Gasless single-port laparoscopic cholecystectomy.

Purpose: Single-port laparoscopic surgery is anticipated to become the future of minimally invasive surgery. We have devised an alternative approach for laparoscopic cholecystectomy by inserting a single port at the umbilicus and using the abdominal wall-lifting method, without establishing pneumoperitoneum. Methods: Retrospective analysis of 130 patients undergoing laparoscopic cholecystectomy was done to compare the conventional laparoscopic cholecystectomy (CLC) (n = 69) and the novel single-port laparoscopic cholecystectomy (SLC) using the abdominal wall-lifting method (n = 61). The surgical procedures were as follows. A 2- to 3-cm transumbilical incision was made, and a wound retractor was inserted into the abdomen without difficulty. Abdominal distension was obtained using a fan-shaped retractor without the use of carbon dioxide insufflations. A 5-mm flexible scope and modified curved graspers and dissectors were used to give the feeling of triangulation during dissection. Results: The SLC group consisted of 25 males and 36 females with a mean age of 58.1 ± 7.2 years and a mean body mass index of 23.1 ± 3.2 kg/m2. The two groups were comparable for mean age, sex, disease, American Society of Anesthesiologists physical status classification, and comorbidity. Likewise, the duration of operation, postoperative hospital stays, complications, the number of use of analgesics, and conversion rate to open technique were not significantly different in the two groups. Conclusion: The impaired view in single-port laparoscopic surgery can be improved by using articulating instruments that can be rotated out of the field of view. This novel gasless method is cost-effective and produces minimal postoperative discomfort with no additional scars.

Type 2 inositol 1,4,5-trisphosphate receptor inhibits the progression of pulmonary arterial hypertension via calcium signaling and apoptosis.

Pulmonary arterial hypertension (PAH) is a progressive disease associated with vasoconstriction and remodeling. Intracellular Ca2+ signaling regulates the contraction of pulmonary arteries and the proliferation of pulmonary arterial smooth muscle cells (PASMCs); however, it is not clear which molecules related to Ca2+ signaling contribute to the progression of PAH. In this study, we found the specific expression of type 2 inositol 1,4,5-trisphosphate receptor (IP3R2), which is an intracellular Ca2+ release channel, on the sarco/endoplasmic reticulum in mouse PASMCs, and demonstrated its inhibitory role in the progression of PAH using a chronic hypoxia-induced PAH mouse model. After chronic hypoxia exposure, IP3R2-/- mice exhibited the significant aggravation of PAH, as determined by echocardiography and right ventricular hypertrophy, with significantly greater medial wall thickness by immunohistochemistry than that of wild-type mice. In IP3R2-/- murine PASMCs with chronic hypoxia, a TUNEL assay revealed the significant suppression of apoptosis, whereas there was no significant change in proliferation. Thapsigargin-induced store-operated Ca2+ entry (SOCE) was significantly enhanced in IP3R2-/- PASMCs in both normoxia and hypoxia based on in vitro fluorescent Ca2+ imaging. Furthermore, the enhancement of SOCE in IP3R2-/- PASMCs was remarkably suppressed by the addition of DPB162-AE, an inhibitor of the stromal-interacting molecule (STIM)-Orai complex which is about 100 times more potent than 2-APB. Our results indicate that IP3R2 may inhibit the progression of PAH by promoting apoptosis and inhibiting SOCE via the STIM-Orai pathway in PASMCs. These findings suggest a previously undetermined role of IP3R in the development of PAH and may contribute to the development of targeted therapies.

Comparison of diffusion-weighted MRI and anti-Stokes Raman scattering (CARS) measurements of the inter-compartmental exchange-time of water in expression-controlled aquaporin-4 cells.

