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Clarifying the factors related to decreased physical activity in post-stroke patients is essential for effective disease management. This study aimed to examine the factors influencing the amount of daily steps taken by post-stroke patients in a convalescent rehabilitation ward during activities other than rehabilitation (non-rehabilitation steps).Eighty-nine post-stroke patients (60.8±14.4 years; 55 men) were enrolled. The inclusion criteria were walking independently within the ward and having a walking speed of ≥24 m/min. Data on patient clinical characteristics including age, sex, body mass index, stroke type, hemiparetic side, and time from stroke onset were collected. Stroke impairment and motor and cognitive functional disabilities were assessed using the Stroke Impairment Assessment Set and the Functional Independence Measure, respectively. The non-rehabilitation steps were calculated by subtracting the steps during the rehabilitation activities from the total steps using Fitbit Flex2.The average number of non-rehabilitation steps was 4,523±2,339 steps/day. The hierarchical multiple regression analysis revealed that sex, motor disability, and the interaction term of stroke impairment with cognitive disability were significantly related to non-rehabilitation steps. Simple slope analysis demonstrated that the stroke impairment slope was steeper at lower levels than at higher levels of cognitive disability for non-rehabilitation steps.In addition to independent effects of sex and motor disability, this study found that stroke impairment and cognitive disability were interactively related to non-rehabilitation steps in post-stroke patients in a convalescent rehabilitation ward. These findings may provide useful information for managing physical activity in post-stroke patients after hospital discharge.

Adipose-derived stem cells (ADSCs) and the stromal vascular fraction (SVF) promote nerve regeneration. Biodegradable nerve conduits are used to treat peripheral nerve injuries, but their efficiencies are lower than those of autologous nerve grafts. This study developed biodegradable nerve conduits containing ADSCs and SVF and evaluated their facial nerve regenerating abilities in a rat model with a 7-mm nerve defect. SVF and ADSCs were individually poured into nerve conduits with polyglycolic acid-type I collagen as a scaffold (ADSCs and SVF groups). The conduits were grafted on to the nerve defects. As the control, the defect was bridged with polyglycolic acid-collagen nerve conduits without cells. At 13 weeks, after transplantation, the regenerated nerves were evaluated physiologically and histologically. The compound muscle action potential of the SVF group was significantly higher in amplitude than that of the control group. Electron microscopy showed that the axon diameter of the SVF group was the largest, followed by the ADSC group and control group with significant differences among them. The SVF group had the largest fiber diameter, followed by the ADSC group and control group with significant differences among them. The ADSC group had the highest myelin thickness, followed by the SVF group and control group with significant differences among them. Identical excellent promoting effects on nerve regeneration were observed in both the ADSC and SVF groups. Using SVF in conduits was more practical than using ADSCs because only the enzymatic process was required to prepare SVF, indicating that SVF could be more suitable to induce nerve regeneration.

Aldehyde dehydrogenase (ALDH) is an enzyme that plays an important role in retinoid metabolism and highly expressed in stem cells. This study isolated ALDH-expressing cells from subcutaneous adipose tissue and investigated their potential to enhance healing in a full-thickness skin wound in rats by co-implanting them with collagen-glycosaminoglycan (c-GAG) scaffolds. ALDH-positive cells were isolated by a fluorescence-activated cell sorting (FACS) technique from Lewis rat's Stromal-Vascular-Fraction (SVF) and transplanted with c-GAG scaffolds in a rat full-thickness skin wound model. At seven days after surgery, the microscopic appearance of c-GAG scaffolds seeded with ALDH-positive was compared with those of uncultured-SVF, and cultured- adipose-derived stromal cells (ASCs). The thickness of cellular ingrowth in the ASC group (630 ± 180 μm) was significantly thicker than that in the control (390 ± 120 μm) or SVF (380 ± 140 μm) groups, but non-significantly thicker than that in the ALDH-positive group (570 ± 220 μm). The thickness of regenerated collagen layer was significantly thicker in the ALDH-positive group (160 ± 110 μm) than in the ASCs (81 ± 41 μm), the control (65 ± 24 μm), or SVF (64 ± 34 μm) groups. Immunofluorescent staining with CD31 proved that transplanted ALDH-positive cells differentiated into vascular endothelial cells in c-GAG scaffolds. Combined transplantation with c-GAG scaffolds and adipose-derived ALDH-positive cells promoted dermal regeneration, giving a possibility that ALDH-positive cells would greatly shorten the waiting period before secondary autologous skin grafting was possible. This article is protected by copyright. All rights reserved.

