Hypercholesterolemic Dysregulation of Calpain in Lymphatic Endothelial Cells Interferes With Regulatory T-Cell Stability and Trafficking.

BACKGROUND: Although hypercholesterolemia reportedly counteracts lymphocyte trafficking across lymphatic vessels, the roles of lymphatic endothelial cells (LECs) in the lymphocyte regulations remain unclear. Previous studies showed that calpain-an intracellular modulatory protease-interferes with leukocyte dynamics in the blood microcirculation and is associated with hypercholesterolemic dysfunction in vascular endothelial cells. METHODS: This study investigated whether the calpain systems in LECs associate with the LEC-lymphocyte interaction under hypercholesterolemia using gene-targeted mice. RESULTS: Lipidomic analysis in hypercholesterolemic mice showed that several lysophospholipids, including lysophosphatidic acid, accumulated in the lymphatic environment. Lysophosphatidic acid enables the potentiation of calpain systems in cultured LECs, which limits their ability to stabilize regulatory T cells (Treg) without altering Th1/Th2 (T helper type1/2) subsets. This occurs via the proteolytic degradation of MEKK1 (mitogen-activated protein kinase kinase kinase 1) and the subsequent inhibition of TGF (transforming growth factor)-ß1 production in LECs. Targeting calpain systems in LECs expanded Tregs in the blood circulation and reduced aortic atherosclerosis in hypercholesterolemic mice, concomitant with the reduction of proinflammatory macrophages in the lesions. Treg expansion in the blood circulation and atheroprotection in calpain-targeted mice was prevented by the administration of TGF-ß type-I receptor inhibitor. Moreover, lysophosphatidic acid-induced calpain overactivation potentiated the IL (interleukin)-18/NF-κB (nuclear factor κB)/VCAM1 (vascular cell adhesion molecule 1) axis in LECs, thereby inhibiting lymphocyte mobility on the cells. Indeed, VCAM1 in LECs was upregulated in hypercholesterolemic mice and human cases of coronary artery disease. Neutralization of VCAM1 or targeting LEC calpain systems recovered afferent Treg transportation via lymphatic vessels in mice. CONCLUSIONS: Calpain systems in LECs have a key role in controlling Treg stability and trafficking under hypercholesterolemia.

Calpain-mediated proteolytic production of free amino acids in vascular endothelial cells augments obesity-induced hepatic steatosis.

Free amino acids that accumulate in the plasma of patients with diabetes and obesity influence lipid metabolism and protein synthesis in the liver. The stress-inducible intracellular protease calpain proteolyzes various substrates in vascular endothelial cells (ECs), although its contribution to the supply of free amino acids in the liver microenvironment remains enigmatic. In the present study, we showed that calpains are associated with free amino acid production in cultured ECs. Furthermore, conditioned media derived from calpain-activated ECs facilitated the phosphorylation of ribosomal protein S6 kinase (S6K) and de novo lipogenesis in hepatocytes, which were abolished by the amino acid transporter inhibitor, JPH203, and the mammalian target of rapamycin complex 1 inhibitor, rapamycin. Meanwhile, calpain-overexpressing capillary-like ECs were observed in the livers of high-fat diet-fed mice. Conditional KO of EC/hematopoietic Capns1, which encodes a calpain regulatory subunit, diminished levels of branched-chain amino acids in the hepatic microenvironment without altering plasma amino acid levels. Concomitantly, conditional KO of Capns1 mitigated hepatic steatosis without normalizing body weight and the plasma lipoprotein profile in an amino acid transporter-dependent manner. Mice with targeted Capns1 KO exhibited reduced phosphorylation of S6K and maturation of lipogenic factor sterol regulatory element-binding protein 1 in hepatocytes. Finally, we show that bone marrow transplantation negated the contribution of hematopoietic calpain systems. We conclude that overactivation of calpain systems may be responsible for the production of free amino acids in ECs, which may be sufficient to potentiate S6K/sterol regulatory element-binding protein 1-induced lipogenesis in surrounding hepatocytes.

Age Classification of Rice Seeds in Japan Using Gradient-Boosting and ANFIS Algorithms.