We performed multi-b and multi-diffusion-time diffusion-weighted magnetic resonance imaging on aquaporin-4-expressing (AQ) and -non-expressing (noAQ) cells, and demonstrated a clear difference between the signals from the two cell types. The data were interpreted using a two-compartment (intra and extracellular spaces) model including inter-compartmental exchange. It was also assumed that restricted diffusion of water molecules inside the cells leads to the intracellular diffusion coefficient being inversely proportional to the diffusion-time. Estimates of the water-exchange-times obtained with this model are compared to those measured using an independent optical imaging technique (coherent anti-Stokes Raman scattering imaging, CARS). For both techniques it was found that the exchange-time estimated for the noAQ cells was significantly longer than that for the AQ cells.

The binding property of a monoclonal antibody against the extracellular domains of aquaporin-4 directs aquaporin-4 toward endocytosis.

Neuromyelitis optica (NMO), an autoimmune disease of the central nervous system, is characterized by an autoantibody called NMO-IgG that recognizes the extracellular domains (ECDs) of aquaporin-4 (AQP4). In this study, monoclonal antibodies (mAbs) against the ECDs of mouse AQP4 were established by a baculovirus display method. Two types of mAb were obtained: one (E5415A) recognized both M1 and M23 isoforms, and the other (E5415B) almost exclusively recognized the square-array-formable M23 isoform. While E5415A enhanced endocytosis of both M1 and M23, followed by degradation in cells expressing AQP4, including astrocytes, E5415B did so to a much lesser degree, as determined by live imaging using fluorescence-labeled antibodies and by Western blotting of lysate of cells treated with these mAbs. E5415A promoted cluster formation of AQP4 on the cell surface prior to endocytosis as determined by immunofluorescent microscopic observation of bound mAbs to astrocytes as well as by Blue native PAGE analysis of AQP4 in the cells treated with the mAbs. These observations clearly indicate that an anti-AQP4-ECDs antibody possessing an ability to form a large cluster of AQP4 by cross-linking two or more tetramers outside the AQP4 arrays enhances endocytosis and the subsequent lysosomal degradation of AQP4.

Dynamic subcellular localization of aquaporin-7 in white adipocytes.

Aquaporin-7 (AQP7) is expressed in adipose tissue, permeated by water and glycerol, and is involved in lipid metabolism. AQP7-null mice develop obesity, insulin resistance, and adipocyte hypertrophy. Here, we show that AQP7 is expressed in adipocyte plasma membranes, and is re-localized to intracellular membranes in response to catecholamine in mouse white adipose tissue. We found that internalization of AQP7 was induced by PKA activation and comparative gene identification 58 (CGI-58). This relocation was confirmed by functional studies in 3T3-L1 adipocytes. Collectively, these results suggest that AQP7 makes several contributions to adipocyte metabolism, in both cortical and intracellular membranes.

Unprecedented cell-selection using ultra-quick freezing combined with aquaporin expression.

Freezing is usually used for preservation and storage of biological samples; however, this process may have some adverse effects such as cell membrane damage. Aquaporin (AQP), a water channel protein, has been suggested to play some roles for cryopreservation although its molecular mechanism remains unclear. Here we show that membrane damage caused by ultra-quick freezing is rescued by the expression of AQP4. We next examine if the expression of AQP combined with ultra-quick freezing can be used to select cells efficiently under freezing conditions where most cells are died. CHO cells stably expressing AQP4 were exclusively selected from mixed cell cultures. Having identified the increased expression of AQP4 during ES cell differentiation into neuro-ectoderm using bioinformatics, we confirmed the improved survival of differentiated ES cells with AQP4 expression. Finally we show that CHO cells transiently transfected with Endothelin receptor A and Aqp4 were also selected and concentrated by multiple cycles of freezing/thawing, which was confirmed with calcium imaging in response to endothelin. Furthermore, we found that the expression of AQP enables a reduction in the amount of cryoprotectants for freezing, thereby decreasing osmotic stress and cellular toxicity. Taken together, we propose that this simple but efficient and safe method may be applicable to the selection of mammalian cells for applications in regenerative medicine as well as cell-based functional assays or drug screening protocols.

Direct visualization and quantitative analysis of water diffusion in complex biological tissues using CARS microscopy.