We aimed to examine the rate of force development (RFD) of knee extensors on both sides in independently ambulant patients with acute stroke with mild paresis compared with that in age-matched healthy adults. A total 31 patients with acute stroke history (patient group: 67 ± 12 years) and 54 age-matched healthy, community-dwelling adults (control group: 67 ± 8 years) were included. Maximum voluntary contraction (MVC) and RFD were assessed1 month post-stroke during isometric knee extension (sitting position; 90° knee flexion) using a hand-held dynamometer. RFD was measured as the average slope of the torque-time curve over time intervals of 0-50 ms and 0-200 ms from contraction onset. In the patient group, MVC and RFD for 0-50 ms were significantly lower on the affected side than on the unaffected side (p 0.01). RFD was significantly decreased in the patient group, to 32%-38% and 62%-71% of that in the control group, over 0-50 ms and 0-200 ms, respectively, regardless of the affected side (p 0.01). No significant differences in MVC between patient and control groups were observed for either side. RFD of the knee extensors significantly decreased without MVC reduction in patients with acute stroke history compared with that in age-matched healthy adults in both the affected and unaffected sides. These results suggest that decrease in RFD was initiated from the acute phase of stroke, even in patients with stroke who had good motor function.

PURPOSE Interpositional jump-graft (IPJG) technique with the hypoglossal nerve for supercharging can be applied in a facial nerve paresis case. In IPJG, an autologous nerve is required, and the donor site morbidity is unavoidable. Biodegradable nerve conduits are made from polyglycolic acid (PGA) and used recently without donor site complications after providing autologous grafts. Hybrid artificial nerve conduits with adipose-derived stem cells (ASCs) also attract attention as a nerve-regeneration enhancing agent. This study examined the effect of hybrid artificial nerve conduit on IPJG. MATERIALS AND METHODS A total of 34 Lewis rats were used and divided into 4 groups by the bridge materials: autograft (n = 8), PGA nerve conduit (n = 8), hybrid PGA nerve conduit with ASCs (n = 8), and the nontreated control groups (n = 8). ASCs were collected from 2 rats and cultured. The animals were assessed physiologically and histopathologically at 13 weeks after surgery. RESULTS In compound muscle action potential, the amplitude of hybrid PGA group (3,222 ± 1,779 μV) was significantly higher than that of PGA group (1,961 ± 445 μV, P

PURPOSE This study investigated the potential of collagen-coated polyglycolic acid (PGA) tube with interpositional jump graft (IPJG) in rat. MATERIALS AND METHODS A total of 16 Lewis rats were used in this study. Facial nerve paralysis was created by ligating facial nerve trunk with a ligature clip. The rats were divided into 3 groups. Nerve conduit group (n = 6) were treated by IPJG with collagen-coated PGA tubes between the facial nerve trunks and the hypoglossal nerves. Autograft group (n = 6) were treated by IPJG with the greater auricular nerves. As the control group (n = 4), non-treated-model rats with facial nerve paralysis were used. The number of myelinated fibers, fiber diameter, axon diameter, myelin thickness, and g-ratio, were analyzed histologically at 13 weeks after surgery. Compound muscle action potential (CMAP) and retrograde tracing were measured. RESULT Although the number of myelinated fibers in autograft group (1957 ± 775) had significantly higher than that of nerve conduit group (90 ± 41, P

Elevated reactive oxygen species (ROS) and oxidative damage occur in the red blood cells (RBCs) of SOD1-deficient C57BL/6 mice. This leads to autoimmune responses against RBCs in aged mice that are similar to autoimmune hemolytic anemia (AIHA). We examined whether a SOD1 deficiency and/or the human SOD1 transgene (hSOD1) would affect phenotypes of AIHA-prone New Zealand Black (NZB) mice by establishing three congenic strains: those lacking SOD1, those expressing hSOD1 under a GATA-1 promoter, and those lacking mouse SOD1 but expressing hSOD1. Levels of intracellular ROS and oxidative stress markers increased, and the severity of the AIHA phenotype was aggravated by a SOD1 deficiency. In contrast, the transgenic expression of hSOD1 in an erythroid cell-specific manner averted most of the AIHA phenotype evident in the SOD1-deficient mice and also ameliorated the AIHA phenotype in the mice possessing intrinsic SOD1. These data suggest that oxidative stress in RBCs may be an underlying mechanism for autoimmune responses in NZB mice. These results were consistent with the hypothetical role of reactive oxygen species in triggering the autoimmune reaction in RBCs and may provide a novel approach to mitigating the progression of AIHA by reducing oxidative stress.