The rapidly changing climate affects an extensive spectrum of human-centered environments. The food industry is one of the affected industries due to rapid climate change. Rice is a staple food and an important cultural key point for Japanese people. As Japan is a country in which natural disasters continuously occur, using aged seeds for cultivation has become a regular practice. It is a well-known truth that seed quality and age highly impact germination rate and successful cultivation. However, a considerable research gap exists in the identification of seeds according to age. Hence, this study aims to implement a machine-learning model to identify Japanese rice seeds according to their age. Since agewise datasets are unavailable in the literature, this research implements a novel rice seed dataset with six rice varieties and three age variations. The rice seed dataset was created using a combination of RGB images. Image features were extracted using six feature descriptors. The proposed algorithm used in this study is called Cascaded-ANFIS. A novel structure for this algorithm is proposed in this work, combining several gradient-boosting algorithms such as XGBoost, CatBoost, and LightGBM. The classification was conducted in two steps. First, the seed variety was identified. Then, the age was predicted. As a result, seven classification models were implemented. The performance of the proposed algorithm was evaluated against 13 state-of-the-art algorithms. Overall, the proposed algorithm has a higher accuracy, precision, recall, and F1-score than the others. For the classification of variety, the proposed algorithm scored 0.7697, 0.7949, 0.7707, and 0.7862, respectively. The results of this study confirm that the proposed algorithm can be employed in the successful age classification of seeds.

Endothelial calpain systems orchestrate myofibroblast differentiation during wound healing.

The transformation of fibroblasts to myofibroblasts plays a major role in fibrogenic responses during dermal wound healing. We show a contribution of calpain systems (intracellular regulatory protease systems) in vascular endothelial cells (ECs) to myofibroblast differentiation in wound sites. Dermal wound healing experiments in mice found that calpastatin (an endogenous inhibitor of calpains) is enriched in preexisting vessels but not in newly formed capillaries. Transgenic overexpression of calpastatin in ECs delayed wound healing in mice as well as reducing the keratinocyte layer, extracellular matrix deposition, and myofibroblast accumulation in wound sites. EC and leukocyte markers, however, remain unchanged. Calpastatin overexpression reduced the expression of genes encoding platelet-derived growth factor-B and PDGF receptor-ß (PDGFR-ß). Topical application of platelet-derived growth factor-BB-containing ointment to wounds accelerated healing in control mice, but calpastatin overexpression prevented this acceleration. In cultured human dermal fibroblasts, α-smooth muscle actin and PDGFR-ß were up-regulated by coculturing with ECs, but this action was inhibited by suppression of EC calpain activity. EC-driven transformation of mouse dermal fibroblasts was also suppressed by calpastatin overexpression in ECs. These results suggest that endothelial calpain systems influence PDGFR-ß signaling in fibroblasts, EC-driven myofibroblast differentiation, and subsequent fibrogenic responses in wounds.-Miyazaki, T., Haraguchi, S., Kim-Kaneyama, J.-R., Miyazaki, A. Endothelial calpain systems orchestrate myofibroblast differentiation during wound healing.

Hypothalamic Monoaminergic Pathology in a Neurodevelopmental Rat Model Showing Prenatal 5-Bromo-2'-Deoxyuridine Treatment-Induced Hyperactivity and Hyporeproductivity.

OBJECTIVE: Prenatal treatment of rats with 5-bromo-2'-deoxyuridine (BrdU) is a neurodevelopmental model showing hyperactivity and impaired sexual activity. Human neurodevelopmental disorders, such as autism, exhibit sex-related pathology, but sex-related neurodevelopment has not been fully investigated in this model. We conducted this study to facilitate the understanding of the pathophysiology of neurodevelopmental disorders. METHODS: Pregnant rats received 50 mg/kg BrdU on gestational days 9-15. The tissue content of dopamine (DA), serotonin (5-HT), and their metabolites dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid were measured in male and female offspring at 3 weeks (juveniles) and 10 weeks (adults) of age. RESULTS: Prenatally BrdU-treated rats had reduced DA metabolism or DA content in the hypothalamus from the juvenile through the adult period without sex differences, but sex-specific striatal DA abnormalities emerged after maturation. A reduction in 5-HT metabolism was measured in the hypothalamus without sex differences throughout development. Developmental alterations in the striatal 5-HT states were sex-dependent. Temporal changes in DA or 5-HT metabolism were found in the frontal cortex and midbrain. CONCLUSION: The sex-specific influence of a genotoxic factor on the development of the DA and 5-HT systems was clarified in the hypothalamus and striatum. The results suggest that the observed sex dependence and region specificity are related to the pathology of social dysfunction in neurodevelopmental disorders.

