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Background and Aim The pathogenic process underlying the development of hepatocellular carcinoma (HCC) is not yet clear in patients with hepatitis C virus (HCV) who have received direct‐acting antiviral (DAA) therapy and achieved sustained virological response (SVR). This study validated a composite predictive model for HCC in these patients. Methods This study included 3058 patients in whom HCV was eradicated with DAA therapy. After DAAs recommendation for surveillance (ADRES) score, which is based on sex, FIB‐4 index, and α‐fetoprotein, was used as a composite predictive model for HCC development. Results The 1‐, 3‐, and 5‐year cumulative incidence rates of HCC were 0.9, 4.5, and 15.2%, respectively. Multivariate analysis with Cox proportional hazards models showed that male sex (hazard ratio [HR], 2.646; 95% confidence interval [CI], 1.790–3.911), FIB‐4 index >3.25 (HR, 2.891; 95% CI, 1.947–4.293), and α‐fetoprotein >5 ng/mL (HR, 2.835; 95% CI, 1.914–4.200) are independently associated with HCC development. The incidence of HCC differed significantly by ADRES score (P, This study aimed to validate the utility of the after direct‐acting antivirals recommendation for surveillance (ADRES) score for predicting hepatocellular carcinoma (HCC) development in patients who had received direct‐acting antiviral therapy and achieved sustained virological response. The incidence of HCC differed significantly by ADRES score in this patient population. ADRES score had higher predictive power for the development of HCC than the FIB‐4 index and a‐fetoprotein according to time‐dependent receiver operating characteristic analysis.

The identification of patients with advanced fibrosis who do not need any further hepatocellular carcinoma (HCC) surveillance after the eradication of hepatitis C is pivotal. In this study, we developed a simple serum-based risk model that could identify patients with low-risk HCC. This was a nationwide multicenter study involving 16 Hospitals in Japan. Patients with advanced fibrosis (1,325 in a derivation cohort and 508 in a validation cohort) who achieved sustained virological responses at 24weeks after treatment (SVR24) were enrolled. The HCC risk model at any point after SVR24 and its change were evaluated, and subsequent HCC development was analyzed. Based on the multivariable analysis, patients fulfilling all of the factors (GAF4 criteria: gamma-glutamyl transferase&nbsp

The real-world virological efficacy and safety of interferon-free direct-acting antiviral (DAA) therapy with sofosbuvir (SOF) and velpatasvir (VEL) were assessed in hepatitis C virus (HCV) genotype 1- and 2-infected patients with decompensated cirrhosis. A total of 65 patients with HCV-related decompensated cirrhosis (Child-Pugh score of 7 points or more) who were treated with the SOF/VEL regimen were enrolled. The sustained virological response (SVR) rate and safety profile were analyzed. SVR was defined as undetectable serum HCV RNA at 12 weeks after the end of treatment (SVR12). The percentages of patients with undetectable HCV RNA at 4, 8, and 12 weeks after the start of therapy were 81.2% (95% confidence interval [CI], 69.5-89.9) (52/64), 98.4% (95% CI, 91.2-100.0) (60/61), and 98.5% (95% CI, 91.7-100.0) (64/65), respectively. The overall SVR rate was 92.3% (95% CI, 83.0-97.5) (60/65). Albumin-bilirubin (ALBI) scores decreased during and after treatment (p

Background It is unclear whether the fibrosis 4 index (FIB-4), a marker of liver fibrosis, at baseline and change in FIB-4 after sustained virological response (SVR) is associated with incident hepatocellular carcinoma (HCC) risk. In this study, we examined the association of incident HCC risk with baseline FIB-4 and sustained high FIB-4 (>3.25) at any time point after SVR. Methods A total of 3823 patients who received direct-acting antiviral treatment and achieved SVR were enrolled. The FIB-4 was measured 24 weeks after the end of direct-acting antiviral treatment and achievement of SVR (SVR24), and 1, 2, and 3 years after SVR24, after which subsequent HCC development was investigated. Results In patients with an FIB-4 >3.25 at SVR24 and 1, 2, and 3 years after SVR24, subsequent HCC development was significantly higher than in those with an FIB-4 ≤3.25 at each point. The rates of HCC development 1, 2, 3, and 4 years after SVR24 were significantly higher in patients with sustained FIB-4 >3.25 than in those whose FIB-4 decreased to ≤3.25 (5.4%, 9.2%, 11.7%, and 16.0%, respectively, vs 2.2%, 3.1%, 3.7%, and 4.4%; P 3.25 at SVR24 and 1, 2, and 3 years later were 3.38 (2.4–4.8), 2.95 (1.9–4.7), 2.62 (1.3–5.1), and 3.37 (1.4–9.8), respectively. Conclusions The FIB-4 could be used to assess HCC development risk at any time after SVR, and changes in FIB-4 were associated with changes in the HCC development risk. Repeated assessments of FIB-4 could serve as a prognostic indicator of a high-risk HCC cohort that may require more intensive HCC surveillance strategy.

Nucleos(t)ide analogs (NA) suppress hepatitis B virus (HBV) replication and reduce the risk of hepatocellular carcinoma (HCC). However, NA cannot suppress carcinogenesis completely in patients with chronic hepatitis B. The aims of this study were to identify risk factors for HCC and develop a refined carcinogenesis prediction model. Patients receiving NA therapy (n = 1,183) were recruited retrospectively from the 16 hospitals. All patients had been receiving NA continuously for more than 1 year until the end of the follow-up. During a median follow-up of 4.9 (1.0–12.9) years, 52 (4.4%) patients developed HCC. A multivariate analysis revealed that male gender, older age, lower platelet counts at the baseline, and detectable HBV DNA during NA therapy were independent predictive factors of HCC development. The PAGE-B score was calculated by using these factors. 240 (20.3%), 661 (55.9%), and 282 (23.8%) patients were classified into low-, intermediate-, and high-risk groups, respectively. In the intermediate- and high-risk group, detectable HBV DNA was significantly associated with a higher risk of HCC development compared with continuously undetectable HBV DNA, respectively (HR 3.338; 95% CI 1.045–10.66/HR 3.191; 95% CI 1.543–6.597). PAGE-B–DNA, which is the combined PAGE-B and HBV DNA status, was valuable for a more refined stratification of PAGE-B.