Superoxide dismutase (SOD) plays a role in antioxidation, and SOD1-knockout (KO) mice show moderate phenotypes. Primary cultured mouse embryonic fibroblasts (MEFs) lead to growth failure and eventual death under normoxic culture (20% oxygen). We attempted to elucidate the molecular mechanisms responsible for the oxygen toxicity in SOD1-KO MEFs. Increases in reactive oxygen species, lipid peroxidation products, and senescence-associated β-galactosidase activity were observed in SOD1-KO MEFs. Hypoxic culture (2% oxygen) averted immediate cell death but could not recover the proliferative ability of the SOD1-KO cells. The cell cycles of SOD1-deficient MEFs were arrested at the G2 and M phases, leading to the accumulation of tetraploid cells under hypoxic culture. The suppressed expression of cyclin A2 and B1 and the concomitant induction of p21(Waf1) were evident in SOD1-KO cells. The phosphorylation of p53 and histone H2Ax and the induction of the two proapoptotic genes Bax and Noxa were evident in SOD1-deficient MEFs and more enhanced under normoxic culture than under hypoxic culture. We concluded that low levels of oxygen consumption moderately activates the p53 pathway, and leads to cellular senescence, but that high levels of oxygen consumption hyperactivates the p53 pathway, which results in cell death in SOD1-deficient MEFs.

The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.

Superoxide dismutase 1 (SOD1) is an important antioxidative enzyme that protects skin from oxidative stress. SOD1 (-/-) mice with a genetic background of b129Sv mice showed facial skin damage after 15 weeks of age. Eyelid swelling occurred as the initial symptom and caused impairment by triggering self-scratching. The period required for wound healing in the back was markedly delayed in 20-week SOD1 (-/-) mice. Oxidative stress markers, 4-hydroxynonenal and thiobarbituric acid-reactive substances, were unexpectedly lower in SOD1 (-/-) mice at day 1 after wounding. The decay rate of electron paramagnetic resonance signal intensity of intravenously injected nitroxide radical indicated that the half-life of the signal intensity was significantly prolonged in the wounded skin of SOD1 (+/+) mice. However, while the half-life of the signal intensity in control skin was a little longer in SOD1 (-/-) mice, it did not change in wounded skin. Taken together, these data suggest that the skin of SOD1 (-/-) mice is in redox imbalance and prone to damage by wounding.

Oxidative stress has been implicated as a cause of various diseases such as anaemia. We found that the SOD1 [Cu,Zn-SOD (superoxide dismutase)] gene deficiency causes anaemia, the production of autoantibodies against RBCs (red blood cells) and renal damage. In the present study, to further understand the role of oxidative stress in the autoimmune response triggered by SOD1 deficiency, we generated mice that had the hSOD1 (human SOD1) transgene under regulation of the GATA-1 promoter, and bred the transgene onto the SOD1(-/-) background (SOD1(-/-);hSOD1(tg/+)). The lifespan of RBCs, levels of intracellular reactive oxygen species, and RBC content in SOD1(-/-);hSOD1(tg/+) mice, were approximately equivalent to those of SOD1(+/+) mice. The production of antibodies against lipid peroxidation products, 4-hydroxy-2-nonenal and acrolein, as well as autoantibodies against RBCs and carbonic anhydrase II were elevated in the SOD1(-/-) mice, but were suppressed in the SOD1(-/-);hSOD1(tg/+) mice. Renal function, as judged by blood urea nitrogen, was improved in the transgenic mice. These results rule out the involvement of a defective immune system in the autoimmune response of SOD1-deficient mice, because SOD1(-/-);hSOD1(tg/+) mice carry the hSOD1 protein only in RBCs. Metabolomic analysis indicated a shift in glucose metabolism to the pentose phosphate pathway and a decrease in the energy charge potential of RBCs in SOD1-deficient mice. We conclude that the increase in reactive oxygen species due to SOD1 deficiency accelerates RBC destruction by affecting carbon metabolism and increasing oxidative modification of lipids and proteins. The resulting oxidation products are antigenic and, consequently, trigger autoantibody production, leading to autoimmune responses.