A member of cation diffusion facilitator family, MTP11, is required for manganese tolerance and high fertility in rice.

MAIN CONCLUSION: Rice MTP11 is the trans-Golgi-localized transporter that is involved in Mn tolerance with MTP8.1, and it is required for normal fertility. Rice (Oryza sativa L.) is one of the most manganese (Mn)-tolerant species, and it is able to accumulate high levels of this metal in the leaves without showing toxic symptoms. The metal tolerance protein 8.1 (MTP8.1), a member of the Mn-cation diffusion facilitator (CDF) family, has been shown to play a central role in high Mn tolerance by sequestering Mn into vacuoles. Recently, rice MTP11 was identified as an Mn transporter that is localized to Golgi-associated compartments, but its exact role in Mn tolerance in planta has not yet been understood. Here, we investigated the role of MTP11 in rice Mn tolerance using knockout lines. Old leaves presented higher levels of constitutively expressed MTP11 than other tissues and MTP11 expression was also found in reproductive organs. Fused MTP11:green fluorescent protein was co-localized to trans-Golgi markers and differentiated from other Golgi-associated markers. Knockout of MTP11 in wild-type rice did not affect tolerance and accumulation of Mn and other heavy metals, but knockout in the mtp8.1 mutant showed exacerbated Mn sensitivity at the vegetative growth stage. Knockout of MTP11 alone resulted in decreased grain yield and fertility at the reproductive stage. Thus, MTP11 is a trans-Golgi localized transporter for Mn, which plays a role in Mn tolerance through intracellular Mn compartmentalization. It is also required for maintaining high fertility in rice.

Emerging roles of calpain proteolytic systems in macrophage cholesterol handling.

Calpains are Ca2+-dependent intracellular proteases that play central roles in the post-translational processing of functional proteins. In mammals, calpain proteolytic systems comprise the endogenous inhibitor calpastatin as well as 15 homologues of the catalytic subunits and two homologues of the regulatory subunits. Recent pharmacological and gene targeting studies in experimental animal models have revealed the contribution of conventional calpains, which consist of the calpain-1 and -2 isozymes, to atherosclerotic diseases. During atherogenesis, conventional calpains facilitate the CD36-dependent uptake of oxidized low-density lipoprotein (LDL), and block cholesterol efflux through ATP-binding cassette transporters in lesional macrophages, allowing the expansion of lipid-enriched atherosclerotic plaques. In addition, calpain-6, an unconventional non-proteolytic calpain, in macrophages reportedly potentiates pinocytotic uptake of native LDL, and attenuates the efferocytic clearance of apoptotic and necrotic cell corpses from the lesions. Herein, we discuss the recent progress that has been made in our understanding of how calpain contributes to atherosclerosis, in particular focusing on macrophage cholesterol handling.

Calpastatin counteracts pathological angiogenesis by inhibiting suppressor of cytokine signaling 3 degradation in vascular endothelial cells.