Aim To evaluate the virologic responses and clinical course of daclatasvir plus asunaprevir treatment in non-hemodialysis (non-HD) and hemodialysis (HD) patients infected with genotype 1 hepatitis C virus (HCV). Methods A total of 1113 non-HD patients and 67 HD patients were assessed. To evaluate pretreatment factors contributing to sustained virological response at 12 weeks (SVR12), univariate and multivariate analyses were carried out. To adjust for differences in patient background, propensity score matching was undertaken. Results The overall SVR12 rates were 91.6% in non-HD patients and 95.5% in HD patients. Compared with non-HD patients, HD patients were younger, were more likely to be male, were less likely to have received interferon-based pretreatment, had a lower viral load, and had lower levels of alanine transaminase, hemoglobin, and α-fetoprotein. Multivariate analysis revealed that viral load, α-fetoprotein, L31 substitution negative, and Y93 substitution negative were independent predictive factors for SVR12 in non-HD patients. The proportion of patients with undetectable HCV-RNA during the initial 4 weeks was significantly higher in HD patients than in non-HD patients. The SVR12 rate was clearly higher in HD patients than in non-HD patients, although the difference was not statistically significant. After propensity score matching to adjust for viral load, α-fetoprotein, L31 substitution, and Y93 substitution, these trends disappeared. Conclusions For treatment of HCV genotype 1 infection, daclatasvir plus asunaprevir is useful not only in non-HD patients but also in HD patients. Viral load, α-fetoprotein levels, L31 substitution, and Y93 substitution influence treatment course and outcome.

AIM The present study aimed to determine the real-world efficacy and safety of the non-structural protein (NS)5A inhibitor elbasvir (EBR) combined with the NS3/4A protease inhibitor grazoprevir (GZR) in patients with hepatitis C virus (HCV) genotype 1 (GT1) infection. METHODS This study retrospectively evaluated the rate of sustained virologic response at 12 weeks post-treatment (SVR12) and the safety of EBR/GZR treatment in 159 men and 194 women with a median age of 72 years, and it assessed factors associated with the SVR12 rate. The attending physicians were responsible for selecting candidate patients for EBR/GZR in this retrospective study. RESULTS Treatment outcomes for EBR/GZR were good in direct-acting antiviral (DAA)-naive patients, of whom 99.4% achieved SVR. Of 353 patients, 10 (2.9%) had treatment failure. Of these patients, eight previously underwent DAA therapy, and the remaining two had NS5A-L31/Y93 double mutation. The SVR rate was 50% (8/16 patients) in patients who previously underwent DAA therapy, and 18.2% (2/11 patients) in patients with NS5A-L31/Y93 double mutation. On multivariate logistic regression analysis, NS5A-Y31/Y93 double mutation (odds ratio 356.3; 95% confidence interval, 23.91-16 940; P

We aimed to describe the real-world efficacy and safety of combination therapy with ledipasvir and sofosbuvir (LDV/SOF) for chronic hepatitis C virus (HCV) genotype 1 (GT1) infection. This retrospective analysis of a prospective, nationwide, multicenter registry included GT1-infected patients treated with LDV/SOF for 12 weeks. We assessed the rate of sustained virological response at 12 weeks post-treatment (SVR12), incidence of adverse events, and serum markers of hepatocellular carcinoma (HCC). Among the 1461 patients included (mean age, 69 years; 29.5% aged > 75 years; cirrhosis, 23.8%; history of treatment for HCC, 10.9%), the overall SVR12 rate was 98.4% (1438/1461). Factors associated with treatment failure were cirrhosis (odds ratio, 4.19; p = 0.014) and resistance-associated substitutions (RASs) in NS5A at baseline (odds ratio, 7.78; p = 0.0004). The SVR12 rate in patients with cirrhosis and NS5A RASs was 93.0% compared to 100% in patients without cirrhosis or NS5A RASs. In patients with SVR, the levels of alpha-fetoprotein (AFP), AFP-L3, and Mac-2 binding protein glycosylation isomer (M2BPGi) decreased from baseline to end of treatment (from 13.4 ± 37.6 to 6.0 ± 10.6 ng/mL, p

To evaluate the virologic responses and clinical course of daclatasvir plus asunaprevir treatment in non-hemodialysis (non-HD) and hemodialysis (HD) patients infected with genotype 1 hepatitis C virus (HCV).A total of 1113 non-HD patients and 67 HD patients were assessed. To evaluate pretreatment factors contributing to sustained virological response at 12 weeks (SVR12), univariate and multivariate analyses were carried out. To adjust for differences in patient background, propensity score matching was undertaken.The overall SVR12 rates were 91.6% in non-HD patients and 95.5% in HD patients. Compared with non-HD patients, HD patients were younger, were more likely to be male, were less likely to have received interferon-based pretreatment, had a lower viral load, and had lower levels of alanine transaminase, hemoglobin, and α-fetoprotein. Multivariate analysis revealed that viral load, α-fetoprotein, L31 substitution negative, and Y93 substitution negative were independent predictive factors for SVR12 in non-HD patients. The proportion of patients with undetectable HCV-RNA during the initial 4 weeks was significantly higher in HD patients than in non-HD patients. The SVR12 rate was clearly higher in HD patients than in non-HD patients, although the difference was not statistically significant. After propensity score matching to adjust for viral load, α-fetoprotein, L31 substitution, and Y93 substitution, these trends disappeared.For treatment of HCV genotype 1 infection, daclatasvir plus asunaprevir is useful not only in non-HD patients but also in HD patients. Viral load, α-fetoprotein levels, L31 substitution, and Y93 substitution influence treatment course and outcome.