Prx (peroxiredoxin) is a multifunctional redox protein with thioredoxin-dependent peroxidase activity. Prx4 is present as a secretory protein in most tissues, whereas in sexually mature testes it is anchored in the ER (endoplasmic reticulum) membrane of spermatogenic cells via an uncleaved N-terminal hydrophobic peptide. We generated a Prx4 knockout mouse to investigate the function of Prx4 in vivo . Prx4 −/ y mice lacking Prx4 expression in all cells were obtained by mating Prx4 flox /+ female mice with Cre-transgenic male mice that ubiquitously expressed Cre recombinase. The resulting Prx4 −/ y male mice were fertile, and most organs were nearly normal in size, except for testicular atrophy. The number of deoxynucleotidyl transferase-mediated dUTP nick end labelling-positive spermatogenic cells was higher in Prx4 −/ y mice than in Prx4 +/ y mice and increased remarkably in response to warming the lower abdomen at 43 °C for 15 min. Cells reactive to antibodies against 4-hydroxynonenal and 8-hydroxyguanine were high in the Prx4 −/ y mice and concomitant with elevated oxidation of lipid and protein thiols. The cauda epididymis of Prx4 −/ y mice contained round spermatocytes, which were not found in Prx4 +/ y mice, and displayed oligozoospermia. However, mature spermatozoa from the epididymis of Prx4 −/ y mice exhibited normal fertilization In vitro . Taken together, these results indicate that spermatogenic cells lacking Prx4 are more susceptible to cell death via oxidative damage than their wild-type counterparts. Our results suggest that the presence of Prx4, most likely the membrane-bound form, is important for spermatogenesis, but not an absolute requisite.

Because glutathione scavenges reactive oxygen species (ROS) and also donates electrons to antioxidative systems, it may compensate for the oxidative stress caused by SOD1 deficiency. The cystine/glutamate transporter, which consists of two proteins, xCT and 4F2hc, has been designated system x (c) (-) . This transporter system plays a role in the maintenance of glutathione levels in mammalian cells. In the present study, we created SOD1 (-/-); xCT (-/-) double-knockout mice by intercrossing xCT-knockout and SOD1-knockout animals. We determined if the double-knockout mice express the phenotypic characteristics unique to SOD1 (-/-) mice-increased oxidative stress and the production of autoantibodies against erythrocytes. We also compared the phenotype of the double-knockout mice with those of the single-knockout and wild-type mice. Although two major antioxidative systems were found to be defective in the SOD1 (-/-); xCT (-/-) mice, relative to the SOD1 (-/-) mice, no functional deficits were observed. Based on these results, it appears that defects in system x (c) (-) do not exacerbate the phenotypic consequences of SOD1 deficiency in postnatal mice under ordinary breeding conditions.

Channelrhodopsins (CHR1 and CHR2) are light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. In neuroscience, they are used to trigger action potentials by light in neuronal cells, tissues, or living animals. Here, we demonstrate that Chlamydomonas cells with low CHR2 content exhibit photophobic and phototactic responses that strictly depend on the availability of CHR1. Since CHR1 was described as a H+-channel, the ion specificity of CHR1 was reinvestigated in Xenopus laevis oocytes. Our experiments show that, in addition to H+, CHR1 also conducts Na+, K+, and Ca2+. The kinetic selectivity analysis demonstrates that H+ selectivity is not due to specific translocation but due to selective ion binding. Purified recombinant CHR1 consists of two isoforms with different absorption maxima, CHR1505 and CHR1463, that are in pH-dependent equilibrium. Thus, CHR1 is a photochromic and protochromic sensory photoreceptor that functions as a light-activated cation channel mediating phototactic and photophobic responses via depolarizing currents in a wide range of ionic conditions.