RATIONALE: Janus kinase/signal transducer and activator of transcription (JAK/STAT) signals and their endogenous inhibitor, suppressor of cytokine signaling 3 (SOCS3), in vascular endothelial cells (ECs) reportedly dominate the pathological angiogenesis. However, how these inflammatory signals are potentiated during pathological angiogenesis has not been fully elucidated. We suspected that an intracellular protease calpain, which composes the multifunctional proteolytic systems together with its endogenous inhibitor calpastatin (CAST), contributes to the JAK/STAT regulations. OBJECTIVE: To specify the effect of EC calpain/CAST systems on JAK/STAT signals and their relationship with pathological angiogenesis. METHODS AND RESULTS: The loss of CAST, which is ensured by several growth factor classes, was detectable in neovessels in murine allograft tumors, some human malignant tissues, and oxygen-induced retinopathy lesions in mice. EC-specific transgenic introduction of CAST caused downregulation of JAK/STAT signals, upregulation of SOCS3 expression, and depletion of vascular endothelial growth factor (VEGF)-C, thereby counteracting unstable pathological neovessels and disease progression in tumors and oxygen-induced retinopathy lesions in mice. Neutralizing antibody against VEGF-C ameliorated pathological angiogenesis in oxygen-induced retinopathy lesions. Small interfering RNA-based silencing of endogenous CAST in cultured ECs facilitated µ-calpain-induced proteolytic degradation of SOCS3, leading to VEGF-C production through amplified interleukin-6-driven STAT3 signals. Interleukin-6-induced angiogenic tube formation in cultured ECs was accelerated by CAST silencing, which is suppressible by pharmacological inhibition of JAK/STAT signals, antibody-based blockage of VEGF-C, and transfection of calpain-resistant SOCS3, whereas transfection of wild-type SOCS3 exhibited modest angiostatic effects. CONCLUSIONS: Loss of CAST in angiogenic ECs facilitates µ-calpain-induced SOCS3 degradation, which amplifies pathological angiogenesis through interleukin-6/STAT3/VEGF-C axis.

Hic-5 deficiency attenuates the activation of hepatic stellate cells and liver fibrosis through upregulation of Smad7 in mice.

BACKGROUND & AIM: Hydrogen peroxide-inducible clone-5 (Hic-5), also named as transforming growth factor beta-1-induced transcript 1 protein (Tgfb1i1), was found to be induced by TGF-ß. Previous studies have shown that TGF-ß is a principal mediator of hepatic stellate cell (HSC) activation in liver fibrosis. However, this process remains elusive. In this study, we aimed to define the role of Hic-5 in HSC activation and liver fibrosis. METHODS: We examined the expression levels of Hic-5 during HSCs activation and in fibrotic liver tissues by quantitative real-time reverse transcriptase polymerase chain reaction, Western blot and immunohistochemistry. Hic-5 knockout (KO) and wild-type (WT) mice were subjected to bile duct ligation (BDL) or carbon tetrachloride (CCl4) injection to induce liver fibrosis. RESULTS: Hic-5 expression was strongly upregulated in activated HSCs of the human fibrotic liver tissue and BDL or CCl4-induced mouse liver fibrosis. Hic-5 deficiency significantly attenuated mouse liver fibrosis and HSC activation. Furthermore, Hic-5 knockdown by siRNA in vivo repressed CCl4-induced liver fibrosis in mice. Mechanistically, the absence of Hic-5 significantly inhibited the TGF-ß/Smad2 signaling pathway, proved by increasing Smad7 expression, resulting in reduced collagen production and α-smooth muscle actin expression in the activated HSCs. CONCLUSION: Hic-5 deficiency attenuates the activation of HSCs and liver fibrosis though reducing the TGF-ß/Smad2 signaling by upregulation of Smad7. Thus, Hic-5 can be regarded as a potential therapeutic target for liver fibrosis.

Fibroblast growth factor-23 induces cellular senescence in human mesenchymal stem cells from skeletal muscle.

Although muscle wasting and/or degeneration are prevalent in patients with chronic kidney disease, it remains unknown whether FGF-23 influences muscle homeostasis and regeneration. Mesenchymal stem cells (MSCs) in skeletal muscle are distinct from satellite cells and have a known association with muscle degeneration. In this study we sought to investigate the effects of FGF-23 on MSCs isolated from human skeletal muscle in vitro. The MSCs expressed FGF receptors (1 through 4) and angiotensin-II type 1 receptor, but no traces of the Klotho gene were detected. MSCs and satellite cells were treated with FGF-23 and angiotensin-II for 48 h. Treatment with FGF-23 significantly decreased the number of MSCs compared to controls, while treatment with angiotensin-II did not. FGF-23 and angiotensin-II both left the cell counts of the satellite cells unchanged. The FGF-23-treated MSCs exhibited the senescent phenotype, as judged by senescence-associated ß-galactosidase assay, cell morphology, and increased expression of p53 and p21 in western blot analysis. FGF-23 also significantly altered the gene expression of oxidative stress regulators in the cells. In conclusion, FGF-23 induced premature senescence in MSCs from skeletal muscle via the p53/p21/oxidative-stress pathway. The interaction between the MSCs and FGF-23 may play a key role in the impaired muscle reparative mechanisms of chronic kidney disease.

