Correction: Real-space evidence of the equilibrium ordered bicontinuous double diamond structure of a diblock copolymer.

Correction for 'Real-space evidence of the equilibrium ordered bicontinuous double diamond structure of a diblock copolymer' by C. Y. Chu et al., Soft Matter, 2015, 11, 1871-1876.

Real-space evidence of the equilibrium ordered bicontinuous double diamond structure of a diblock copolymer.

The ordered bicontinuous double diamond (OBDD) structure has long been believed to be an unstable ordered network nanostructure, which is relative to the ordered bicontinuous double gyroid (OBDG) structure for diblock copolymers. Using electron tomography, we present the first real-space observation of the thermodynamically stable OBDD structure in a diblock copolymer composed of a stereoregular block, syndiotactic polypropylene-block-polystyrene (sPP-b-PS), in which the sPP tetrapods are interconnected via a bicontinuous network with Pn3̄m symmetry. The OBDD structure underwent a thermally reversible order-order transition (OOT) to OBDG upon heating, and the transition was accompanied with a slight reduction of domain spacing, as demonstrated both experimentally and theoretically. The thermodynamic stability of the OBDD structure was attributed to the ability of the configurationally regular sPP block to form helical segments, even above its melting point, as the reduction of internal energy associated with the helix formation may effectively compensate the greater packing frustration in OBDD relative to that in the tripods of OBDG.

Surface curvatures of trabecular bone microarchitecture.

Microstructure of trabecular bone has been examined with a particular emphasis on surface curvatures in two-phase (trabecular and intertrabecular space- i.e., marrow space) structures. Three trabecular bone samples, quantified as "plate-like," "rod-like," and a mixture of these two structural elements according to the structure model index (SMI), were subjected to analysis based on (differential) geometry. A correspondence between the SMI and the mean curvature was found. A method to measure surface curvatures is proposed. The gaussian curvatures averaged over the surfaces for the three analyzed bone structures were all found to be negative, demonstrating their surfaces to be, on average, hyperbolic. In addition, the Euler-Poincaré characteristics and the genus, both characterizing topological features of bone connectivity, were estimated from integral gaussian curvature (Gauss-Bonnet theorem). The three bone microstructures were found to be topologically analogous to spheres with one to three handles.

Interfacial and topological measurements of bicontinuous polymer morphologies.

Bicontinuous morphologies are ubiquitous in nature and occur at various length scales. Topological features of two such morphologies arising in an ordered block copolymer at equilibrium and a polymer blend during spinodal decomposition are measured from three-dimensional images. Interfacial curvature, coordination number, and interjunction distance distributions exhibit remarkable similarity in these systems, despite vastly different length scales. A channel coordination of 3 is dominant in both morphologies, and topological measurements such as the Euler-Poincaré characteristic and genus are reported.

Morphological characterization of bicontinuous structures in polymer blends and microemulsions by the inverse-clipping method in the context of the clipped-random-wave model.

A method is proposed to determine the spectral function of the clipped-random-wave (CRW) model directly from scattering data. The spectral function f(k) (k is a wave number) gives the distribution of the magnitude of wave vectors of the sinusoidal waves that describes the essential features of the two-phase morphology. The proposed method involves "inverse clipping" of a correlation function to obtain f(k) and does not require any a priori assumptions for f(k). A critical test of the applicability of the inverse-clipping method was carried out by using three-component bicontinuous microemulsions. The method was then used to determine f(k) of the bicontinuous structure of a phase-separating polymer blend. f(k) for the polymer blend turned out to be a multipeaked function, while f(k) for the microemulsions exhibits a single broad maximum representing periodicity of the morphology. These results indicate the presence of the long-range regularity in the morphology of the polymer blend. Three-dimensional (3D) morphology corresponding to the scattering data of the polymer blend was generated using the CRW model together with the multipeaked f(k). Interface curvatures of the 3D morphology calculated from f(k) were measured and compared with those experimentally determined directly from the laser scanning confocal microscopy in the same blend.

Direct measurement of interfacial curvature distributions in a bicontinuous block copolymer morphology.