Recent reports have described the involvement of the diacylglycerol kinase (DGK) family in various pathological conditions. In an animal model of transient ischemia, DGKζ containing a nuclear localization signal (NLS) is shown to translocate quickly from the nucleus to the cytoplasm in hippocampal neurons and to disappear gradually after reperfusion. Those neurons die a delayed neuronal death because of glutamate excitotoxicity. This study investigated the molecular mechanism and functional relation linking DGKζ and neuronal death. In primary cultured neurons, transient exposure to excitotoxic concentration of glutamate led to cytoplasmic accumulation of DGKζ followed by its down-regulation. Results showed that DGKζ down-regulation was caused by proteolytic degradation through the ubiquitin–proteasome system (UPS) rather than transcriptional inhibition. DGKζ polyubiquitination was inhibited in the presence of nuclear export inhibitor leptomycin B. Furthermore, NLS-deleted mutant DGKζΔNLS, which mainly localizes to the cytoplasm, was ubiquitinated more heavily than wild-type DGKζ. From a functional perspective, in vitro gene silencing of DGKζ via specific siRNA enhanced DNA fragmentation in cultured neurons after glutamate exposure. At the organismal level, hippocampal neurons of DGKζ-deficient mice showed vulnerability to kainate-induced seizures. In addition, DGKζ-deficient hippocampus exhibited a significant increase in Ser807/811 phosphorylated retinoblastoma protein levels together with up-regulation of the expression of type D and E cyclins, indicative of cell cycle reentry. Collectively, these results suggest that 1) glutamate excitotoxicity induces nucleocytoplasmic translocation of DGKζ followed by its degradation through the cytoplasmic UPS in hippocampal neurons and that 2) DGKζ-deficient neurons do not succumb directly to apoptosis, although they are more vulnerable to excitotoxicity because of aberrant cell cycle reentry.

Diacylglycerol kinase (DGK) plays an important role in phosphoinositide signaling cascade by regulating the intracellular level of diacylglycerol and phosphatidic acid. The DGK family is involved in various pathophysiological responses that are mediated through unique binding partners in different tissues and cells. In this study, we identified a small GTPase effector protein, IQGAP1, as a novel DGKζ-associated complex protein. A bacterial endotoxin, lipopolysaccharide (LPS), facilitated the complex formation in macrophages. Both proteins co-localized at the edge and phagocytic cup of the cell. Furthermore, RNA interference-mediated knockdown of DGKζ or IQGAP1 impaired LPS-induced Rac1 activation. Primary macrophages derived from DGKζ−/− mice attenuated LPS-induced phagocytosis of bacteria. These results suggest that DGKζ is involved in IQGAP1/Rac1-mediated phagocytosis upon LPS stimulation in macrophages.

Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGKζ, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGKζ. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGKζ binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGKζ and NAP1Ls prohibits nuclear import of DGKζ because binding of NAP1Ls to DGKζ blocks import carrier proteins, Qip1 and NPI1, to interact with DGKζ, leading to cytoplasmic tethering of DGKζ. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGKζ and provide a clue to examine functional significance of its translocation under pathological conditions.

The effects of geometric properties of membranes on the structure of the phospholipase C-delta1 (PLC-delta1) pleckstrin homology (PH) domain were investigated using solid state (13)C NMR spectroscopy. Conformations of the PLC-delta1 PH domain at the surfaces of multilamellar vesicles (MLV), small unilamellar vesicles (SUV), and micelles were examined to evaluate the effects of membrane curvature on the PH domain. An increase in curvature of the water-hydrophobic layer interface hinders membrane-penetration of the amphipathic alpha2-helix of the PH domain that assists the membrane-association of the PH domain dominated by the phosphatidylinositol 4,5-bisphosphate (PIP(2)) specific lipid binding site. The solid state (13)C NMR signal of Ala88 located at the alpha2-helix indicates that the conformation of the alpha2-helix at the micelle surface is similar to the solution conformation and significantly different from those at the MLV and SUV surfaces which were characterized by membrane-penetration and re-orientation. The signal of Ala112 which flanks the C-terminus of the beta5/beta6 loop that includes the alpha2-helix, showed downfield displacement with decrease in the interface curvature of the micelles, SUV and MLV. This reveals that the conformation of the C-terminus of the beta5/beta6 loop connecting the beta-sandwich core containing the PIP(2) binding site and the amphipathic alpha2-helix is sensitive to alterations of the curvature of lipid bilayer surface. It is likely that these alterations in the conformation of the PLC-delta1 PH domain contribute to the regulatory mechanisms of the intracellular localization of PLC-delta1 in a manner dependent upon the structure of the molecular complex containing PIP(2).

START-GAP2, also termed as DLC2, is a START domain-containing RhoGAP and a negative regulator of RhoA and Cdc42. Although it was reported as a tumor suppresser gene product, the molecular basis for function of START-GAP2 remains to be clarified. Here, we demonstrate that START-GAP2 is localized in focal adhesions through a "FAT (focal adhesion targeting)" region in the N-terminal half. START-GAP2 competes with START-GAP1/DLC1, another START domain-containing RhoGAP, in focal adhesion targeting. Moreover, the C-terminus of tensin2, one of focal adhesion components and reported to bind START-GAP1, also directly interacts with START-GAP2. These results suggest that START-GAP2 and START-GAP1 share the same molecular mechanism in targeting to focal adhesions.

