Showing papers by "Kagawa University published in 2009"

Pharmacokinetics of bevacizumab after topical, subconjunctival, and intravitreal administration in rabbits.

Abstract: Purpose To investigate the pharmacokinetics of bevacizumab in rabbits for three different routes of administrations: intravitreal injection, subconjunctival injection, and eye drops. Methods Pigmented rabbits received bevacizumab in one eye by topical eye drops (1.25 mg/0.05 mL six times daily for the first 7 days), single subconjunctival injection (1.25 mg/0.05 mL), or single intravitreal injection (1.25 mg/0.05 mL). Bevacizumab concentrations in plasma and ocular tissues in the treated and fellow eyes were determined by sandwich enzyme-linked immunosorbent assay at 1, 2, 4, and 12 weeks after administration. Results After intravitreal injection in the treated eye, the mean maximum concentrations (C(max)) of bevacizumab in the iris/ciliary body and retina/choroid were 109,192.6, and 93,990.0 ng/g, respectively, whereas after subconjunctival injection, the C(max) was 1418.7 and 295.8 ng/g, respectively. In the fellow eyes, when the drug was administered by intravitreal injection, the C(max) was 753.6 ng/g in the iris/ciliary body and 224.2 ng/g in the retina/choroid and by subconjunctival injection was 1192.9 and 187.0 ng/g, respectively. With eye drops, only a small level of bevacizumab was detected in the iris/ciliary body and retina/choroid. Systemic exposure to bevacizumab was at the same level when administered by intravitreal or subconjunctival injection. Conclusions Intravitreal injection of bevacizumab was the most effective route of administration for intraocular tissue. Also, bevacizumab injected subconjunctivally was transported into the intraocular tissues of the treated eyes at an effective level. Both intravitreal and subconjunctival injections of bevacizumab resulted in high plasma concentrations. Bevacizumab was distributed into the intraocular tissues in fellow eyes via the systemic circulation. This treatment may be effective for blocking vascular endothelial growth factor activity.

ROS and innate immunity.

Abstract: Oxygen is converted into reactive oxygen (RO) by radiation, light, the electron transport system in mitochondria, or by other enzymes and is regulated by the action of antioxidative enzymes which convert RO into an inactive state. Reactive oxygen species (ROS) have a biocidal effect on invading bacteria and they can also injure the cells of the host. For this reason, RO is considered as a general cause of aging and contributes to lifestyle-related diseases and cancer. However, for any organism that uses oxygen as an energy source, RO is inevitably produced and has important biological significance. Apart from the direct activity of RO, recent studies have shown that it functions as a second messenger of signal transduction. In this review, the recent findings related to ROS/nitric oxide (NO) and especially of its relationship to innate immunity are summarized.

Stress-induced changes of methylglyoxal level and glyoxalase I activity in pumpkin seedlings and cDNA cloning of glyoxalase I gene.

Abstract: Abiotic stresses cause extensive losses to agricultural production worldwide. In this study, the effects of various abiotic stresses on the upregulation of methylglyoxal levels and glyoxalase I activities in pumpkin seedlings (Cucurbita maxima Duch.) were investigated. Most of the stresses caused significant increases in methylglyoxal level and glyoxalase I activity, white light causing the highest induction followed by salinity, chemical, drought, and heavy metal stresses. We showed that accumulation of methylglyoxal in plants under various stressful conditions is a common phenomenon, and methylglyoxal could therefore act as a signal for plants to respond to stress. The stress-induced increases in methylglyoxal level, glyoxalase I activity and Gly I transcript found in the present study suggest an important role of glyoxalase I in conferring tolerance to plants under stress conditions and showed that the glyoxalase pathway is the main detoxification pathway of methylglyoxal in plants. The multistress response of glyoxalase I gene indicates its future utility in developing tolerance to various stresses in crop plants. A cDNA encoding glyoxalase I has been isolated, subcloned and nucleotide sequence was determined. The pumpkin glyoxalase I cDNA consists of 975-bp nucleotides encoding a polypeptide of 185 amino acids having a predicted molecular weight of 20,772.14 Da. Based on the number of amino acids, it is categorized as short-type glyoxalase I and the nucleotide sequence of pumpkin glyoxalase I showed significant homology with other known glyoxalase I sequences of plants.

Sequential signaling in plasma-membrane domains during macropinosome formation in macrophages.

Abstract: Macropinosomes are large endocytic vesicles that form in ruffling regions of plasma membrane. To analyze signal organization relative to ruffle closure into circular ruffles and cup closure into macropinosomes, this study used quantitative microscopy to measure 3′ phosphoinositides and small-GTPase activities in a representative subset of forming macropinosomes. Macropinocytosis was stimulated by the addition of macrophage colony-stimulating factor (M-CSF) to macrophages expressing fluorescent reporter proteins. Ratiometric and fluorescence resonance energy transfer (FRET) microscopy determined that Rac1 activity and phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] levels increased transiently, peaking 26-30 seconds after ruffle closure. Three-dimensional reconstruction of cells labeled with the fluorescent dye FM4-64 showed that PtdIns(3,4,5)P3 was restricted to open, circular cups in the plasma membrane. Quantitative fluorescence microscopic methods determined the timing of cup closure, which followed 40-100 seconds after Rac1 and PtdIns(3,4,5)P3 deactivation and coincided with accumulation of phosphatidylinositol 3-phosphate and Rab5a. Thus, ruffle closure creates a circular domain of plasma membrane that localizes the activation and deactivation of Rac1 and phosphoinositide 3-kinase (PI3K), followed by recruitment of Rab5a and the contractile activities of cup closure.