Self-consistent field theory predicts that the complex phase behavior of block copolymers does not originate solely from the interface seeking constant mean curvature as once thought, but instead reflects competing minimization of interfacial tension and packing frustration. To test this prediction, we directly measure interfacial curvature distributions from a 3D image reconstruction of the bicontinuous gyroid morphology. Results obtained here reveal that the gyroid interface is not constant mean curvature and confirm the importance of packing frustration in the stabilization of such complex nanostructures.

Phase separation with multiple length scales in polymer mixtures induced by autocatalytic reactions.

A ternary polymer blend with two components photo-cross-linked independently in its miscible region undergoes phase separation, exhibiting morphology with multiple length scales. Contrary to the case of thermally induced phase separation, the morphology exhibits a unimodal-->multimodal transition. It is shown that these multiple length scales are caused by the inhomogeneous freezing kinetics of the cross-linking process. This inhomogeneity arises from the autocatalytic feedback driven by the couplings between concentration fluctuations and the photo-cross-linking reactions.

Characterization of phospholipase D activation by GM2 activator in a cell-free system.

Phospholipase D (PLD) activator which synergistically activates the enzyme with ADP ribosylation factor has recently been shown homologous to GM2 activator (Nakamura, S. et al.: Proc. Natl. Acad. Sci. USA 1998. 95, 12249/12253). The present studies were undertaken to further clarify the identity of the activator by immunological technique and to characterize the mechanism of activation of PLD by enzymological approach. The activator was further confirmed as GM2 activator by immunoblot analysis. Kinetic analysis showed Vmax for the PLD reaction was 16 fold elevated by GM2 activator, whereas Km for phosphatidylcholine remained constant by GM2 activator. These results strongly suggest that GM2 activator might activate enzyme by protein-protein interaction not by substrate modification. These results facilitate the understanding how the metabolism of both phospholipids and gangliosides is regulated by the same protein.

A new preoperative immunochemotherapy for the treatment of locally advanced esophageal cancer.

A promising preoperative immunochemotherapy regimen for locally advanced esophageal cancer is herein described. A 67-year-old man suffering from severe dysphagia was diagnosed with unresectable esophageal cancer at initial examination because of a tumor of 11 cm in length and suspicion of trachea invasion. Neoadjuvant immunochemotherapy was undertaken for the down-staging. Interleukin-2 (IL-2) (3.5 x 10(5) Japan reference units), nedaplatin (7 mg/m2) and 5-FU (300 mg/m2) were administered intravenously daily for 5 days a week for three weeks. The gross findings of a barium esophagogram and esophagoscopy revealed significant tumor regression in both size and shape. The patient underwent an esophagectomy through a laparotomy followed by a right thoracotomy. The surgical specimens were serially sectioned and examined microscopically. All of the surgical margins were clear (upper and lower margins as well as the adventitia), and there was no evidence of lymph node metastasis. The surgical specimen revealed neoplastic squamous ghost cells surrounding significant lymphocyte infiltration. This appears to be a unique feature of this particular neoadjuvant immunochemotherapy.

Requirement of GM2 ganglioside activator for phospholipase D activation.

Sequence analysis of a heat-stable protein necessary for the activation of ADP ribosylation factor-dependent phospholipase D (PLD) reveals that this protein has a structure highly homologous to the previously known GM2 ganglioside activator whose deficiency results in the AB-variant of GM2 gangliosidosis. The heat-stable activator protein indeed has the capacity to enhance enzymatic conversion of GM2 to GM3 ganglioside that is catalyzed by beta-hexosaminidase A. Inversely, GM2 ganglioside activator purified separately from tissues as described earlier [Conzelmann, E. & Sandhoff, K. (1987) Methods Enzymol. 138, 792-815] stimulates ADP ribosylation factor-dependent PLD in a dose-dependent manner. At higher concentrations of ammonium sulfate, the PLD activator protein apparently substitutes for protein kinase C and phosphatidylinositol 4,5-bisphosphate, both of which are known as effective stimulators of the PLD reaction. The mechanism of action of the heat-stable PLD activator protein remains unknown.

Purification of a heat-stable activator protein for ADP-ribosylation factor-dependent phospholipase D.