High isolation rate of MDR group B streptococci with reduced penicillin susceptibility in Japan.

OBJECTIVES: In Japan, the isolation rate of group B Streptococcus (GBS) with reduced penicillin susceptibility (PRGBS) was 2.3% between 2005 and 2006. However, no data on this have been available since then; moreover, the isolation rate of MDR-PRGBS has never been reported. The aim of this study was to obtain recent data on the PRGBS isolation rate and to investigate, for the first time, the isolation rate of MDR-PRGBS. METHODS: We collected 306 clinical GBS isolates from various regions in Japan between January 2012 and July 2013. The MICs of penicillin G, oxacillin, ceftizoxime and ceftibuten for all GBS isolates and the MICs of erythromycin and levofloxacin for PRGBS isolates were determined by the agar dilution method. The mutations in the genes involved in antibiotic resistance and antibiotic resistance genes were analysed by PCR and DNA sequencing. RESULTS: We detected 45 clinical PRGBS isolates, with a PRGBS isolation rate among GBS of 14.7% (45/306). Among the PRGBS isolates, 32 (32/45, 71.1%) and 43 (43/45, 95.6%) were resistant/non-susceptible to erythromycin and levofloxacin, respectively. Furthermore, 31 PRGBS isolates, which constituted 68.9% of the PRGBS (31/45) and 10.1% of the GBS (31/306), respectively, were resistant/non-susceptible to both macrolides and fluoroquinolones, indicating multidrug resistance. CONCLUSIONS: These results suggest that the number of clinical PRGBS isolates with a tendency to multidrug resistance increased rapidly between 2005-06 and 2012-13 in Japan.

NADPH oxidase deficiency exacerbates angiotensin II-induced abdominal aortic aneurysms in mice.

OBJECTIVE: Although nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) is reportedly essential for phagocyte host defenses, it has been found to aggravate atherosclerosis in apolipoprotein E (Apoe)-null mice through excess production of superoxide. We therefore assessed the role of NOX2 in an experimental model of abdominal aortic aneurysm (AAA) and assessed the mechanism of NOX2 action in AAA. APPROACH AND RESULTS: AAA was induced in low-density lipoprotein receptor-null (Ldlr(-/-)) mice by infusing angiotensin II. Nox2 expression was elevated in the abdominal aortae of these mice during infusion of angiotensin II, with enhanced Nox2 expression mainly because of the recruitment of NOX2-enriched macrophages into AAA lesions. Unexpectedly, systemic Nox2 deficiency promoted AAA development but reduced the level of reactive oxygen species in AAA lesions. Nox2 deficiency stimulated macrophage conversion toward the M1 subset, enhancing expression of interleukin (IL)-1ß and matrix metalloproteinase-9/12 mRNA. Administration of neutralizing antibody against IL-1ß abolished AAA development in Nox2-deficient mice. Bone marrow transplantation experiments revealed that AAA aggravation by Nox2 deficiency is because of bone marrow-derived cells. Isolated bone marrow-derived macrophages from Nox2-null mice could not generate reactive oxygen species. In contrast, IL-1ß expression in peritoneal and bone marrow-derived macrophages, but not in peritoneal neutrophils, was substantially enhanced by Nox2 deficiency. Pharmacological inhibition of Janus kinase/signal transducers and activators of transcription signaling inhibited excess IL-1ß expression in Nox2-deficient macrophages, whereas matrix metalloproteinase-9 secretion was constitutively stimulated via nuclear factor-κB signals. CONCLUSIONS: Nox2 deficiency enhances macrophage secretion of IL-1ß and matrix metalloproteinase-9, disrupting tissue-remodeling functions in AAA lesions. These actions are unfavorable if NOX2 is to serve as a molecular target for AAA.

Non-significant impact of proteinuria on renal function in Japanese patients with metastatic renal cell carcinoma treated with axitinib.

