvement; on the other hand, 15 lymph node metastases were detected. Then, we added deep sectioning of the whole tumoral area at a thickness of 20 µm and immunohistochemical analyses. As the result, an isolated lymphatic capillary involvement of the extremely superficial submucosa was identified in a single histological section, and pathological diagnosis was corrected to ly1. She received postoperative adjuvant chemotherapy with an S-1 oral agent and had no recurrence under strict surveillance for 1 year postoperatively. CONCLUSIONS: When we perform ESD for undifferentiated-type gastric cancer, deep sectioning of the whole tumoral area into thin slices and immunohistochemical staining using D2-40 should be practically considered.
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As an essential step toward cryopreservation of fish embryos, we examined the chilling sensitivity of medaka (Oryzias latipes) embryos at various developmental stages. Embryos at the 2-4 cell, 8-16 cell, morula, blastula, and early gastrula stages were suspended in Hanks solution. They were chilled to various temperatures (usually 0 degrees C), kept for various periods (usually 20 min), then cultured for up to 14 d to determine survival (assessed by the ability to hatch). Embryos at the 2-4 cell stage were the most sensitive to chilling to 0 degrees C, but sensitivity decreased as development proceeded. The survival rate of 2-4 cell embryos was affected after 2 min of chilling at 0 degrees C; although the rate decreased gradually as the duration of chilling increased, 38% of them still survived after 40 min of chilling. Embryos at the 2-4 cell stage were sensitive to chilling at 0 or -5 degrees C, but much less sensitive at 5 or 10 degrees C. The survival rate of 2-4 cell embryos subjected to repeated rapid cooling and warming was similar to that of those kept chilled. When early gastrula embryos were preserved at 0 or 5 degrees C, the hatching rate did not decrease after 12 and 24h of chilling, respectively, but then decreased gradually as storage was prolonged; however, 3-10% of the embryos hatched even after storage for 10 d. In conclusion, although later-stage medaka embryos would be suitable for cryopreservation (from the perspective of chilling sensitivity), chilling injury may not be serious in earlier stage embryos.
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Temperatura Baixa , Oryzias/embriologia , Preservação de Tecido/veterinária , Animais , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário , Gástrula , Fatores de TempoRESUMO
Highly efficient acetylcholinesterase (AChE) and serotonin transporter (SERT) dual inhibitors, (S)-4 and (R)-13 were designed and synthesized on the basis of the hypothetical model of AChE active site. Both compounds showed potent inhibitory activities against AChE and SERT. [structure: see text]
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Doença de Alzheimer/tratamento farmacológico , Inibidores da Colinesterase/síntese química , Desenho de Fármacos , Inibidores Seletivos de Recaptação de Serotonina/síntese química , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Sítios de Ligação , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Serotonina/metabolismoRESUMO
INTRODUCTION: Hydrocele of the canal of Nuck is a rarely encountered entity. We report a case underwent laparoscopic totally extraperitoneal (TEP) treatment for a hydrocele of the canal of Nuck extending in the extraperitoneal space mainly. PRESENTATION OF CASE: A 37-year-old woman complained of painless and reducible swelling in her left groin, and referred to our hospital for surgical management against left inguinal hernia with the incarcerated ovary. Ultrasonography and MR images revealed a cystic mass in the retroperitoneal space, and we diagnosed as an unusual type of hydrocele of the canal of Nuck. The patient was scheduled for laparoscopic treatment. Laparoscopic findings on pneumoperitoneum showed an extraperitoneal cystic tumor with no contact with the left ovary. The fascia and peritoneum of the port site were closed, and then an extraperitoneal space was created. The cystic tumor with the round ligament of the uterus was dissected and resected by the TEP technique. The extended deep inguinal ring was repaired with polypropylene mesh. Postoperative course was uneventful. DISCUSSION: Hydrocele of the canal of Nuck in the adult female is a rare condition. The accurate diagnosis of an inguinal hydrocele in a female is seldom made. Laparoscopic examination provides surgeons with information of inguinal swelling accompanied with retroperitoneal cyst, and consecutive treatment by laparoscopic technique, especially TEP, is useful in regard to minimal damage of the peritoneum. CONCLUSION: Laparoscopic diagnosis and TEP treatment offers a useful alternative in selected patients with hydrocele of the canal of Nuck.
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To identify a stage feasible for the cryopreservation of zebrafish oocytes, we investigated the permeability to water and cryoprotectants of immature (stage III) and mature (stage V) oocytes. The permeability to water (microm/min/atm) of immature oocytes at 25 degrees C (0.37) was significantly higher than that of mature oocytes (0.10). The permeability (x10(-3)cm/min) of immature oocytes to ethylene glycol, propylene glycol, and Me(2)SO (1.49-3.03) at 25 degrees C was substantially higher than that of mature oocytes approximately 0. The permeability of immature oocytes to glycerol was also high (1.75), although the permeability could not be measured in mature oocytes. Immature oocytes would be more suitable than mature oocytes for conservation of the zebrafish.
