Macromolecular crowded conditions strengthen contacts between mouse oocytes and companion granulosa cells during in vitro growth.

Macromolecular crowded culture medium formed by addition of polyvinylpyrrolidone (PVP; molecular weight = 360 000), positively influences the viability, growth, and development of bovine oocytes. Owing to its apparently various effects, uncovering the specific mechanisms of crowding responsible for these outcomes is important. The present study was conducted to determine the effects of crowding on oocytes with a particular focus on the intimacy of contacts between oocyte and cumulus/granulosa cells. Growing mouse oocyte-granulosa cell complexes were cultured for 10 days in a modified α-minimum essential medium, supplemented with PVP at a concentration of 0%, 1%, 2%, or 3% (w/v). Although the complexes developed in all groups, 2% and 3% PVP medium induced a substantial morphological modification, and a larger proportion of oocytes associated with cumulus cells survived in 3% PVP medium than in the 0% or 1% PVP medium. No significant difference was found in the frequencies of polar body extrusion (78-88%) and blastocyst formation (approximately 40%) after in vitro fertilization among the experimental groups. Confocal laser scanning microscopy indicated a higher number of transzonal processes reaching the oocyte from cumulus cells in 2% PVP medium than in 0% PVP medium. Transmission electron microscopy depicted close adhesion of the oocyte with cumulus cells in 2% PVP medium -bearing a resemblance to their in vivo counterparts- and loose adhesion in 0% PVP medium. In conclusion, we found that a mechanism for the action of crowded conditions involves the strengthening of contacts and communication between oocytes and companion cumulus/granulosa cells.

Erratum: J Reprod Dev, Vol. 64, No. 2, p. 153-160, Macromolecular crowded conditions strengthen contacts between mouse oocytes and companion granulosa cells during in vitro growth.

Figure 7 have been corrected.

Endoplasmic reticulum stress-mediated apoptosis contributes to a skeletal dysplasia resembling platyspondylic lethal skeletal dysplasia, Torrance type, in a novel Col2a1 mutant mouse line.

In humans, mutations in the COL2A1 gene encoding the α1(II) chain of type II collagen, create many clinical phenotypes collectively termed type II collagenopathies. However, the mechanisms generating this diversity remain to be determined. Here we identified a novel Col2a1 mutant mouse line by screening a large-scale N-ethyl-N-nitrosourea mutant mouse library. This mutant possessed a p.Tyr1391Ser missense mutation in the C-propeptide coding region, and this mutation was located in positions corresponding to the human COL2A1 mutation responsible for platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T). As expected, p.Tyr1391Ser homozygotes exhibited lethal skeletal dysplasias resembling PLSD-T, including extremely short limbs and severe dysplasia of the spine and pelvis. The secretion of the mutant proteins into the extracellular space was disrupted, accompanied by an abnormally expanded endoplasmic reticulum (ER) and the up-regulation of ER stress-related genes in chondrocytes. Chondrocyte apoptosis was severely induced in the growth plate of the homozygotes. These findings strongly suggest that ER stress-mediated apoptosis caused by the accumulated mutant proteins in ER contributes to skeletal dysplasia in Co12a1 mutant mice and PLSD-T patients.

Three-Dimensional Ultrastructure of Flower-Spray Nerve Endings in the Rat Carotid Sinus.

The flower-spray nerve endings are afferent nerve terminals in the carotid sinus that arise from carotid sinus nerve of glossopharyngeal nerve. However, the three-dimensional ultrastructural characteristics of flower-spray nerve endings and spatial relationships between the terminal parts and other cellular elements have not been fully understood. To elucidate their detailed relationship, backscattered electron imaging of serial sections was performed with a scanning electron microscope to produce a three-dimensional reconstruction of the flower-spray endings. The terminal parts of flower-spray endings were distributed horizontally approximately 5 µm outside the external elastic membrane in the tunica adventitia of the internal carotid artery. The three-dimensional reconstruction showed that the terminal parts of flower-spray endings were flat with irregular contours and were partially covered by the thin cytoplasmic processes of Schwann cells. The complex consisting of the nerve terminals and associated Schwann cells was surrounded by a multilayered basement membrane. The terminal parts of the endings were also surrounded by fibroblasts with elastic fibers and collagen fibrils. Secretory vesicles without an electron-dense core were observed in the terminal parts of the endings. The accumulation of vesicles just below the axonal membrane was observed in terminal parts not covered by Schwann cell cytoplasmic processes on both the luminal and basal sides. Swollen mitochondria, concentric membranous structures, and glycogen granule-like electron-dense materials were often noted in some of the terminal parts of the endings and the parent axon. Collectively, the present results suggest that flower-spray endings are baroreceptors because their morphology was similar to other mechanoreceptors. Furthermore, flower-spray endings may be affected by glutamate secreted in an autocrine manner.