There is a class of GTPase activating proteins for the Rho family GTPases (RhoGAPs) that contain the steroidogenic acute regulatory protein (STAR)-related lipid transfer (START) domain. In mammals three genes encode such proteins and they are designated START-GAP1-3 or deleted in liver cancer 1-3 (DLC1-3). In this study, we examined the intracellular localization and roles of START-GAP1/DLC1 in cell motility. Immunofluorescence microscopic analysis of NRK cells and HeLa cells revealed that START-GAP1 was localized in focal adhesions. Amino acid residues 265-459 of START-GAP1 were found to be necessary for focal adhesion targeting and we name the region "the focal adhesion-targeting (FAT) domain." It was previously known that ectopic expression of START-GAP1 induced cell rounding. We demonstrated that the FAT domain of START-GAP1 was partially required for this morphological change. Furthermore, expression of this domain in HeLa cells resulted in dissociation of endogenous START-GAP1 from focal adhesions as a dominant negative modulator, reducing cell migration and spreading. Taken together, START-GAP1 is targeted to focal adhesions via the FAT domain and regulates actin rearrangement through down-regulation of active RhoA and Cdc42. Its absence from focal adhesions could, therefore, cause abnormal cell motility and spreading.

Dynamin 2 has been reported to be implicated in phagocytosis. However, the mode of action of dynamin is poorly understood. In this study, we examined whether dynamin 2 participates in actin assembly during phagocytosis in Sertoli cells. In the presence of dynasore, a dynamin inhibitor, phagocytosis was reduced by 60-70% in Sertoli cells and macrophages. Scanning electron microscopy revealed that Sertoli cells treated with dynasore were unable to form phagocytic cups. In addition, dysfunction of dynamin 2 reduced both actin polymerization and recruitment of actin and dynamin 2 to phosphatidylinositol (4,5) bisphosphate [PI(4,5)P{sub 2}]-containing liposomes. The formation of dynamin 2-positive ruffles of Sertoli cells was decreased by 60-70% by sequestering PI(4,5)P{sub 2} either by expression of PH domain of PLC{delta} or treatment with neomycin. These results strongly suggest that dynamin 2 is involved in actin dynamics and the formation of dynamin 2-positive ruffles during phagocytosis.

Backgrounds & Aims We aimed to clarify the characteristics of resistance-associated substitutions (RASs) after treatment failure with NS5A inhibitor, daclatasvir (DCV) in combination with NS3/4A inhibitor, asunaprevir (ASV), in patients with chronic hepatitis C virus genotype 1b infection. Methods This is a nationwide multicenter study conducted by the Japanese Red Cross Liver Study Group. The sera were obtained from 68 patients with virological failure after 24 weeks of DCV/ASV treatment. RASs in NS5A and NS3 were determined by population sequencing. Results The frequency of signature RASs at position D168 of NS3 was 68%, and at positions L31 and Y93 of NS5A was 79 and 76%, respectively. The frequency of dual signature RASs in NS5A (L31-RAS and Y93-RAS) was 63%. RASs at L28, R30, P32, Q54, P58, and A92 in addition to dual signature RAS were detected in 5, 5, 1, 22, 2, and 0 patients, respectively. In total, triple, quadruple, and quintuple RASs in combination with dual signature RAS were detected in 35, 10, and 1.5% patients, respectively. These RASs were detected in patients without baseline RASs or who prematurely discontinued therapy. Co-existence of D168 RAS in NS3 and L31 and/or Y93 RAS in NS5A was observed in 62% of patients. Conclusion Treatment-emergent RASs after failure with DCV/ASV combination therapy are highly complex in more than 50% of the patients. The identification of complex RAS patterns, which may indicate high levels of resistance to NS5A inhibitors, highlights the need for RAS sequencing when considering re-treatment with regimens including NS5A inhibitors.

To evaluate the role of contrast-enhanced ultrasonography (CEUS) in the diagnosis of ruptured hepatocellular carcinoma (HCC).CEUS and angiography were performed in ten cases of ruptured HCC. We evaluated whether this technique allowed us to determine the bleeding point by observing an extravasation of contrast media into the ascites.In four of the ten cases, CEUS demonstrated an extravasation of Levovist into ascites. Angiography showed an extravasation of contrast medium in three of these four cases. In three of the remaining six cases, in which CEUS did not show the presence of contrast medium in ascites, angiography demonstrated an extravasation. In eight cases, it yielded cessation of bleeding. In two cases, embolization was not successful. The bleeding point was not determined by CEUS or angiography in one case.CEUS allows us to differentiate active bleeding (presence of contrast medium in the ascites) from nonactive bleeding.

この章では，まず膵の解剖学的位置関係，特に周囲脈管系との関係を述べ，次いで，膵超音波検査で伝統的に用いられてきた走査法に，そして最後に我々が推奨する走査法の工夫について解説する．通常の走査法では，一断面で膵全体を観察するのは困難で，幾つかの走査面を組み合わせざるを得ない．大まかには，次の4走査法が中心となる．a）上腹部横走査：脾静脈を指標として膵頭体部を観察する．b）上腹部矢状走査：この断面では腹腔動脈と上腸間膜動脈に挟まれた膵が見える．これらは全て呼吸性に移動する．c）右上腹部斜走査：門脈本幹を指標にその腹側の頭部と背部の釣部を観察するのに適している．d）左上腹部斜走査：脾動静脈を指標にその腹側の膵尾部を観察するのに適している．我々は，走査法の工夫として，1）プローブを回転させる（回外運動），2）船頭の船こぎ様に手首を背屈させる（背屈運動），3）たすきがけの様に，膵尾部を見る時は頭部に，頭部を見る時は尾部に，プローブを移動させ，目的箇所を斜めに観察する．4）必要に応じ，プローブを円弧状に移動させるなど，1）‐3）を組み合わせたり変法を加える．なお，今まで引水法や体位の変換を推賞しているテキストもあったが，被検者にかかる負担が大きく，その割に効果がなかったのであまりお薦め出来ない．

Splenic inflammatory pseudotumor is a rare pathology, and the literature stresses the difficulty of preoperative diagnosis. There are no previous reports of contrast-enhanced ultrasound findings for this tumor in the literature. Our case appears to be the first to be examined using this technique. In our case, contrast-enhanced ultrasound showed the mass to be homogeneously and less enhanced than the surrounding parenchyma in all phases, and it included fine enhanced spots. We also briefly review the literature.