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Permeabilidade da Membrana Celular , Criopreservação/métodos , Crioprotetores/metabolismo , Oócitos/metabolismo , Água/metabolismo , Peixe-Zebra/metabolismo , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Propilenoglicol/farmacologia , Fatores de TempoRESUMO
Movement of water and cryoprotectants through the plasma membrane needs to be accelerated for successful cryopreservation of zebrafish oocytes/embryos, which are much larger than their mammalian counterparts. Aquaporin-3 is a water/solute channel that can transport not only water but also various cryoprotectants. In this study, we attempted to increase the permeability of immature zebrafish oocytes at stage III to water and cryoprotectants by exogenous expression of rat aquaporin-3. Immature zebrafish oocytes were injected with rat aquaporin-3 cRNA and cultured for 5-12 h. Permeability to water and cryoprotectants was then determined based on changes in the volumes of the oocytes in a hypertonic sucrose solution and various cryoprotectant solutions at 25 C. The permeability to water of the aquaporin-3 cRNA-injected oocytes was three times higher than that of intact and water-injected oocytes. The permeability of the aquaporin-3 cRNA-injected oocytes to ethylene glycol, glycerol, propylene glycol, and DMSO was also 2-4 times higher than that of intact oocytes. Thus, the permeability of immature zebrafish oocytes to water and cryoprotectants was enhanced by exogenous expression of aquaporin-3. Cryopreservation of teleost oocytes may be realized through a further increase in permeability.
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Aquaporina 3/metabolismo , Permeabilidade da Membrana Celular/genética , Crioprotetores/metabolismo , Oócitos/metabolismo , Água/metabolismo , Peixe-Zebra/metabolismo , Animais , Aquaporina 3/genética , Expressão Gênica , Microinjeções , RNA Complementar , Ratos , Transformação Genética , Peixe-Zebra/genéticaRESUMO
The permeability to water and cryoprotectants of the plasma membrane is crucial to the successful cryopreservation of embryos. Previously, we have shown in mouse morulae that water and glycerol move across the plasma membrane by facilitated diffusion, and we have suggested that aquaporin 3 plays an important role in their movement. In the present study, we clarify the contribution of aquaporin 3 to the movement of water and various cryoprotectants in mouse morulae by measuring the Arrhenius activation energies for permeability to cryoprotectants and water, through artificial expression of aquaporin 3 using Aqp3 cRNA in mouse oocytes, and by suppressing the expression of aquaporin 3 in morulae by injecting double-stranded RNA of Aqp3 at the one-cell zygote stage. The results show that aquaporin 3 plays an important role in the facilitated diffusion of water, glycerol, and ethylene glycol, but not of acetamide and dimethylsulfoxide. On the other hand, in a propylene glycol solution, aquaporin 3 in morulae transported neither propylene glycol nor water by facilitated diffusion, probably because of strong water-solute interactions. These results provide important information for understanding the permeability of the plasma membrane of the mouse embryo.
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Aquaporina 3/fisiologia , Crioprotetores/metabolismo , Mórula/fisiologia , Água/metabolismo , Animais , Aquaporina 3/genética , Permeabilidade da Membrana Celular , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mórula/ultraestrutura , Gravidez , Propilenoglicol/metabolismo , RNA de Cadeia Dupla/genética , Soluções , Termodinâmica , TransfecçãoRESUMO
The first successful cryopreservation of fish embryos was reported in the Japanese flounder by vitrification [Chen and Tian, Theriogenology, 63, 1207-1219, 2005]. Since very high concentrations of cryoprotectants are needed for vitrification and fish embryos have a large volume, Japanese flounder embryos must have low sensitivity to cryoprotectant toxicity and high permeability to water and cryoprotectants. So, we investigated the sensitivity and the permeability of Japanese flounder embryos. In addition, we assessed the survival of flounder embryos after vitrification with solutions containing methanol and propylene glycol, following Chen and Tian's report. The embryos were relatively insensitive to the toxicity of individual cryoprotectants at lower concentrations, especially methanol and propylene glycol as their report. Although their permeability to water and cryoprotectants could not be measured from volume changes in cryoprotectant solutions, the embryos appeared to be permeable to methanol but less permeable to DMSO, ethylene glycol, and propylene glycol. Although vitrification solutions containing methanol and propylene glycol, which were used in Chen and Tian's report, were toxic to embryos, a small proportion of embryos did survived. However, when vitrified with the vitrification solutions, no embryos survived after warming. The embryos became opaque during cooling with liquid nitrogen, indicating the formation of intracellular ice during cooling. When embryos had been kept in vitrification solutions for 60 min after being treated with the vitrification solution, some remained transparent during cooling, but became opaque during warming. This suggests that dehydration and/or permeation by cryoprotectants were insufficient for vitrification of the embryos even after they had been over-treated with the vitrification solutions. Thus, Chen and Tian's cryopreservation method lacks general application to Japanese flounder embryos.