Three-dimensional architecture of the subepithelial corpuscular nerve ending in the rat epiglottis reconstructed by array tomography with scanning electron microscopy.

In the rat laryngeal mucosa, subepithelial corpuscular nerve endings, called laminar nerve endings, are distributed in the epiglottis and arytenoid region and are activated by the pressure changes of the laryngeal cavity. They are also suggested to play a role in efferent regulation because of secretory vesicles in the axoplasm. In the present study, the laminar nerve endings in the rat laryngeal mucosa were analyzed by 3D reconstruction from serial ultrathin sections in addition to immunohistochemistry for synapsin 1. In the light microscopy, synapsin 1-immunoreactive flattened or bulbous terminal parts of the laminar endings were also immunoreactive with VGLUT1, and were surrounded by S100- or S100B-immunoreactive Schwann cells and vimentin-immunoreactive fibroblasts. In the electron microscopy, 3D reconstruction views showed that laminar endings were composed of flattened terminal parts sized 2-5 µm in longitudinal length, overlapping in three to five multiple layers. The terminal parts of the endings were incompletely wrapped by flat cytoplasmic processes of the Schwann cells. In addition, the fibroblast network surrounded the complex of nerve endings and the Schwann cells. Several terminal parts entered through the basement membrane into the epithelial layer and attached to the basal epithelial cells, suggesting that interaction between epithelial cells and laminar nerve endings plays an important role in sensing the pressure changes in the laryngeal cavity. Secretory vesicles were unevenly distributed throughout the terminal part of the laminar nerve endings. The secretory vesicles were frequently observed in the peripheral limb of the terminal parts. It suggests that the laminar nerve endings in the larynx may release glutamate to maintain continuous discharge during the stretching of the laryngeal mucosa.

Vascular anatomical study of persistent descending mesocolon in patients undergoing laparoscopic surgery for colorectal cancer.

INTRODUCTION: Persistent descending mesocolon (PDM) is a rare congenital atypia of fixation of the descending colon, and currently, very few detailed studies exist on its vascular anatomy. This study was conducted to evaluate the features of the vascular anatomy of PDM to help avoid intraoperative lethal injury and subsequent postoperative complications in laparoscopic colorectal surgery. METHODS: We retrospectively analyzed the data of 534 patients who underwent laparoscopic left-sided colorectal surgery. PDM was diagnosed using preoperative axial computed tomography (CT) view. The vascular anatomical features were compared between PDM and non-PDM cases based on three-dimensional (3D)-CT angiography findings. Additionally, the perioperative short-term outcomes of laparoscopic surgery in the 534 patients were also compared between PDM and non-PDM cases. RESULTS: Of the total 534 patients, 13 patients (2.4%) presented with PDM. No branching pattern of the inferior mesenteric artery (IMA) specific to PDM was found. In the running direction of the IMA and sigmoidal colic artery (SA), the midline-shift of IMA and the right-shift of SA were significantly more in PDM than in non-PDM cases, respectively (38.5% vs. 2.5%, P ≤ .0001; 61.5% vs. 4.6%, P ≤ .0001). The perioperative short-term outcomes of laparoscopic surgery in the 534 patients were similar between PDM and non-PDM cases. CONCLUSION: Because changes in the direction of the vascular running are often observed due to adhesions and shortening of the mesentery in PDM cases, performing a detailed preoperative evaluation of vascular anatomy using imaging modalities such as 3D-CT angiography is important.

Slightly acidic electrolyzed water inhibits inflammation induced by membrane vesicles of Staphylococcus aureus.