BACKGROUND: To investigate the impact of proteinuria on the renal function of patients with metastatic renal cell carcinoma (mRCC) who received axitinib, a tyrosine kinase inhibitor specifically targeting vascular endothelial growth factor receptors. METHODS: This study included 65 consecutive Japanese patients who were diagnosed with mRCC and were subsequently treated with axitinib for at least 12 weeks. The association between the changes in the estimated glomerular filtration rate (eGFR) and proteinuria in these 65 patients was retrospectively assessed. RESULTS: Of the 65 patients, 41 (63.1 %) were judged to be positive for proteinuria. There were no significant differences between the eGFR value before the introduction of axitinib and that at the last clinic visit in either group with or without proteinuria. Furthermore, no significant correlation was noted between the changes in eGFR and the urine protein to creatinine ratio in the group positive for proteinuria, and there was no significant effect of the duration of treatment with axitinib on the changes in eGFR in the proteinuria group. Of several factors examined, univariate analysis identified age, eGFR prior to the introduction of axitinib, and timing of axitinib introduction, but not the presence of proteinuria, as predictors of a decrease in eGFR of >10 %; however, only age appeared to be independently associated with a decrease in eGFR of >10 %. CONCLUSIONS: These findings suggest that treatment with axitinib may not have a significant adverse impact on the renal function in patients with mRCC, irrespective of the presence of proteinuria.

Performance status as a significant prognostic predictor in patients with urothelial carcinoma of the bladder who underwent radical cystectomy.

OBJECTIVES: To assess the significance of performance status as a prognostic factor after radical cystectomy for urothelial carcinoma of the bladder. METHODS: The present study included 730 consecutive patients with urothelial carcinoma of the bladder who underwent radical cystectomy. Clinicopathological outcomes in these patients were analyzed focusing on the impact of performance status, which was assessed using the Karnofsky Performance Status scale before surgery. Patients were classified into groups with Karnofsky Performance Status ≥90 and ≤80. RESULTS: A total of 561 (76.8%) and 169 (23.2%) patients were judged to have Karnofsky Performance Status ≥90 and ≤80, respectively. During a mean of 52.0 months, disease recurrence and mortality occurred in 257 (35.2%) and 249 (34.1%) patients, respectively, and the 5-year recurrence-free and overall survival rates were 64.1 and 65.3%, respectively. There were significant differences in age, hemoglobin, albumin, estimated glomerular filtration rate, pathological T stage and nodal involvement between the Karnofsky Performance Status ≥90 and ≤80 groups. Multivariate analysis showed independent impacts of Karnofsky Performance Status, pathological T stage, nodal involvement and lymphovascular invasion on recurrence-free survival, as well as independent impacts of Karnofsky Performance Status, age, body mass index, hemoglobin, pathological T stage, nodal involvement and lymphovascular invasion on overall survival. CONCLUSIONS: The results suggest a significant association between impaired performance status and unfavorable prognosis in patients with urothelial carcinoma of the bladder undergoing radical cystectomy.

Anomalous metallic-like transport of Co-Pd ferromagnetic nanoparticles cross-linked with π-conjugated molecules having a rotational degree of freedom.

We investigated the electron transport in Co-Pd ferromagnetic nanoparticles (Co 16%) cross-linked with oligo(phenyleneethynylene)diethanethiolate, which consists of three rotary phenylene moieties bridged by two acetylene groups, or icosane-1,20-dithiol, which consists of one alkane chain. Although the nanoparticles cross-linked with the alkane dithiols (the latter) have extremely high electrical resistance in electron transport, the resistance of the nanoparticles cross-linked with the conjugated molecules (the former) demonstrates a linear temperature dependence from room temperature to ca. 20 K; below that temperature, it has a weak temperature-dependent residual contribution with a resistance minimum around 7 K. Computational simulations suggest that the apparent metallic-like temperature dependence at high temperatures can be explained in terms of the rotational degree of freedom of the linker molecule. The rotational motion of the constituent phenylene groups, which hinders π-conjugation along the linker molecule, becomes less excited as the temperature is lowered. The successive development of a ballistic transport path through the π-conjugated linker molecule with decreasing temperature yields the metallic-like temperature dependence observed for the bridged nanoparticles. The low-temperature resistance behaviour with a minimum is a consequence of carrier scattering by the localized Co spins of Co-Pd nanoparticles randomly ordered in a ferromagnetic state that develops below the temperature of the resistance minimum.