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Criopreservação/métodos , Crioprotetores/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Linguados/embriologia , Animais , Cristalização , Dimetil Sulfóxido/toxicidade , Etilenoglicol/toxicidade , Metanol/toxicidade , Permeabilidade , Propilenoglicol/toxicidadeRESUMO
The permeability of the plasma membrane plays a crucial role in the successful cryopreservation of oocytes and embryos. Several efforts have been made to facilitate the movement of water and cryoprotectants across the plasma membrane of fish oocytes/embryos because of their large size. Aquaporin-3 is a water/solute channel that can also transport various cryoprotectants. In this study, we tried to improve the permeability of immature medaka (Oryzias latipes) oocytes to water and cryoprotectants by artificially expressing aquaporin-3. The oocytes were injected with aquaporin-3 cRNA and cultured for 6-7 h. Then, hydraulic conductivity (L(P)) and cryoprotectant permeability (P(S)) were determined from volume changes in a hypertonic sucrose solution and various cryoprotectant solutions, respectively, at 25 degrees C. The L(P) value of the cRNA-injected oocytes was 0.22+/-0.04 microm/min/atm, nearly twice larger than that of intact or water-injected oocytes (0.14+/-0.02 and 0.14+/-0.03 microm/min/atm, respectively). P(S) values of intact oocytes for ethylene glycol, propylene glycol, and DMSO were 1.36+/-0.34, 1.97+/-0.20, and 1.17+/-0.52 x 10(-3) cm/min, respectively. The permeability to glycerol could not be calculated because oocytes remained shrunken in the glycerol solution. On the other hand, cRNA-injected oocytes had significantly higher P(S) values (glycerol, 2.20+/-1.29; ethylene glycol, 2.98+/-0.36; propylene glycol, 3.93+/-1.70; DMSO, 3.11+/-0.74 x 10(-3) cm/min) than intact oocytes. When cRNA-injected oocytes were cultured for 12-14 h, 51% matured to the metaphase II stage, and 43% of the matured oocytes were fertilized and hatched following in vitro fertilization and 14 days of culture. Thus, the permeability of medaka oocytes to water and cryoprotectants was improved by the artificial expression of aquaporin-3, and the oocytes retained the ability to develop to term.
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Aquaporina 3/biossíntese , Membrana Celular/metabolismo , Criopreservação/métodos , Crioprotetores/farmacologia , Oócitos/metabolismo , Animais , Oryzias , Permeabilidade , RNA Complementar/metabolismo , Temperatura , Água/químicaRESUMO
The permeability of the plasma membrane plays a crucial role in the successful cryopreservation of oocytes/embryos. To identify a stage feasible for the cryopreservation of teleost oocytes, we investigated the permeability to water and various cryoprotectants of medaka (Oryzias latipes) oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. In sucrose solutions, the volume changes were greater in GV oocytes than MII oocytes. Estimated values for osmotically inactive volume were 0.41 for GV oocytes and 0.74 for MII oocytes. Water-permeability (microm/min/atm) at 25 degrees C was higher in GV oocytes (0.13+/-0.01) than MII oocytes (0.06+/-0.01). The permeability of MII oocytes to various cryoprotectants (glycerol, propylene glycol, ethylene glycol, and DMSO) was quite low because the oocytes remained shrunken during 2 h of exposure in the cryoprotectant solutions at 25 degrees C. When the chorion of MII oocytes was removed, the volume change was not affected, except in DMSO solution, where dechorionated oocytes shrunk and then regained their volume slowly; the P(DMSO) value was estimated to be 0.14+/-0.01x10(-3) cm/min. On the other hand, the permeability of GV oocytes to cryoprotectants were markedly high, the P(s) values (x10(-3) cm/min) for propylene glycol, ethylene glycol, and DMSO being 2.21+/-0.29, 1.36+/-0.18, and 1.19+/-0.01, respectively. However, the permeability to glycerol was too low to be estimated, because GV oocytes remained shrunken after 2 h of exposure in glycerol solution. These results suggest that, during maturation, medaka oocytes become less permeable to water and to small neutral solutes, probably by acquiring resistance to hypotonic conditions before being spawned in fresh water. Since such changes would make it difficult to cryopreserve mature oocytes, immature oocytes would be more suitable for the cryopreservation of teleosts.
Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Oócitos/patologia , Preservação de Órgãos/métodos , Água/farmacologia , Animais , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Feminino , Glicerol/química , Oócitos/metabolismo , Oryzias , Osmose , Permeabilidade , Propilenoglicol/farmacologia , Temperatura , Fatores de TempoRESUMO
Alzheimer's disease (AD) has been treated with acetylcholinesterase (AChE) inhibitors such as donepezil. However, the clinical usefulness of AChE inhibitors is limited mainly due to their adverse peripheral effects. Depression seen in AD patients has been treated with serotonin transporter (SERT) inhibitors. We considered that combining SERT and AChE inhibition could improve the clinical usefulness of AChE inhibitors. In a previous paper, we found a potential dual inhibitor, 1, of AChE (IC50=101 nM) and SERT (IC50=42 nM), but its AChE inhibition activity was less than donepezil (IC50=10 nM). Here, we report the conformationally restricted (R)-18a considerably enhanced inhibitory activity against AChE (IC50=14 nM) and SERT (IC50=6 nM).