Staphylococcus aureus grows in the skin of patients with atopic dermatitis and the associated symptoms are induced by membrane vesicles (MVs). This study explored the effects of slightly acidic electrolyzed water (SAEW) on the expression of virulence factors of S. aureus and MV-induced inflammation to uncover the potential of SAEW as a new treatment method for atopic dermatitis. Expression levels of genes related to virulence factors in S. aureus was assessed and S. aureus-derived MVs were characterized. Moreover, expression level of MV-induced Type I allergic reaction-related genes in RBL2H3 cells was also assessed. Significantly decreased staphylococcal enterotoxin A production and decreased virulence factor-related gene expression were observed after culturing S. aureus in broth supplemented with SAEW at ratios of 1, 2, and 5 per broth. MVs prepared by culturing S. aureus in SAEW-supplemented broth exhibited altered particle size and markedly reduced staphylococcal enterotoxin A content under all addition conditions; moreover, those obtained at a ratio of 1:5 (broth:SAEW) exhibited a reduction in the expression of several proteins associated with hemolytic activity and free iron uptake. The MVs prepared in SAEW-supplemented broth also exhibited remarkably reduced allergy-related gene expression levels in rat cell lines derived from basophilic leukemia-2H3 cells. Overall, SAEW is expected to suppress atopic dermatitis symptoms through the alteration of the properties of S. aureus-derived MVs.

[A case report of complete response to low-dose S-1 monotherapy for pancreatic cancer in elderly patient].

A 87-year-old woman was diagnosed with pancreatic body cancer(Stage II ). Because of her age and history of open distal partial gastrectomy, it seemed that having her undergo an invasive surgery would be difficult. Therefore, S-1 was administered orally at a dose of 50mg/day for 28 consecutive days followed by a 14-day rest course(low-dose S-1 monotherapy). After 2 courses, the tumor marker(CA19-9)dramatically decreased to within the normal range. After 5 courses, the tumor could not be identified on the abdominal CT image, and it was judged to be a complete response. Low-dose S-1 monotherapy may be useful for improving the prognosis of pancreatic cancer without causing intolerable toxicity, especially for elderly patients.

Effects of Growth Stage on the Characterization of Enterotoxin A-Producing Staphylococcus aureus-Derived Membrane Vesicles.

Virulence factors, such as staphylococcal enterotoxin A (SEA), are contained within membrane vesicles (MVs) in the cell membrane of Staphylococcus aureus. In this study, the effects of the growth stage on quantitative and qualitative changes in the components contained in the MVs of S. aureus SEA-producing strains were examined. Changes in the expression levels of S. aureus genes were examined at each growth stage; phenol-soluble modulin (PSM) gene reached a maximum after 8 h, and the expression of cell membrane-related genes was decreased after 6 h. Based on these gene expression patterns, MVs were prepared at 6, 17, and 24 h. The particle size of MVs did not change depending on the growth stage. MVs prepared after culture for 17 h maintained their particle size when stored at 23 °C. The amount of SEA in the culture supernatant and MVs were not correlated. Bifunctional autolysin, a protein involved in cell wall biosynthesis/degradation, was increased in MVs at 17 h. The expression pattern of inflammation-related genes in human adult low calcium high temperature (HaCaT) cells induced by MVs was different for each growth stage. The inclusion components of S. aureus-derived MVs are selective, depend on the stage of growth, and may play an important role in toxicity.

[A case of huge omental-origin liposarcoma controlled by a combination of ifosfamide and adriamycin].

A 51-year-old woman came to our hospital after a medical check -up. She suffered from abdominal distension. Abdominal CT revealed the abdominal cavity filled with omental cake. The preoperative diagnosis was unassertive. Laparotomy through midline incision was performed. The bulk of the omental tumor occupied the abdominal cavity, but infiltrated the bowel or abdominal wall less. Fractional resection of the tumor, right hemicolectomy, sigmoidectomy, wedge resection of small intestine and left ovariectomy were performed in parallel. Resected specimens weighed 6. 6 kg in total. The operation was considered palliative because of the peritoneal dissemination. Postoperative intraabdominal administration of cisplatin(50mg) made the ascites disappear. She was discharged 3 weeks after the operation. The final diagnosis was liposarcoma(myxoid type), and 5 courses of adjuvant chemotherapy with adriamycin and ifosfamide were performed. No relapse was found 11 months after the operation, but tumor regrowth occurred in the thoracic and abdominal cavity and the patient died 14 months after the operation.

Arf GTPase-activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor.

Arf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors (Arfs), which are critical to form transport intermediates. ArfGAPs have been thought to be negative regulators of Arfs; however, accumulating evidence indicates that ArfGAPs are important for cargo sorting and promote membrane traffic. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPB biogenesis at the Golgi was reported to be regulated by Arf and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the role of ArfGAPs in the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. The results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. On the other hand, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA) or histamine stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected roles of ArfGAPs in vWF transport.

[A case of diffuse peritoneal malignant mesothelioma--intraabdominal administration of cisplatin is useful for diminishing ascites].