ErbB3 receptor tyrosine kinase has been shown to induce tumor progression in several types of cancer through heterodimerization with ErbB2. However, the role of ErbB3 and its ligand heregulin (HRG) in tumor metastasis remains poorly understood. In the present study, we tried to clarify their contributions to the metastasis of ErbB3-overexpressing B16-BL6 melanoma cells. Stimulation with HRG induced phosphorylation of ErbB3 and metastatic properties including MMP-9 expression, invasion, adhesion and experimental lung metastasis in vivo. These cellular responses were blocked by inhibiting the tyrosine kinase activity of EGFR with PD153035. In addition, phosphorylation of EGFR was rapidly induced by HRG, suggesting that EGFR is a possible heterodimeric counterpart of ErbB3. RNA interference demonstrated that subcutaneous tumor growth and angiogenesis was attenuated by inactivation of ErbB3 in cancer cells. Although experimental pulmonary metastasis was not affected by the knockdown of ErbB3, spontaneous metastasis was, even when primary tumors in the foot pad were amputated at a similar size. These results indicate that HRG-induced activation of ErbB3 via EGFR promotes tumor growth and metastasis of melanoma cells.

An influence of content of Ni and Ag in a Sn-Ag-Cu-Ni-Ge lead-free solder has been investigated on microstructure and joint strength of the soldered joint under heat exposure conditions. The growth kinetics of the reaction layer formed at the joint interface has been investigated, and the apparent activation energy of the reaction layer growth has been also examined. Moreover, the soldered joints with Sn-Ag and Sn-Ag-Cu solders were prepared and were compared with the joints with the Sn-Ag-Cu-Ni-Ge solders.

Fibroblast growth factor (FGF)-2 has been considered to play a critical role in neovascularization in several tumors; however, its precise role in tumor progression is not fully understood. In the present study, we have characterized the role of FGF-2 in B16-BL6 mouse melanoma cells, focusing on effects during the initial phase of tumor growth. FGF-2 was injected at the tumor inoculation site of dorsal skin during the initial phase. FGF-2 induced marked tumor growth and lymph node metastasis. This was well correlated with an increase in neovascularization in the host stroma. FGF-2 also recruited inflammatory and mesenchymal cells in host stroma. Marked tumor growth, pulmonary metastasis and intensive neovascularization in tumor parenchyma were also observed after a single injection of FGF-2 into the footpad inoculation site. In contrast, repeated injections of FGF-2 at a site remote from the footpad tumor were ineffective in promoting tumor growth and metastasis. These promoting activities of FGF-2 were blocked by local injections of a glucocorticoid hormone, suggesting that host inflammatory responses induced by FGF-2 are associated with FGF-2-induced tumor progression. In addition, although FGF-2 did not promote cellular proliferation and vascular endothelial growth factor A (VEGFA) mRNA expression in B16-BL6 cells in vitro, FGF-2 induced VEGFA expression in host stroma rather than tumor tissue, and local injections of a neutralizing antibody against VEGFA inhibited these activities of FGF-2 in vivo. These results indicate that abundant FGF-2 during the initial phase of tumor growth induces VEGFA-dependent intensive neovascularization in host stroma, and supports marked tumor growth and metastasis.

Accumulation of pathological tau protein is a major hallmark of Alzheimer’s disease. Tau protein spreads from the entorhinal cortex to the hippocampal region early in the disease. Microglia, the primary phagocytes in the brain, are positively correlated with tau pathology, but their involvement in tau propagation is unknown. We developed an adeno-associated virus–based model exhibiting rapid tau propagation from the entorhinal cortex to the dentate gyrus in 4 weeks. We found that depleting microglia dramatically suppressed the propagation of tau and reduced excitability in the dentate gyrus in this mouse model. Moreover, we demonstrate that microglia spread tau via exosome secretion, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo. These data suggest that microglia and exosomes contribute to the progression of tauopathy and that the exosome secretion pathway may be a therapeutic target.

The reliability of a solder ball joint with a Sn-Ag-Cu-Ni-Ge lead-free alloy, which is expected to be an advanced lead-free solder, was investigated under heat exposure conditions. Solder ball joints with a eutectic Sn-Ag alloy and a ternary Sn-Ag-Cu alloy were also prepared to compare with that of the Sn-Ag-Cu-Ni-Ge alloy. Microstructual observations of the cross sections of the solder ball joints were conducted to investigate microstructural evolutions in the solders and the growth kinetics of reaction layers formed at joint interfaces. The influence of heat exposure treatment on joint strength was investigated by ball shear test. Moreover, the influence of surface treatment of a Cu pad on the reliability of the solder joint was also investigated.