A heat-stable activator for ADP-ribosylation factor (ARF)-dependent phospholipase D (PLD) was purified to near homogeneity from rat kidney cytosol by a sequential column chromatography. The purified activator has a molecular mass of 23 kDa on SDS-PAGE. Using a partially purified ARF-dependent PLD from rat kidney, the activator synergistically stimulates PLD with ARF in time- and dose-dependent manner. In the absence of ARF, the activator has little or no effect. The purified activator also stimulates PLD under several conditions including permeabilized cell system, suggesting that the activator is a physiologically relevant regulator of PLD.

Interleukin 12 augments the liver-associated immunity and reduces liver metastases.

BACKGROUND/AIMS: It has been reported recently that in vivo administration of interleukin-12 (IL-12) augments the cytotoxic activity of natural killer (NK)/T cells and shows a powerful anti-tumor activity. In this study, we evaluated that the IL-12 effect on liver-associated immunity and in vivo efficacy on the hepatic metastases in a rat model. MATERIALS AND METHODS: Varying amounts of mouse recombinant IL-12 were injected intraperitoneally for 5 days to adult male Fischer rats and hepatic sinusoidal lymphocytes (HSL) were collected. Purified HSL are spontaneously cytolytic to both conventional NK-sensitive target (YAC-1) and NK-resistant target (RCN-H4) tumor cells. RESULTS: IL-12 was found to increase the number of HSL and the cytolytic activity against these target cells in a dose-dependent fashion. Flow cytometric analysis showed that IL-12 caused an increase of CD8+ subpopulation in HSL and a double staining study revealed that the increased subpopulation was not CD3+8+ (cytotoxic T cell) fraction, but actually CD3-8+ (NK cell) fraction. Experimental liver metastases was markedly reduced in rats treated intraperitoneally with IL-12. CONCLUSION: These results demonstrate that IL-12 augments the cytolytic activity of HSL and suggests this cytokine as an attractive choice for liver metastases therapy.

Reconstitution of GTP-gamma-S-dependent phospholipase D activity with ARF, RhoA, and a soluble 36-kDa protein.

For activation of kidney membrane phospholipase D (PLD), cytosol is absolutely needed in addition to GTP-gamma-S. The active component of cytosol consists of three protein factors: ADP-ribosylation factor, RhoA, and a soluble 36-kDa protein. Any combination of these two factors synergistically activates PLD to some extent, but the presence of the three factors causes full activation. The 36-kDa protein is stable at 60 degrees C but inactivated at 80 degrees C for 10 min. Tissue distribution of the 36-kDa protein roughly coincides with that of PLD, suggesting physiological relevance of the protein in the regulation of PLD.

Mammalian phospholipase D: phosphatidylethanolamine as an essential component.

Bovine kidney phospholipase D (PLD) was assayed by measuring the formation of phosphatidylethanol from added radioactive phosphatidylcholine (PtdCho) in the presence of ethanol, guanosine 5'-[gamma-thio]triphosphate, ammonium sulfate, and cytosol factor that contained small GTP-binding regulatory proteins. The PLD enzyme associated with particulate fractions was solubilized by deoxycholate and partially purified by chromatography on a heparin-Sepharose column. This PLD preferentially used PtdCho as substrate. After purification, the enzyme per se showed little or practically no activity but required an additional factor for the enzymatic reaction. This factor was extracted with chloroform/methanol directly from particulate fractions of various tissues, including kidney, liver, and brain, and identified as phosphatidylethanolamine (PtdEtn), although this phospholipid did not serve as a good substrate. Plasmalogen-rich PtdEtn, dioleoyl-PtdEtn, and L-alpha-palmitoyl-beta-linoleoyl-PtdEtn were effective, but dipalmitoyl-PtdEtn was inert. Sphingomyelin was 30% as active as PtdEtn. The results suggest that mammalian PLD reacts nearly selectively with PtdCho in the form of mixed micelles or membranes with other phospholipids, especially PtdEtn.

IL-2 perfusion to the liver augments the hepatic extraction rate of accompanying anticancer drugs.