Prognostic factors and clinical outcomes in patients with leptomeningeal metastasis from solid tumors

Abstract: Background Leptomeningeal metastasis (LM) occurs in 4–15% of patients with solid tumors. Although the clinical outcomes in cancer patients have been improving recently, no standard treatment for LM has been established as yet. The purpose of this study was to identify the prognostic factors in patients with solid tumors with cytologically proven LM. Methods We retrospectively analyzed a series of 85 consecutive patients with cytologically proven LM who were treated between 1997 and 2005. Results The primary diseases were as follows; lung cancer (n = 36), breast cancer (n = 33), gastric cancer (n = 8), and others (n = 8). Forty-nine patients had brain metastasis at the time of diagnosis of the LM, and in 51 patients, MRI revealed meningeal dissemination in the brain or spine. The performance status (PS) was 0–1 in 26 patients and 2–4 in 59 patients. Thirty-one patients, including 19 with breast cancer, four with lung cancer, five with gastric cancer and three with other cancers, were treated by intrathecal (IT) chemotherapy. The response rate to the IT was 52% (95% confidence interval (CI): 41.4–62.6%). The median survival was 51 days (range, 3–759 days). A univariate analysis identified breast cancer, good PS (0–1), time to development of the LM (>1 year), and treatment by IT chemotherapy as being associated with a good prognosis, and multivariate analysis identified poor PS (HR: 1.72 (95% CI, 1.04–2.86) P = 0.04) and MRI-proven LM (HR: 1.82 (95% CI, 1.11–2.98) P = 0.02) as being associated with a poor prognosis. Conclusion In patients with poor prognostic factors, such as poor PS or MRI-proven LM, palliative therapy might be the most suitable treatment strategy.

Attenuation of the Systemic Inflammatory Response and Infectious Complications After Gastrectomy with Preoperative Oral Arginine and ω-3 Fatty Acids Supplemented Immunonutrition

Abstract: Past trials have shown perioperative immunonutrition to improve the outcome for patients with gastric cancer. The present study was designed to evaluate the effect of preoperative oral immunonutrition on cellular immunity, the duration of the systemic inflammatory response syndrome (SIRS), and detailed postoperative complications in patients with gastric cancer. Sixty patients with gastric cancer were randomly assigned to two groups: one group received immune-enhanced formulas supplemented with arginine and ω-3 fatty acids (immune-enhancing diet (ID) group, n = 30); the other received standard formulas (conventional diet (CD) group, n = 30) for 7 days before the operation. These groups were well matched in terms of age, sex, operations, cancer stages, and intraoperative variables. The postoperative outcome was evaluated based on clinical variables, including postoperative infectious complications, noninfectious complications, and SIRS duration. In addition, the perioperative state of cellular immunity was evaluated and compared between the two groups. The incidence of postoperative infectious complications in the ID group (6%) was significantly (p < 0.05) lower than that of the CD group (28%). The duration of SIRS in the ID group (0.77 ± 0.9 days) was significantly (p < 0.05) shorter than that in the CD group (1.34 ± 1.45 days). The postoperative lymphocyte and CD4+T-cell counts significantly decreased (p < 0.05) in both groups. However, the number of CD4+T-cells on preoperative day 1 and postoperative day 7 was significantly (p < 0.05) higher in the ID group than in the CD group. Preoperative oral immune-enhanced formulas supplemented with arginine and ω-3 fatty acids enhanced the immune status of the patients, reduced the duration of SIRS, and decreased the incidence of postoperative infectious complications. CD4+T-cell immunity likely played an important role in the modulation of the postoperative immune and inflammatory response after gastrectomy.

Sphingosine-1-phosphate and modulation of vascular tone

Abstract: Sphingosine-1-phosphate (S1P) is a phosphorylated product of sphingosine, the core structure of the class of lipids termed sphingolipids. S1P is a naturally occurring lipid metabolite, and usually is present at a concentration of a few 100 nanomolar in human sera. S1P has been found to exert a diverse set of physiological and pathophysiological responses in mammalian tissues through the activation of heterotrimeric G-proteins that in turn modulate the activity of various downstream effecter molecules. In blood vessels, vascular endothelial cells and smooth muscle cells express specific receptors for S1P that modulate vascular tone. This article will provide a brief overview of S1P metabolism in the vasculature and will discuss some of the pathways whereby S1P regulates intracellular signal transduction pathways in endothelial and smooth muscle cells, leading to the activation of both vasorelaxation and vasoconstriction responses.