We present two cases of subcutaneous hematoma in which color Doppler US revealed colored areas under probe compression, and Doppler waveforms obtained from these areas changed rapidly (from a constant flow to a pulsatile flow, and from a high-velocity flow to a slow-velocity flow) according to the degree of probe compression. In our cases, a sufficient number of blood cells within the lesion moving under probe compression was thought to give rise to Doppler signals and changes in Doppler waveforms.

Refractive artifacts are frequently encountered in clinical settings, and they have been analyzed on the basis of conventional two-dimensional (2-D) ultrasound (US) images, but this method is restricted to monoplane data and is limited by its inability to assess the three-dimensional (3-D) structure of refractive artifacts. The aim of this study was to evaluate the role of reconstructed 3-D US images in the analysis of refractive artifacts. The following representative refractive artifacts were analyzed on the basis of reconstructed 3-D US images: (a) a distorted image of a fine tube behind a cyst (balloon); (b) a deformed image of the bottom of a balloon; and (c) a duplication artifact due to the acoustic lens effect. (a) A tube was imaged as a fine echogenic line with two points of sudden interruption, unlike a curved needle, which was imaged without interruption. (b) 3-D US allowed us to visualize the mode of deformity in the image of the bottom of a fluid-filled balloon in a water bath. When the acoustic velocity in the fluid was greater than that in the surrounding water, the bottom of the balloon appeared to be shrunken. When the acoustic velocity in the fluid was less than that in the surrounding water, the bottom of the balloon appeared to be swollen. (c) When we placed two pieces of white chicken meat in front of a fine needle, the needle was duplicated in the resulting image. In this case, the needle appeared to be vague and fuzzy. In this case, 3-D US did not add further information to the 2-D images. Our study suggests that reconstructed 3-D US images provide a better understanding of the mode of refractive artifacts than do 2-D US images.

Phosphoinositides (PIs) and proteins involved in the PI signaling pathway are distributed in the nucleus as well as at the plasma membrane and in the cytoplasm, although their nuclear localization mechanisms have not been clarified in detail. Generally, proteins that shuttle between the cytoplasm and nucleus contain nuclear localization signal (NLS) and nuclear export signal (NES) sequences for nuclear import and export, respectively. They bind to specific carrier proteins of the importin/exportin family and are transported to and from the nucleus. Thus there is a steady state shuttling of the cargo molecules to and from the nucleus, and the shift in equilibrium determines their nuclear or cytoplasmic localization. Our previous studies have shown that phospholipase C (PLC)-δ1, regarded as having cytoplasmic- or plasma membrane-bound localization, accumulates in the nucleus when its NES sequence is disrupted. In addition, a cluster of positively charged residues on the surface of the catalytic barrel is important for nuclear import. In quiescent cells, the shuttling equilibrium seems to be shifted to the nuclear export of PLCδ1. In this review, recent findings regarding the molecular machineries and mechanisms of the nucleocytoplasmic shuttling of PLCδ1 will be discussed. It is important to know when and how they are regulated. A shift in the equilibrium in a certain stage of the cell cycle or by external stimuli is possible and resulting changes in the intra-nuclear environments (or architectures) may alter proliferation and differentiation patterns. Evidences support the idea that an increase in the levels of intracellular Ca2+ shifts the equilibrium to the nuclear import of PLCδ1. A myriad of external stimuli have also been reported to change the nuclear PI metabolism following accelerated accumulation in the nucleus of other phospholipases such as phospholipase A2 and phospholipase D in addition to PLC isoforms such as PLCβ1 and PLCγ1. The consequence of the nuclear accumulation of PLC is also discussed. J. Cell. Biochem. © 2005 Wiley-Liss, Inc.

Phospholipase D (PLD), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline, plays key roles in cellular signal transduction by mediating extracellular stimuli including hormones, growth factors, neurotransmitters, cytokines and extracellular matrix molecules. The molecular mechanisms by which domains regulate the activity of PLD - especially the phox homology (PX) domain - have not been fully elucidated. In this study, we have examined the properties of the PX domains of PLD1 and PLD2 in terms of phosphoinositide binding and PLD activity regulation. Interestingly, the PX domain of PLD1, but not that of PLD2, was found to specifically interact with phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3). We found that mutation of the conserved arginine at position 179 of the PLD1 PX domain to lysine or to alanine (R179A or R179K, respectively) disrupts PtdIns(3,4,5)P3 binding. In NIH-3T3 cells, the EGFP-PLD1 PX wild-type domain, but not the two mutants, localized to the plasma membrane after 5-minute treatment with platelet-derived growth factor (PDGF). The enzymatic activity of PLD1 was stimulated by adding PtdIns(3,4,5)P3 in vitro. Treatment with PDGF resulted in the significant increase of PLD1 activity and phosphorylation of the downstream extracellular signal-regulated kinases (ERKs), which was blocked by pre-treatment of HEK 293 cells with phosphoinositide 3-kinase (PI3K) inhibitor after the endogenous PLD2 had been depleted by siRNA specific for PLD2. Nevertheless, both PLD1 mutants (which cannot interact with PtdIns(3,4,5)P3) did not respond to treatment with PDGF. Moreover, PLD1 was activated in HepG2 cells stably expressing the Y40/51 mutant of PDGF receptor that is required for the binding with PI3K. Our results suggest that the PLD1 PX domain enables PLD1 to mediate signal transduction via ERK1/2 by providing a direct binding site for PtdIns(3,4,5)P3 and by activating PLD1.