To date, it has not been possible to measure microscopic diffusive water movements in epithelia and in the interstitial space of complex tissues and organs. Diffusive water movements are essential for life because they convey physiologically important small molecules, e.g. nutrients and signaling ligands throughout the extracellular space of complex tissues. Here we report the development of a novel method for the direct observation and quantitative analysis of water diffusion dynamics in a biologically organized tissue using Coherent Anti-Stokes Raman Scattering (CARS) microscopy. Using a computer simulation model to analyze the CARS O-H bond vibration data during H2O/D2O exchange in a 3D epithelial cyst, we succeeded in measuring the diffusive water permeability of the individual luminal and basolateral water pathways and also their response to hormonal stimulation. Our technique will be applicable to the measurement of diffusive water movements in other structurally complex and medically important tissues and organs.

PKN-1, a homologue of mammalian PKN, is involved in the regulation of muscle contraction and force transmission in C. elegans.

To examine the in vivo functions of protein kinase N (PKN), one of the effectors of Rho small guanosine triphosphatases (GTPases), we used the nematode Caenorhabditis elegans as a genetic model system. We identified a C. elegans homologue (pkn-1) of mammalian PKN and confirmed direct binding to C. elegans Rho small GTPases. Using a green fluorescent protein reporter, we showed that pkn-1 is mainly expressed in various muscles and is localized at dense bodies and M lines. Overexpression of the PKN-1 kinase domain and loss-of-function mutations by genomic deletion of pkn-1 resulted in a loopy Unc phenotype, which has been reported in many mutants of neuronal genes. The results of mosaic analysis and body wall muscle-specific expression of the PKN-1 kinase domain suggests that this loopy phenotype is due to the expression of PKN-1 in body wall muscle. The genomic deletion of pkn-1 also showed a defect in force transmission. These results suggest that PKN-1 functions as a regulator of muscle contraction-relaxation and as a component of the force transmission mechanism.

Perioperative immune responses in cancer patients undergoing digestive surgeries.

BACKGROUND: Th1/Th2 cell balance is thought to be shifted toward a Th2-type immune response not only by malignancy but also by surgical stress. The aim of this study was to estimate perioperative immune responses with respect to the Th1/Th2 balance in patients with gastrointestinal cancer. METHODS: Ninety-four patients who underwent abdominal surgeries were divided into three groups: gastric resection (n = 40), colorectal resection (n = 34) and hepatic resection (n = 20). Twelve patients undergoing laparoscopic cholecystectomy and 20 healthy subjects were served as control groups. Intracellular cytokine staining in CD4+ T lymphocytes was identified to characterize Th1/Th2 balance. Th1/Th2 balance was evaluated before operation and until postoperative days (POD) 14. RESULTS: The preoperative Th1/Th2 ratio was significantly lower in patients with malignancy compared with control. The Th1/Th2 ratio of patients in all groups decreased significantly postoperatively. Th1/Th2 balance on POD 2 in patients with malignancy was significantly decreased compared to patients with laparoscopic cholecystectomy, but there were no significant differences among the four groups on POD 14. CONCLUSION: Patients with malignancy showed an abnormal perioperative Th1/Th2 balance suggesting predominance of a type-2 immune response. Major abdominal surgeries induce a marked shift in Th1/Th2 balance toward Th2 in the early postoperative stage.

Clinical implications of thymidylate synthetase, dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase activity levels in colorectal carcinoma following radical resection and administration of adjuvant 5-FU chemotherapy.

BACKGROUND: A number of studies have investigated whether the activity levels of enzymes involved in 5-fluorouracil (5-FU) metabolism are prognostic factors for survival in patients with colorectal carcinoma. Most reports have examined thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in unresectable or metastatic cases, therefore it is unclear whether the activity of these enzymes is of prognostic value in colorectal cancer patients treated with radical resection and adjuvant chemotherapy with 5-FU. METHODS: This study examined fresh frozen specimens of colorectal carcinoma from 40 patients who had undergone curative operation and were orally administered adjuvant tegafur/uracil (UFT) chemotherapy. TS, DPD and orotate phosphoribosyl transferase (OPRT) activities were assayed in cancer tissue and adjacent normal tissue and their association with clinicopathological variables was investigated. In addition, the relationships between TS, DPD and OPRT activities and patient survival were examined to determine whether any of these enzymes could be useful prognostic factors. RESULTS: While there was no clear relationship between pathological findings and TS or DPD activity, OPRT activity was significantly lower in tumors with lymph node metastasis than in tumors lacking lymph node metastasis. Postoperative survival was significantly better in the groups with low TS activity and/or high OPRT activity. CONCLUSION: TS and OPRT activity levels in tumor tissue may be important prognostic factors for survival in Dukes' B and C colorectal carcinoma with radical resection and adjuvant chemotherapy with UFT.