The oxidative modification of gametes by a reactive oxygen species is a major deleterious factor that decreases the successful rate of in vitro fertilization. Superoxide dismutase 1 (SOD1) plays a pivotal role in antioxidation by scavenging the superoxide anion, and its deficiency causes infertility in female mice, but the significance of the enzyme in male mice remains unclear. In the present study, we characterized Sod1(-/-) (Sod1-KO) male reproductive organs and compiled the first report of the impaired fertilizing ability of Sod1-KO sperm in in vitro fertilization. Insemination of wild-type oocytes with Sod1-KO sperm exhibited lower rates of fertility compared with insemination by wild-type sperm. The low fertilizing ability found for Sod1-KO sperm was partially rescued by reductant 2-mercaptoethanol, which suggested the oxidative modification of sperm components. The numbers of motile and progressive sperm decreased during the in vitro fertilization process, and a decline in ATP content and elevation in lipid peroxidation occurred in the Sod1-KO sperm in an incubation time-dependent manner. Tyrosine phosphorylation, which is a hallmark for sperm capacitation, was also impaired in the Sod1-KO sperm. These results collectively suggest that machinery involved in sperm capacitation and motility are vulnerable to oxidative damage during the in vitro fertilization process, which could increase the rate of inefficient fertilization.

The oxidative modification of gametes by a reactive oxygen species is a major deleterious factor that decreases the successful rate of in vitro fertilization. Superoxide dismutase 1 (SOD1) plays a pivotal role in antioxidation by scavenging the superoxide anion, and its deficiency causes infertility in female mice, but the significance of the enzyme in male mice remains unclear. In the present study, we characterized Sod1(-/-) (Sod1-KO) male reproductive organs and compiled the first report of the impaired fertilizing ability of Sod1-KO sperm in in vitro fertilization. Insemination of wild-type oocytes with Sod1-KO sperm exhibited lower rates of fertility compared with insemination by wild-type sperm. The low fertilizing ability found for Sod1-KO sperm was partially rescued by reductant 2-mercaptoethanol, which suggested the oxidative modification of sperm components. The numbers of motile and progressive sperm decreased during the in vitro fertilization process, and a decline in ATP content and elevation in lipid peroxidation occurred in the Sod1-KO sperm in an incubation time-dependent manner. Tyrosine phosphorylation, which is a hallmark for sperm capacitation, was also impaired in the Sod1-KO sperm. These results collectively suggest that machinery involved in sperm capacitation and motility are vulnerable to oxidative damage during the in vitro fertilization process, which could increase the rate of inefficient fertilization.

Oxidative stress characterized by elevated reactive oxygen species is a well-known cause of developmental arrest and cellular fragmentation in the development of in vitro-produced embryos. To investigate the effects of intrinsic oxidative stress on the early development of embryos, oocytes from superoxide dismutase 1 (SOD1)-deficient mice resulting from in vitro fertilization, followed by culture for 4 days, were examined. Development of all embryos from SOD1-deficient oocytes was arrested at the 2-cell stage under conventional culture conditions with atmospheric oxygen (20% O(2)). Significantly higher levels of superoxide were detected in SOD1-deficinet embryos cultured under 20% O(2) using dihydroethidium. Among treatments with antioxidants, only hypoxic culture with 1% O(2) negated the 2-cell arrest and advanced the development of the embryos with efficacy similar to that in wild-type embryos. Mitochondrial function was investigated because its malfunction was a suspected cause of 2-cell arrest. However, respiratory activity, ATP content and mitochondrial membrane potential in the 2-cell embryos were not markedly affected by culture with 20% O(2). When embryos from SOD1-deficient oocytes were first developed to the 4-cell stage under 1% O(2) culture and were then transferred to 20% O(2), most of them developed to the morula stage but underwent total degeneration thereafter. Thus, oxidative stress was found to damage embryos differentially, depending on the developmental stage. These results suggest that embryos derived from SOD1-deficient mouse oocytes are an ideal model to investigate intrinsic oxidative stress-induced developmental abnormality.