Phospholipase C (PLC)delta1 shuttles between the nucleus and the cytoplasm. Here, we demonstrate that treatment of MDCK cells and PC12 cells with ionomycin causes nuclear accumulation of ectopically expressed and endogenous PLCdelta1, respectively, suggesting that signals that increase [Ca2+]i trigger nuclear translocation. To clarify the molecular mechanisms involved in this translocation, we have examined whether PLCdelta1 binds with importins. PLCdelta1 interacted with importin beta1 in a Ca2+-dependent manner in vitro even in the absence of importin alpha. A PLCdelta1 mutant E341A, which lacks Ca2+-binding to the catalytic core, did not show this interaction at any physiological Ca2+ concentration and did not translocate into the nucleus after ionomycin treatment when expressed in MDCK cells. These results suggested that the nuclear import of PLCdelta1 is mediated by its Ca2+-dependent interaction with importin beta1.

We have established a new system for studying phytic acid, myo-inositol hexakisphosphate (InsP6) synthesis in suspension-cultured cells of Catharanthus. InsP6 and other intermediates of myo-inositol (Ins) phosphate metabolism were measured using an ion chromatography method. The detection limit for InsP6 was less than 50 nm, which was sufficient to analyze Ins phosphates in living cells. Synthesis of Ins phosphates was induced by incubation in high inorganic phosphate medium. InsP6 was mainly accumulated in vacuoles and was enhanced when cells were grown in high concentration of inorganic phosphates with the cations K+, Ca2+, or Zn2+. However, there was a strong tendency for InsP6 to accumulate in the vacuole in the presence of Ca2+ and in nonvacuolar compartments when supplied with Zn2+, possibly due to precipitation of InsP6 with Zn2+ in the cytosol. A vesicle transport inhibitor, brefeldin A, stimulated InsP6 accumulation. The amounts of both Ins(3)P1myo-inositol monophosphate synthase, a key enzyme for InsP6 synthesis, and Ins(1,4,5)P3 kinase were unrelated to the level of accumulation of InsP6. The mechanisms for InsP6 synthesis and localization into vacuoles in plant cells are discussed.

Fertilization elicits a dramatic, transient rise in Ca2+ within the egg which is an essential component of egg activation and consequent initiation of development. In the sea urchin egg, three distinct Ca2+ stores have been identified which could, either individually or in combination, initiate Ca2+ release at fertilization. Inositol 1,4,5-trisphosphate (IP3) production by phospholipase C (PLC) has been suggested as the singular signal in initiating the Ca2+ transient. Other studies indicate that Ca2+ stores gated by cyclic adenosine diphosphate ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) are also necessary. We have examined the temporal relationship between the Ca2+ rise and IP3 production at fertilization in vivo within individual eggs using a green fluorescent protein (GFP) coupled to a pleckstrin homology (PH) domain that can detect changes in IP3. Translocation of the probe occurred after the Ca2+ rise was initiated. Earlier, and possibly smaller, IP3 changes could not be excluded due to limitations in probe sensitivity. High IP3 levels are maintained during the decline in cytoplasmic Ca2+, suggesting that later IP3 metabolism might not be related to regulation of Ca2+, but may function to modulate other PIP2 regulated events such as actin polymerization or reflect other novel phosphoinositide signaling pathways.

PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein with a domain organization similar to phospholipase C-delta1 (PLC-delta1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P3. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P3-mediated Ca2+ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1-/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca2+ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1-/- mice than in those from wild type mice. The relative amounts of [3H]Ins(1,4,5)P3 measured in neurons labeled with [3H]inositol was also lower in cells from PRIP-1-/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,5)P3 is enhanced in cells from PRIP-1-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P3 to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P3-mediated Ca2+ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P3.

Background: Although phospholipase C (PLC)δ1 containing a functional nuclear export signal (NES) is normally localized at the plasma membrane and in the cytoplasm, it shuttles between the nucleus and the cytoplasm. Since nucleocytoplasmic shuttling of a molecule is generally regulated by a balance between its NES and the nuclear localization signal (NLS), we examined whether PLCδ1 contains an NLS sequence. Results: A region corresponding to the C terminus of the X domain and the XY-linker, which contains clusters of basic amino acid residues, was essential for the nuclear import of PLCδ1 in Madin–Darby canine kidney cells. A series of point mutations on lysine residues in this region revealed that K432 and K434 in combination were important for the nuclear import. A short synthetic peptide corresponding to residues 429–442, however, was not able to function as an NLS sequence when they were injected into the cytoplasm in a carrier-conjugated form. Neither a longer peptide equivalent to PLCδ1 412–498 fused to a protein tag consisting of glutathione S-transferase and green fluorescent protein was imported to the nucleus after microinjection into the cytoplasm. Conclusion: The nuclear import of PLCδ1 requires the C-terminus of the X domain, particularly the amino acid residues K432 and K434, and the XY-linker. The region alone, however, cannot serve as a functional NLS. The machinery for nuclear transport may require additional structural component(s) of the enzyme.

Cycloprodigiosin hydrochloride (cPrG.HCl) obtained from a marine bacterium Pseudoalteromonas denitrificans induces apoptotic cell death in various cancerous cell lines. cPrG.HCl alone caused a little cytotoxicity in HeLa cells, but it enhanced the apoptotic process progressively when co-administered with tumor necrosis factor (TNF)alpha. Here we studied the effect of cPrG.HCl on TNFalpha-induced activation of the transcription factor nuclear factor kappaB (NF-kappaB). Luciferase gene reporter assays revealed that cPrG.HCl potently suppressed the TNFalpha- and the phorbol myristate acetate-induced activation of NF-kappaB. The suppression occurred in the presence of imidazole, indicating that it was not related to the intracellular acidification resulting from the intrinsic H(+)/Cl(-) symporter activity of cPrG.HCl. cPrG.HCl inhibited neither the TNFalpha-induced phosphorylation and degradation of inhibitor of nuclear factor-kappaB, nor the subsequent nuclear translocation and DNA binding of NF-kappaB. cPrG.HCl also suppressed NF-kappaB-enhanced gene expression induced by Rac1, Cdc42, MEKK1, inhibitor of nuclear factor-kappaalpha (IKKalpha), IKKbeta, and a subunit of NF-kappaB, p65. These results indicate that cPrG.HCl suppresses NF-kappaB-dependent gene expression through the inhibition of transcriptional activation.