Rho small GTPase regulates the stability of individual focal adhesions: a FRET-based visualization of GDP/GTP exchange on small GTPases.

RhoA and Rac1 are small GTPases primarily involved in cytoskeletal remodeling. Many biochemical studies have suggested that they are also key organizers of cell-substrate adhesion. Recently, fluorescence resonance energy transfer (FRET)-based indicators have been developed to visualize RhoA and Rac1 activity in living cells [Yoshizaki et al., J. Cell Biol. 162, 223 (2003); Pertz et al., Nature 440, 1069 (2006)]. These indicators use one of the interactions between RhoA (Rac1) and the RhoA (Rac1)-binding domain of their effector proteins. However, distribution of RhoA activity in single cells has not yet been observed with micrometer-scale resolution. Here, we employed an approach that detects GDP/GTP exchange on small GTPases by using FRET from YFP-fused small GTPases to a fluorescent analogue of GTP, BODIPY(TR)-GTP. This approach allowed us to visualize confined localization of active (GTP-bound forms of) RhoA and Rac1 in individual focal adhesions. Activated RhoA accumulated in immobile and long-lived focal adhesions but was not evident in unstable and temporary adhesions, while activated Rac1 was observed at every adhesion. Our results suggest that RhoA is the major regulator determining the stability of individual cell adhesion structures.

Epidermal growth factor-regulated activation of Rac GTPase enhances CD44 cleavage by metalloproteinase disintegrin ADAM10.

Invasive tumour cells, such as gliomas, frequently express EGF (epidermal growth factor) receptor at a high level and they exhibit enhanced cell migration in response to EGF. We reported previously that tumour cell migration is associated with ectodomain cleavage of CD44, the major adhesion molecule that is implicated in tumour invasion and metastasis, and that the cleavage is enhanced by ligation of CD44. In the present study, we show that EGF promotes CD44 cleavage and CD44-dependent cell migration. Introduction of a dominant-negative mutant of the small GTPase Rac1 or depletion of Rac1 by RNAi (RNA interference) abrogated CD44 cleavage induced by EGF. Treatment with PD98059, an inhibitor for MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase), also suppressed the CD44 cleavage. Furthermore, RNAi studies showed that EGF induced ADAM10 (a disintegrin and metalloproteinase 10)-dependent CD44 cleavage and cell migration. These results indicate that EGF induces ADAM10-mediated CD44 cleavage through Rac1 and mitogen-activated protein kinase activation, and thereby promotes tumour cell migration and invasion.

Engagement of CD44 promotes Rac activation and CD44 cleavage during tumor cell migration.

CD44 is a major cell surface adhesion molecule for hyaluronan, a component of the extracellular matrix, and is implicated in tumor metastasis and invasion. We reported previously that hyaluronan oligosaccharides induce CD44 cleavage from tumor cells. Here we show that engagement of CD44 promotes CD44 cleavage and tumor cell migration, both of which were suppressed by a metalloproteinase inhibitor KB-R7785 and tissue inhibitor of metalloproteinases-1 (TIMP-1) but not by TIMP-2. We also present evidence that blockade of metalloproteinase-disintegrin ADAM10 (a disintegrin and metalloproteinase 10) by RNA interference suppresses CD44 cleavage induced by its ligation. Engagement of CD44 concurrently induced activation of the small GTPase Rac1 and led to drastic changes in cell morphology and actin cytoskeleton with redistribution of CD44 to newly generated membrane ruffling areas. A fluorescence resonance energy transfer approach to visualize GTP-bound Rac1 in living cells revealed the localization of the active Rac1 in the leading edge of the membrane ruffling areas upon ligation of CD44. Taken together, our results indicate that the cleavage of CD44 catalyzed by ADAM10 is augmented by the intracellular signaling elicited by engagement of CD44, through Rac-mediated cytoskeletal rearrangement, and suggest that CD44 cleavage contributes to the migration and invasion of tumor cells.