Time course-changes in phosphatidylcholine profile during oxidative modification of low-density lipoprotein.

BACKGROUND: Oxidized phosphatidylcholines (oxPC) and lysophosphatidylcholine (lysoPC) generated during the formation of oxidized low-density lipoprotein (oxLDL) are involved in atherosclerotic lesion development. We investigated the time course-changes in phosphatidylcholine (PC) molecular species during oxidation of LDL to determine how those atherogenic PCs are produced. METHODS: Human and rabbit LDLs were pretreated with or without a selective platelet-activating factor acetylhydrolase (PAF-AH) inhibitor. LDL was oxidized by incubation with copper sulfate, and PC profiles were analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: When human LDL was oxidized, the peak areas for polyunsaturated fatty acid (PUFA)-containing PC species dramatically decreased after a short lag period, concomitantly lysoPC species increased sharply. Although a variety of oxPC species containing oxidized fatty acyl groups or cleaved acyl chains are formed during LDL oxidation, only a few oxPC products accumulated in oxLDL: 1-palmitoyl-2-(9-oxo-nonanoyl) PC and long-chain oxPC with two double bonds. Pretreatment of LDL with the PAF-AH inhibitor greatly reduced lysoPC production while it had no effect on lipid peroxidation reactions and oxPC profiles. Rabbit LDL, which has a different composition of PC molecular species and needs a longer time to reach achieve full oxidation than human LDL, also accumulated lysoPC during oxidation. The increase in lysoPC in rabbit oxLDL was suppressed by pretreatment with the PAF-AH inhibitor. The major oxPC species formed in rabbit oxLDL were almost the same as human oxLDL. CONCLUSIONS: These results suggest that lysoPC species are the major products and PAF-AH activity is crucial for lysoPC generation during oxidation of LDL. The oxPC species accumulated are limited when LDL is oxidized with copper ion in vitro.

Clinical outcome of paclitaxel and carboplatin as second-line chemotherapy for advanced urothelial carcinoma resistant to first-line therapy with gemcitabine and cisplatin.

OBJECTIVE: The objective was to investigate the efficacy and tolerability of combined therapy with paclitaxel and carboplatin (TC) in patients with advanced urothelial carcinoma after the failure of first-line chemotherapy with gemcitabine and cisplatin (GC). PATIENTS AND METHODS: This was a retrospective study including a total of 16 patients with advanced urothelial carcinoma who showed evidence of progressive and/or recurrent disease after first-line therapy with GC and subsequently received second-line chemotherapy consisting of paclitaxel (175 mg/m(2)) and carboplatin (area under the curve 5). TC therapy was repeated every 3 weeks and was continued until disease progression or intolerable toxicity was observed. RESULTS: The baseline patient characteristics were rather favorable; only 3 of 16 patients had liver metastases and 7 patients had an Eastern Cooperative Oncology Group performance status of 0. Of these, response to TC therapy was achieved in 5 (31.3%), including 2 with complete and 3 with partial response. The median progression-free and overall survival times in the 16 patients were 7.9 and 17.3 months, respectively. CONCLUSIONS: TC therapy appeared to show modest activity with acceptable tolerability in patients refractory to GC therapy; therefore, TC chemotherapy might be considered as an alternative option as a second-line regimen for advanced urothelial carcinoma following GC therapy.

Hybrid molecular systems containing tetrathiafulvalene and iron-alkynyl electrophores: five-component functional molecules obtained from C-H bond activation.