Here, we used a reductant, N-acetyl-L-cysteine (NAC), to investigate the redox-sensitive step(s) in the signalling pathway from the tumor necrosis factor (TNF) receptor to nuclear factor kappaB (NF-kappaB). We found that NAC suppressed NF-kappaB activation triggered by TNF or by overexpression of either the TNF receptor-associated death domain protein, TNF receptor-associated factor 2, NF-kappaB-inducing kinase (NIK), or IkappaB kinases (IKKalpha and IKKbeta). NAC also suppressed the TNF-induced activation of IKKalpha and IKKbeta, phosphorylation and degradation of IkappaB, and nuclear translocation of NF-kappaB. Furthermore, NAC suppressed the activation of IKKalpha and IKKbeta triggered by the overexpression of NIK. These results indicate that IKKalpha and IKKbeta are subject to redox regulation in the cells, and that NAC inhibits NF-kappaB activation through the suppression of these kinases.

Two types of immunosuppressants, cycloprodigiosin hydrochloride (cPrG) and l -leucyl- l -leucine methyl ester (LeuLeuOMe), both have the ability to selectively inhibit the lysosomal function, and a related compound to cPrG, prodigiosin 25-C, and LeuLeuOMe have been reported to selectively inhibit the T cell function in vitro. We therefore examined the cell-type specificity of cPrG and LeuLeuOMe using murine splenocytes. Concanavalin A (Con A)- and lentil lectin-induced proliferation was suppressed by cPrG more profoundly than lipopolysaccharide-induced proliferation. At the optimal concentration, Con A induced the proliferation of both CD4+ and CD8+ cells, whereas at a supra-optimal concentration Con A induced rather selective proliferation of CD8+ cells. Irrespective of the dose of Con A, CD4+ and CD8+ cells were equally affected by cPrG. In contrast, LeuLeuOMe induced the selective loss of CD8+ cells. cPrG enhanced the apoptosis of murine splenocytes and nylon fiber column-purified T cells cultured in the presence of Con A, as shown by the decrease in cell size and/or DNA fragmentation. Overall, this study revealed that the cell-type specificity of cPrG is different from that of LeuLeuOMe, and that the immunosuppression by cPrG is associated with apoptosis.

The pleckstrin homology (PH) domains of phospholipase C (PLC)-delta1 and a related catalytically inactive protein, p130, both bind inositol phosphates and inositol lipids. The binding to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by PLC-delta1 is proposed to be the critical interaction required for membrane localization to where the substrate resides; it is also required for the Ca(2+)-dependent activation of PLC-delta1 observed in the permeabilized cells. In the proximity of the PH domain, both PLC-delta1 and p130 possess the EF-hand domain, containing classical motifs implicated in calcium binding. Therefore, in the present study we examined whether the binding of the PH domain to PtdIns(4,5)P2 is regulated by changes in free Ca2+ concentration within the physiological range. A Ca2+ dependent increase in the binding to PtdIns(4,5)P2 was observed with a full-length PLC-delta1, while the isolated PH domain did not show any Ca2+ dependence. However, the connection of the EF-hand motifs to the PH domain restored the Ca2+ dependent increase in binding, even in the absence of the C2 domain. The p130 protein showed similar properties to PLC-delta1, and the EF-hand motifs were again required for the PH domain to exhibit a Ca2+ dependent increase in the binding to PtdIns(4,5)P2. The isolated PH domains from several other proteins which have been demonstrated to bind PtdIns(4,5)P2 showed no Ca2+ dependent enhancement of binding. However, when present within a chimera also containing PLC-delta1 EF-hand motifs, the Ca2+ dependent binding was again observed. These results suggest that the binding of Ca2+ to the EF-hand motifs can modulate binding to PtdIns(4,5)P2 mediated by the PH domain.

We have previously observed, using a green fluorescent protein (GFP) fusion system, that PLC-delta1 is localized mainly at the plasma membrane and in the cytosol, whereas little is present in the nucleus in Madin-Darby canine kidney cells (Fujii, M., Ohtsubo, M., Ogawa, T., Kamata, H., Hirata, H., and Yagisawa, H. (1999) Biochem. Biophys. Res. Commun. 254, 284-291). Herein, we demonstrate that PLC-delta1 has a functional nuclear export signal (NES) sequence in amino acid residues 164-177 of the EF-hand domain. The fluorescence of NES-disrupted GFP/PLC-delta1 expressed in Madin-Darby canine kidney cells was present not only at the plasma membrane and in the cytosol but also in the nucleus. Moreover, treatment with leptomycin B, a specific inhibitor of NES-dependent nuclear export, resulted in the accumulation of GFP/PLC-delta1 in the nucleus. A site-directed mutant containing a pleckstrin homology domain, which does not bind inositol 1,4,5-trisphosphate and cannot hydrolyze phosphatidylinositol 4,5-bisphosphate in vitro, accumulated in the nucleus to a much greater extent than wild-type GFP/PLC-delta1 after treatment with leptomycin B. These results suggest that PLC-delta1 is shuttled between the cytoplasm and the nucleus; its nuclear export is dependent on the leucine-rich NES sequence and its active nuclear import is regulated by an unidentified signal(s).

Green fluorescent protein (GFP)-tagged phospholipase C (PLC)-delta1 and its mutants were expressed in Madin-Darby canine kidney (MDCK) cells. GFP-PLC-delta1 or the GFP-tagged pleckstrin homology (PH) domain of PLC-delta1 itself was found to be predominantly localized at the plasma membrane. The DeltaPH mutant or a site-directed mutant containing a PH domain which does not bind inositol 1,4, 5-trisphosphate and cannot hydrolyze phosphatidylinositol 4, 5-bisphosphate in vitro was seen only in the cytosol. In living MDCK cells hypo-osmotic stress caused a rapid dissociation of GFP-PLC-delta1 from the plasma membrane, which coincided with phosphoinositide breakdown. A PLC inhibitor, U73122, blocked this translocation, but depletion of extracellular Ca2+ had no effect. The translocation was reversed by replacement with an iso-osmotic buffer. Our results demonstrate that the PH domain plays a critical role in the membrane targeting of PLC-delta1 and that the intracellular distribution of the enzyme is regulated by osmotic stress-driven phosphoinositide turnover.