A pilot study we conducted on hepatic infusion chemotherapy combined with interleukin-2 (IL-2) for metastatic liver malignancies revealed very encouraging results indicating that this treatment modality is more effective than either of the anticancer drugs used alone. To clarify the mechanisms underlying the synergism of these modalities, the pharmacokinetics of anticancer drugs were examined in a rat model. Adult rats were given 5-fluorouracil (5-FU) or mitomycin C (MMC) combined with various doses of IL-2 up to 7500 JRU/kg per minute for the measurement of hepatic extraction rates (HER). The HER of 5-FU was significantly increased (P < 0.01) in combination with IL-2 in a dose-dependent fashion while that of MMC also showed a tendency to increase. Thus, it is conceivable that the increase of vascular permeability caused by IL-2 results in augmentation of the HER of associated anticancer drugs. This effect may improve the delivery of anticancer drugs to the liver and alleviate general toxicity by reducing the amount of circulating anticancer agents.

Mammalian phospholipase D: activation by ammonium sulfate and nucleotides.

Phospholipase D (PLD) associated with the rat kidney membrane was activated by guanine 5'-[gamma-thio]triphosphate and a cytosol fraction that contained ADP-ribosylation factor. When assayed by measuring the phosphatidyl transfer reaction to ethanol with exogenously added radioactive phosphatidylcholine as substrate, the PLD required a high concentration (1.6 M) of ammonium sulfate to exhibit high enzymatic activity. Other salts examined were far less effective or practically inactive, and this dramatic action of ammonium sulfate is not simply due to such high ionic strength. Addition of ATP but not of nonhydrolyzable ATP analogue adenosine 5'-[beta, gamma-imido]diphosphate further enhanced the PLD activation approximately equal to 2- to 3-fold. This enhancement by ATP needed cytosol, implying a role of protein phosphorylation. A survey of PLD activity in rat tissues revealed that, unlike in previous observations reported thus far, PLD was most abundant in membrane fractions of kidney, spleen, and liver in this order, and the enzymatic activity in brain and lung was low.

A high level of prostaglandin E2 (PGE2) in the portal vein suppresses liver-associated immunity and promotes liver metastases.

Prostaglandin E2 (PGE2) is generally accepted to be an immunosuppressant produced by cancer cells and their surrounding macrophages. Although several investigators have reported detecting high concentrations of PGE2 in the portal veins of patients with colorectal cancer, the relationship between these high concentrations of PGE2 in the portal vein and liver-associated immunity remains unclear. In this study, we attempted to determine if the portal administration of PGE2 suppresses the immune function of the liver in a rat model. Donryu rats were administered PGE2 via the portal vein for 7 days, following which the cytotoxic activity of hepatic sinusoidal lymphocytes (HSL) against natural killer (NK)-sensitive YAC-1 and rat syngeneic AH60C tumor cells was assessed. Purified HSL are spontaneously cytolytic; however, the continuous administration of PGE2 dramatically suppressed the cytotoxic activity of HSLs in a dose-dependent fashion. Flow cytometric analysis showed that the large granular lymphocyte (LGL) fraction, hepatic natural killer (pit) cells, and CD4-8+ killer/suppressor T cells were mainly reduced in number in the HSLs following PGE2 infusion. In this rat AH60C metastasis model, the continuous administration of PGE2 increased the number and size of metastatic tumor nodules in the liver, suggesting that high concentrations of PGE2 in the portal vein suppress liver-associated immunity and promote the formation of hepatic metastasis.

Effect of packed red cell and whole blood transfusion on liver-associated immune function.

In order to clarify the effect of allogeneic blood transfusion on liver metastases from primary cancer, liver-associated immune function after blood transfusion was evaluated in a murine model. Hepatic sinusoidal lymphocytes (HSL) were strongly cytotoxic to conventional natural killer cell-sensitive target (YAC-1), as well as to natural killer cell-resistant solid adenocarcinoma cells (colon 26), compared with splenic lymphocytes. Allogeneic whole blood transfusion strikingly suppressed the cytotoxic activities of HSL. Red blood cell transfusions also suppressed cytotoxicity to the same degree. In an animal model, allogeneic transfusion increased the rate of liver metastases. Flow cytometric analysis showed that transfusion caused a temporary decrease in the class II antigen positive cell fraction, mainly Kupffer's cells. This phenomenon occurred in parallel with changes in hepatic antitumor activity, indicating the possible importance of the involvement of Kupffer's cell in the development of the killer activity of HSL. These results suggest that blood transfusion may be a significant risk factor for hepatic metastasis by transiently suppressing the immunocompetence of the liver.