We have recently shown that deficiency of the superoxide dismutase 1 gene (SOD1) causes anemia and autoimmune responses against red blood cells (RBCs) and that transgenic expression of human SOD1 in erythroid cells rescues them. Because these phenotypes observed in SOD1-deficient mice are similar to autoimmune hemolytic anemia (AIHA), a causal connection between reactive oxygen species (ROS) and AIHA was examined using an AIHA-prone New Zealand Black (NZB) mouse. ROS levels in RBCs were high in young NZB mice, compared to control New Zealand White (NZW) mice, and increased during aging. Methemoglobin and lipid peroxidation products were elevated during aging, consistent with the elevated oxidative stress in RBCs of NZB mice. Severity of anemia and levels of intracellular ROS were positively correlated. Levels of antibodies against 4-hydroxynonenal and acrolein were also elevated in NZB mice. Transgenic expression of human SOD1 protein in RBCs of NZB mice suppressed ROS in RBCs and decreased the death rate. When RBCs from C57BL/6 mice were injected weekly into the same strain of mice, production of anti-RBC antibody was observed only in mice that had been injected with oxidized RBCs. Thus, oxidation-mediated autoantibody production may be a more general mechanism for AIHA and related autoimmune diseases.

Inflammation in the tumor stroma greatly influences tumor development. In the present study, we investigated the roles of fibroblast growth factor (FGF)-2-induced chronic inflammation in the development of 4T1 murine mammary tumors. Administration of FGF-2 into the tumor inoculation site during the initial phase of tumor growth enhanced tumor growth and pulmonary metastasis as well as microvessel density in tumor tissues in normal but not in nude mice. Infiltration of T lymphocytes and macrophages, recruitment of pericytes/vascular mural cells in neovascular walls, and the expression levels of cyclooxygenase (COX)-2 and vascular endothelial growth factor A (VEGFA) were also enhanced in the FGF-2-activated host stroma of normal mice. In addition, FGF-2-induced tumor growth and metastasis was abrogated by administration of either an immunosuppressant, FK506, or a COX-2 inhibitor. FGF-2 enhanced prostaglandin E(2) secretion in cultured T lymphocytes. In addition, VEGFA secretion was increased in a co-culture of T lymphocytes and fibroblasts in vitro. These results indicate that the massive infiltration of T lymphocytes into FGF-2-activated host stroma during the initial phase of tumor growth enhances neovascular stability by regulating endogenous COX-2 and VEGFA levels because both compounds are known to play important roles in marked 4T1 mammary tumor development via FGF-2-induced inflammatory reactions.

NP506, the 3-{2,4-dimethyl-5-[2-oxo-5-(N′-phenylhydrazinocarbonyl)-1,2-dihydro-indol-3-ylidenemethyl]-1H-pyrrol-3-yl}-propionic acid, was designed as FGF receptor 1 inhibitor by computational study and found to be more active against endothelial proliferation of HUVEC after the rhFGF-2 stimulation than SU6668 with minimum effective dose of 10 μM. NP506 inhibited the tyrosine phosphorylation in FGF, VEGF, and PDGF receptors and the activation of extracellular signal-regulated kinase (ERK), c-Jun-N-terminal-kinase (JNK) and AKT after the rhFGF-2 stimulation. The introduction of the phenyl hydrazide motif to the position 5 of the pyrido[2,3-d]pyrimidine scaffold led to the inhibitory effect in two signaling pathways: inhibition of AKT activation in the phosphatidyl inositol 3′-kinase (PI13K)/AKT signaling pathway and the inhibition of ERK and JNK activation in MAPK pathway.

ErbB3 receptor tyrosine kinase has been shown to induce tumor progression in several types of cancer through heterodimerization with ErbB2. However, the role of ErbB3 and its ligand heregulin (HRG) in tumor metastasis remains poorly understood. In the present study, we tried to clarify their contributions to the metastasis of ErbB3-overexpressing B16-BL6 melanoma cells. Stimulation with HRG induced phosphorylation of ErbB3 and metastatic properties including MMP-9 expression, invasion, adhesion and experimental lung metastasis in vivo. These cellular responses were blocked by inhibiting the tyrosine kinase activity of EGFR with PD153035. In addition, phosphorylation of EGFR was rapidly induced by HRG, suggesting that EGFR is a possible heterodimeric counterpart of ErbB3. RNA interference demonstrated that subcutaneous tumor growth and angiogenesis was attenuated by inactivation of ErbB3 in cancer cells. Although experimental pulmonary metastasis was not affected by the knockdown of ErbB3, spontaneous metastasis was, even when primary tumors in the foot pad were amputated at a similar size. These results indicate that HRG-induced activation of ErbB3 via EGFR promotes tumor growth and metastasis of melanoma cells.