A 59-year-old man with a history of exposure to asbestos suffered from abdominal distension and visited our hospital. Abdominal CT revealed vast ascites but there was no obvious primary lesion. Serum tumor markers and hyaluronate were within the normal range. Abdominal puncture was carried out, and cytology of ascites was negative. We suspected diffuse malignant peritoneal mesothelioma because hyaluronate in ascites rose to 10×104 ng/mL. Ga-scintigraphy and FDG-PET were negative. We performed laparoscopic observation for definite diagnosis and found fine white particles at the peritoneum. The result of biopsy was malignant mesothelioma. The patient underwent intraperitoneal administration of cisplatin and his ascites was diminished. He lived for a year with no recurrence but died 23 months after diagnosis because of progression of pleural mesothelioma and liver metastases. Relapse of ascites was not found in the entire clinical course. Cisplatin administration in the peritoneal cavity is thus very effective in preventing progression of ascites.

Effects of thawing methods on the biochemical properties and microstructure of pre-rigor frozen scallop striated adductor muscle.

Postmortem biochemical properties (pH, salt solubility, Ca2+-ATPase activity, ATP-related compounds) and microstructural changes in the striated adductor muscle of pre-rigor frozen Japanese scallops (Patinopecten yessoensis) were studied after thawing and during storage at 4℃. Four thawing methods were used: running water (18℃, R); ice-water (0℃, I); air (4℃, A) and ice-saltwater (-2℃, S). The pH values and salt solubility of R group were lower than the other three thawing groups while I group was highest after thawing. However, no significant difference (P < 0.05) in Ca2+-ATPase activity were detected among 4 groups. The microstructure results indicated that the structure of I group was close to that of fresh scallop. Moreover, ATP decomposition rate was the slowest. Therefore, ice-water thawing is the best method because it induced the least changes in the biochemical properties and microstructures of scallop adductor muscle.

[Case of indwelling catheter dislocation into the duodenum during hepatic arterial infusion chemotherapy].

A 44-year-old man underwent hepatic arterial infusion chemotherapy and systematic chemotherapy using fluorouracil (5-FU) for recurrent liver metastasis of colon cancer. He reported upper back pain 38 weeks later. Arteriography using a port system revealed a dislocated catheter tip in the second part of the duodenum. Conservative therapy using antibiotics was employed without removing the catheter tip. Various complications related to intrahepatic arterial infusion chemotherapy have been reported. Catheter chip dislocation is rare, but can sometimes become a severe complication, thereby warranting careful follow-up after hepatic arterial infusion chemotherapy.

Cell wall structure of secreted laccase-silenced strain in Lentinula edodes.

Laccase1 (Lcc1) is abundantly secreted from vegetative mycelia into culture medium by Lentinula edodes. Down-regulation of lcc1 in L. edodes results in abnormal hyphal structure and thinner cell wall in mycelia. In this study, we observed the effects of Lcc1 on the hyphal morphology and cell wall structure of L. edodes. A thick cell wall and fibrous layer were clearly observed in the lcc1-silenced strain ivrL1#32, when purified Lcc1 (0.1 mU/mL) was added to the culture medium. The ratio of cell wall polysaccharide contents was compared between the ivrL1#32 strain and the wild-type (WT) strain SR-1, revealing that levels of the alkali soluble ß-1,3-1,6-glucan were significantly lower in the lcc1-silenced strain than in the WT strain. Chronological analysis revealed that chitin content in the cell wall did not increase over time, but that the alkali soluble ß-1,3-1,6-glucan content increased after Lcc1 secretion in the WT. Taken together, these data suggest that the increased level of ß-1,3-1,6-glucan induced by Lcc1 in the mycelial cell wall contributes to increased cell wall thickness and strength.

Identification of G protein α subunits in the main olfactory system and vomeronasal system of the Japanese Striped snake, Elaphe quadrivirgata.

In the olfactory system, G proteins couple to the olfactory receptors, and G proteins expressed in the main olfactory system and vomeronasal system vary according to animal species. In this study, G protein α subunits expressed in the main olfactory system and vomeronasal system of the snake were identified by immunohistochemistry. In the olfactory epithelium, only anti-Gαolf/s antibody labeled the cilia of the receptor cells. In the vomeronasal epithelium, only anti-Gαo antibody labeled the microvilli of the receptor cells. In the accessory olfactory bulb, anti-Gαo antibody stained the whole glomerular layer. These results suggest that the main olfactory system and the vomeronasal system of the snake express Gαolf and Gαo as G proteins coupling to the olfactory receptors, respectively.