Treatment of [Cp*(dppe)Fe-C≡C-TTFMe3] (1) with Ag[PF6] (3 equiv) in DMF provides the binuclear complex [Cp*(dppe)Fe=C=C=TTFMe2 =CH-CH=TTFMe2 =C=C=Fe(dppe)Cp*][PF6]2 (2[PF6 ]2) isolated as a deep-blue powder in 69 % yield. EPR monitoring of the reaction and comparison of the experimental and calculated EPR spectra allowed the identification of the radical salt [Cp*(dppe)Fe=C=C=TTFMe2 =CH][PF6]2 ([1-CH][PF6]) an intermediate of the reaction, which results from the activation of the methyl group attached in vicinal position with respect to the alkynyl-iron on the TTF ligand by the triple oxidation of 1 leading to its deprotonation by the solvent. The dimerization of [1-CH][PF6] through carbon-carbon bond formation provides 2[PF6]2. The cyclic voltammetry (CV) experiments show that 2[PF6]2 is subject to two sequential well-reversible one-electron reductions yielding the complexes 2[PF6] and 2. The CV also shows that further oxidation of 2[PF6]2 generates 2[PF6]n (n=3-6) at the electrode. Treatment of 2[PF6]2 with KOtBu provides 2[PF6] and 2 as stable powders. The salts 2[PF6] and 2[PF6]2 were characterized by XRD. The electronic structures of 2(n+) (n=0-2) were computed. The new complexes were also characterized by NMR, IR, Mössbauer, EPR, UV/Vis and NIR spectroscopies. The data show that the three complexes 2[PF6]n are iron(II) derivatives in the ground state. In the solid state, the dication 2(2+) is diamagnetic and has a bis(allenylidene-iron) structure with one positive charge on each iron building block. In solution, as a result of the thermal motion of the metal-carbon backbone, the triplet excited state becomes thermally accessible and equilibrium takes place between singlet and triplet states. In 2[PF6], the charge and the spin are both symmetrically distributed on the carbon bridge and only moderately on the iron and TTFMe2 electroactive centers.

Group IVA phospholipase A2 participates in the progression of hepatic fibrosis.

Group IVA phospholipase A2 (IVA-PLA2) is an enzyme that intiates the arachidonic acid pathway and plays an important role in inflammation. We demonstrate that IVA-PLA2 deficiency suppresses lipid deposition in the liver, which was induced by administration of a high-fat and -cholesterol diet (HFCD) for 16 wk in mice. Herein, we performed 2-dimensional gel-based comparative proteomics to further define the suppressive effect of IVA-PLA2 deficiency on fatty liver formation. In comparisons among 4 groups, wild-type (WT)/normal diet (ND), IVA-PLA2-deficient knockout (KO)/ND, WT/HFCD, and KO/HFCD, 4 proteins, 3 of which are associated with hepatic fibrosis, were identified as molecules, of which altered expression by HFCD was suppressed in KO mice compared to WT mice. Therefore, we assessed the effect of IVA-PLA2 deficiency on hepatic fibrosis induced by HFCD or carbon tetrachloride (CCl4) in mouse models. Biochemical and histological analyses revealed that IVA-PLA2 deficiency markedly reduced overall collagen accumulation in the liver of HFCD- and CCl4-derived mouse models. We found that IVA-PLA2 deficiency prevented activation of hepatic stellate cells and infiltration of F4/80-positive macrophages without affecting other immunocytes such as CD8+ lymphocytes and natural killer cells. In summary, IVA-PLA2 deficiency attenuates not only lipid deposition in the liver but also hepatic fibrosis formation.

Canna starch improves immune functions and the intestinal environment in mice.

The present study was conducted to elucidate the dietary effects of canna starch on the immune functions and intestinal luminal environment in mice. The amylose and resistant starch characteristics were determined for six types of starch, including edible canna. Canna starch was found to be higher in amylose and resistant starch compared with the other starches. BALB/c mice were fed 3.16% (low-canna group) and 6.32% (high-canna group) canna starch for 2 weeks, and then intestinal parameters were measured. Fecal IgA and mucin levels were markedly elevated by canna starch intake. IgA levels in serum and spleen lymphocytes were elevated by canna starch intake in the high-canna group, but not in the low-canna group. When the mice were fed canna starch, the cecum weight increased, and the pH in the cecum decreased. The high-canna group had significantly increased levels of Clostridium subcluster XIVa lactic acid, acetic acid, and n-butyric acid in the cecum compared with the control group. These results suggested that canna starch supplementation changed the intestinal microbiota and enhanced the intestinal immune and barrier functions and cecal organic acids in mice.