NP603, the 6-dimethoxy phenyl indolin-2-one, was designed as FGF receptor 1 inhibitor by computational study. NP603 was synthesized and found to be more active against endothelial proliferation of HUVEC after the rhFGF-2 stimulation than SU6668 with minimum effective dose of 0.4 microM but with similar potency as SU16g. NP603 inhibited the tyrosine phosphorylation in FGF receptor and the activation of extracellular signal-regulated kinase and c-Jun-N-terminal-kinase after the rhFGF-2 stimulation. The increase in activity of NP603 supports the role of Lys514 movement in ligand-receptor binding in modeling study as the movement accommodates the hydrophobic interaction at the receptor pocket leading to the enhancement of binding capacity.

Transforming growth factor-alpha (TGF alpha) and epidermal growth factor receptor (EGF-R) mRNAs were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) in the normal and neoplastic mammary glands of four strains of mice with different mammary tumor potentials (from highest to lowest potential): SHN, GR/A, SLN and C3H/He. At 2 months of age, when the mammary glands of these strains consisted mostly of normal tissue, the samples examined showed the positive expressions of both TGF alpha and EGF-R mRNAs in all strains (4-6 mice per group), except for EGF-R mRNA in the SLN mice, expressed in only 2 of 4 samples associated with no end-bud formation in the mammary glands. At 10 months, all of the samples from all four strains had a positive expression of TGF alpha mRNA. The EGF-R mRNA expression paralleled the degree of the formation of preneoplastic hyperplastic alveolar nodules (HAN) in all strains. These findings indicate that TGF alpha and EGF-R participate in the growth of the mammary glands, and that EGF-R especially contributes to the formation of end-buds at younger ages and to that of preneoplastic HAN at later ages. All of the samples of mammary tumors from four strains had positive expressions of both TGF alpha and EGF-R mRNAs.

Pregnancy-dependent mammary tumors (PDMT) of GR/A mice and transplantable PDMT (TPDMT-4 line) in DDD mice, are exceptionally stable in hormone dependence, continue to grow until parturition and regress soon after delivery. In order to study the regression mechanism of PDMT and TPDMT-4, morphological and biochemical changes were examined in the tumors removed on day 18 (TPDMT-4) or day 20 (PDMT) of pregnancy, and on the expected parturient and the following postpartum days. DNA fragmentation occurred from day 18 (TPDMT-4) or day 20 (PDMT) of pregnancy to the day after parturition. Apoptotic cells were demonstrated by an in situ 3'-end labeling method, and the plateau of the number of apoptotic cells was observed on the parturient day in PDMT and on the day after parturition in TPDMT-4. Reverse transcriptase polymerase chain reaction showed that expression of Fas was slightly increased but that of bcl-2 was decreased during the process of involution of TPDMT-4 and PDMT. These results suggest that both an increase in expression of Fas and decrease in expression of bcl-2 are involved in the apoptosis of pregnancy-dependent mammary tumor cells after parturition.

In the experiment of mouse transforming growth factor alpha (TGF alpha) gene expression in mammary tumors, various sizes of amplified products by reverse transcriptase-polymerase chain reaction (RT-PCR) using mouse TGF alpha primers were detected in addition to a predicted size in four strains of mice. During the further analysis of these RT-PCR products in mouse mammary tumors, the transcript of neurocan gene was detected in the mammary tumor from SHN mice by the cloning and nucleotide sequence analysis after RT-PCR reaction using mouse TGF alpha primers. The 5'-nucleotide sequence of sequential 246bp in the amplified cDNA of 527bp was completely identical to a middle part of mouse neurocan cDNA sequence, one of the chondroitin-sulfate proteoglycan expressed in the nervous tissue.

We compared lipid metabolism in the intestines of Sod1-knockout mice with that found in wild-type mice to elucidate the impact of oxidative stress in vivo. A high-fat diet in wild-type mice induced postprandial hypertriglyceridemia, but this adaptive response was impaired in Sod1-knockout mice. While fewer triglycerides were secreted to the blood in the form of triglyceride-rich lipoprotein, more lipid droplets accumulated in the enterocytes of Sod1-knockout mice fed a high-fat diet. These data collectively suggest that high-fat diet induces oxidative stress, inhibits lipid secretion to the blood, and ultimately leads to dysfunctional lipid metabolism in enterocytes.