The laparoscopic appearance of the liver surface in patients with primary sclerosing cholangitis (PSC) has not been fully delineated. We examined 5 patients with PSC and reviewed 35 previously reported cases. Intense white markings in a mesh-like pattern were characteristic of PSC. The findings varied depending on the histologic stage and affected site. Red patches which have also been seen in primary biliary cirrhosis (PBC), green patches indicating cholestasis, and dilatation of the terminal bile ducts were also observed. The red patches were wider than those in PBC. The white markings were less well defined in the red patches than in other areas. The severity of the fibrosis and histologic damage was judged to be mild at the red sites. The green patches were caused by localized cholestasis. This indicated that PSC unevenly affects the liver. Severe obstruction may be present between the interlobular bile ducts and the septal bile ducts in anicteric stages. (Dig Endosc 1999; 10: 37–41)

We examined whether a phosphotyrosine binding (PTB) domain from the human insulin receptor substrate-1 (hIRS-1) is capable of binding inositol phosphates/phosphoinositides. The binding specificity was compared with that of the pleckstrin homology (PH) domain derived from the same protein because the three dimensional structure was found to be very similar to that of the PH domain, despite the lack of sequence similarity. We also attempted to locate the site of binding of the inositol compounds. The PTB domain bound [3H]Ins(1,4,5)P3, which was displaced most strongly by Ins(1,3,4,5,6)P5 and InsP6, indicating that these inositol polyphosphates show the highest affinity. The PTB domain bound to liposomes containing PtdIns(4,5)P2, PtdIns(3,4,5)P3 and PtdIns(3,4)P2, but not phosphatidylinositol. In contrast, the PH domain showed a preference for Ins(1,4,5)P3, the polar head of PtdIns(4,5)P2. Site-directed mutagenesis studies were performed to map the binding site for inositol phosphates in the PTB domain. Mutation of K169Q, K171Q or K177Q, located in the loop connecting the β1 and β2 strands, which is partially responsible for binding inositol phosphates/phosphoinositides in the PH domains of several other proteins, reduced binding activity, probably because of a reduction in affinity. Mutation of R212Q or R227Q, shown to be involved in the binding of a phosphotyrosine, had little effect on the binding capacity. These results indicate that the PTB domain of hIRS-1 can bind inositol phosphates/phosphoinositides. Therefore signalling through the PTB domain could be regulated by the binding not only of proteins with phosphotyrosine but also of inositol phosphates/phosphoinositides, implying that PTB domains could be involved in a myriad of interconnections between intracellular signalling pathways.

There is a marked paucity of reports on contrast-enhanced ultrasonography (US) for focal splenic disease in the literature, and there are no previously reported contrast-enhanced US findings for splenic granuloma. We report the case of a 77-year-old cirrhotic woman with isolated splenic granulomas. We detail the diagnostic difficulty encountered in this case and briefly review the literature.

The efficacy of Interferon (IFN) therapy against hepatitis C is greatly affected by viral factors such as the level of viremia and the HCV genotype. The prognosis of those with high serum HCV-RNA levels in genotype 1b is poor. In the present report, we carried out a randomized controlled study of two IFN doses for patients with genotype 1b chronic hepatitis C whose serum HCV-RNA levels were at least 10 6 copies/ml as determined by multicyclic polymerase chain reaction (PCR) assay. These patients were randomly divided into two groups. Group A received 6 million units (MIU) of IFN 6 days per week for 8 weeks, followed by the same dose 3 days per week for 16 weeks (total IFN dose: 576 MIU). Group B received 6 MIU of IFN 6 days per week for 2 weeks, followed by the same dose 3 days per week for 22 weeks (total IFN dose: 480 MIU). The number of complete responders was four of 24 (16.6%) in group A and three of 20 (15.0%) in group B. There was no significant difference between the groups. On the basis of these findings, it appears that there is no advantage in lengthening the initial period of consecutive IFN administration to 8 weeks or in increasing the total dose by 100 MIU for those with genotype 1b hepatitis C and high virus levels.

The Rho family of GTPases belongs to the superfamily named “Ras-like” proteins, which consists of over 150 varieties in mammals [1]. Significant progress has recently been made in understanding the biological functions mediated by this family of small (~21 kDa) G proteins (guanine nucleotide-binding proteins). Rho GTPases affect crucial biological processes such as transcriptional regulation, cell cycle progression, apoptosis and membrane trafficking [2, 3]. Thus far, a total of 23 Rho proteins have been identified [4], among which RhoA, Rac1 and Cdc42 are characterized in detail. Rho GTPases are also involved in the cytoskeleton formation of the cell via the regulation of actin dynamics [5, 6]. RhoA induces stress fiber formation and focal adhesion assembly, thereby regulating cell shape, attachment and motility, whereas Rac1 promotes extension of lamellipodia and membrane ruffling [7]. Cdc42 has been shown to play a role in the formation of filopodia [8].

The coexistence of idiopathic thrombocytopenic purpura (ITP) and active ulcerative colitis (UC) has been reported. We herein report a rare case of UC accompanied by ITP and Helicobacter pylori (H. pylori) infection. A female UC patient was diagnosed with ITP. At that time, the UC was almost in remission and we suspected that the ITP was caused by some factor other than UC. Accordingly, we found H. pylori infection and administered H. pylori eradication therapy. Consequently, the patient's serum platelet count recovered dramatically. Our report demonstrates the importance of conducting examinations for H. pylori infection in ITP patients, even those with UC.