Repair of incisional hernia with prolene hernia system.

A 70-year-old woman was admitted to our hospital with a complaint of bulging in the right lower portion of the abdomen. The bulging was in accordance with an old operative scar for appendicitis. The findings of computed tomography (CT) showed defects in the abdominal muscles and the protrusion of the intestine into the subcutaneous fat. The patient was diagnosed with incisional hernia after appendectomy and underwent a repair of the incisional hernia, using the prolene hernia system double-layer mesh. The patient's post-operative course was excellent. Recently, the prolene hernia system, double-layer mesh was reported to be effective for groin hernias due to its advantageous protection the recurrence through reinforcement of the patient's myopectrial orifice. It is suggested that this new device is also useful for small incisional hernias.

A case of adenomatous hyperplasia in the liver with chronic hepatitis C that transformed into hepatocellular carcinoma.

We report a rare case of adenomatous hyperplasia (AH) with malignant transformation, which was observed initially in the liver with chronic hepatitis C. A 47-year-old man was admitted to the hospital and chronic active hepatitis was diagnosed using fine needle biopsy, in addition abdominal computed tomography (CT) and ultrasonography showed multiple and hypovascular liver mass. Needle biopsy of the nodules showed AH. Despite transcatheter arterial embolization and percutaneous ethanol injection therapy (PEIT), the patient was readmitted due to enlargement of the nodules and elevation of alpha-fetoprotein 9 months later. On readmission, the nodules in the liver showed early-stage well-differentiated hepatocellular carcinoma (HCC) histopathologically, that were strongly stained on CT-arteriography and were stained less than the surrounding liver on CT-arterial portography, indicating arterial supply in the nodules and malignant transformation. Thereafter, the patient developed jaundice, and enhanced CT and magnetic resonance imaging showed multiple nodules, which occupied most of the liver, and needle biopsy revealed typical well-differentiated HCC. The patient died of hepatic failure 3 years after the initial admission. In this case, it was confirmed that AH transformed into HCC, which showed a multiple tumor on pathological diagnosis. It has been reported that borderline lesions are curable by less aggressive procedures such as enucleation and PEIT. However, such procedures may not be useful for this type of HCC, which rapidly developed from borderline lesions of the liver.

Giant gastrointestinal stromal tumor, associated with esophageal hiatus hernia.

An 85-year-old woman was admitted to our hospital because of vomiting. An upper gastrointestinal series what showed a large esophageal hiatus hernia, suggesting an association with extrinsic pressure in the middle portion of the stomach. An upper gastrointestinal endoscopic examination showed severe esophagitis and a prominent narrowing in the middle portion of the stomach, however, it showed normal gastric mucosa findings. CT and MRI revealed a large tumor extending from the region of the lower chest to the upper abdomen. From these findings, the tumor was diagnosed as gastrointestinal stromal tumor (GIST), which arose from the gastric wall and complicated with an esophageal hiatus hernia. We performed a laparotomy, however, the tumor showed severe invasion to the circumferential organs. Therefore, we abandoned the excision of the tumor. Histologically, the tumor was composed of spindle shaped cells with marked nuclear atypia and prominent mitosis. The tumor cells were strongly positive for CD34 and c-kit by immunohistochemical examination. From these findings, the tumor was definitely diagnosed as a malignant GIST. As palliative treatment, we implanted a self-expandable metallic stent in the narrow segment of the stomach. The patient could eat solid food and was discharged. In the treatment of esophageal hiatus hernia, the rare association of GIST should be considered.