Protective role of palmitoylethanolamide in contact allergic dermatitis.

Abstract: Background: Palmitoylethanolamide (PEA) is an anti-inflammatory mediator that enhances the activation by anandamide (AEA) of cannabinoid receptors and transient receptor potential vanilloid type-1 (TRPV1) channels, and directly activates peroxisome proliferator-activated receptor-a (PPAR-a). In mice, 2,4-dinitrofluorobenzene (DNFB)-induced contact allergic dermatitis (CAD) in inflamed ears is partly mediated by the chemokine Monocyte Chemotactic Protein-2 (MCP-2) and accompanied by elevation of AEA levels. No datum is available on PEA regulation and role in CAD. Objective: We examined whether PEA is produced during DNFB-induced CAD, and if it has any direct protective action in keratinocytes in vitro. Methods: Eight- to ten-week-old female C57BL/6J wild-type and CB1/CB2 double knock-out mice were used to measure PEA levels and the expression of TRPV1, PPAR-a receptors and enzymes responsible for PEA biosynthesis and degradation. Human keratinocytes (HaCaT) cells were stimulated with polyinosinic polycytidylic acid [poly-(I:C)], and the expression and release of MCP-2 were measured in the presence of PEA and antagonists of its proposed receptors. Results: 2,4-Dinitrofluorobenzene increased ear skin PEA levels and up-regulated TRPV1, PPAR-a and a PEA-biosynthesizing enzyme in ear keratinocytes. In HaCaT cells, stimulation with poly-(I:C) elevated the levels of both PEA and AEA, and exogenous PEA (10 lM) inhibited poly-(I:C)-induced expression and release of MCP-2 in a way reversed by antagonism at TRPV1, but not PPAR-a. PEA (5–10 mg/kg, intraperitoneal) also inhibited DNFB-induced ear inflammation in mice in vivo, in a way attenuated by TRPV1 antagonism. Conclusions: We suggest that PEA is an endogenous protective agent against DNFB-induced keratinocyte inflammation and could be considered for therapeutic use against CAD.

Host plant genome overcomes the lack of a bacterial gene for symbiotic nitrogen fixation

Abstract: Homocitrate is a component of the iron-molybdenum cofactor in nitrogenase, where nitrogen fixation occurs. NifV, which encodes homocitrate synthase (HCS), has been identified from various diazotrophs but is not present in most rhizobial species that perform efficient nitrogen fixation only in symbiotic association with legumes. Here we show that the FEN1 gene of a model legume, Lotus japonicus, overcomes the lack of NifV in rhizobia for symbiotic nitrogen fixation. A Fix(-) (non-fixing) plant mutant, fen1, forms morphologically normal but ineffective nodules. The causal gene, FEN1, was shown to encode HCS by its ability to complement a HCS-defective mutant of Saccharomyces cerevisiae. Homocitrate was present abundantly in wild-type nodules but was absent from ineffective fen1 nodules. Inoculation with Mesorhizobium loti carrying FEN1 or Azotobacter vinelandii NifV rescued the defect in nitrogen-fixing activity of the fen1 nodules. Exogenous supply of homocitrate also recovered the nitrogen-fixing activity of the fen1 nodules through de novo nitrogenase synthesis in the rhizobial bacteroids. These results indicate that homocitrate derived from the host plant cells is essential for the efficient and continuing synthesis of the nitrogenase system in endosymbionts, and thus provide a molecular basis for the complementary and indispensable partnership between legumes and rhizobia in symbiotic nitrogen fixation.

Production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons.

Abstract: In the present study, we describe the production of transgenic silkworms expressing a recombinant mouse mAb in their cocoons. Two transgenic lines, L- and H-, were generated that carried cDNAs encoding the L- and H-chains of a mouse IgG mAb, respectively, under the control of the enhancer-linked sericin-1 promoter. Cocoon protein analysis indicated that the IgG L- or H-chain was secreted into the cocoons of each line. We also produced a transgenic line designated L/H, which carried both cDNAs, by crossing the L- and H-lines. This line efficiently produced the recombinant mAb as a fully assembled H(2)L(2) tetramer in its cocoons, with negligible L- or H-chain monomer and H-chain dimer production. Thus, the H(2)L(2) tetramer was synthesized in, and secreted from, the middle silk gland cells. Crossing of the L/H-line with a transgenic line expressing a baculovirus-derived trans-activator produced a 2.4-fold increase in mAb expression. The recombinant mAb was extracted from the cocoons with a buffer containing 3 m urea and purified by protein G affinity column chromatography. The antigen-binding affinity of the purified recombinant mAb was identical to that of the native mAb produced by a hybridoma. Analysis of the structure of the N-glycans attached to the recombinant mAb revealed that the mAb contained high mannose-, hybrid- and complex-type N-glycans. By contrast, insect-specific paucimannose-type glycans were not detected. Fucose residues alpha-1,3- and alpha-1,6-linked to the core N-acetylglucosamine residue, both of which are found in insect N-glycans, were not observed in the N-glycans of the mAb.

The Indian Ocean Dipole and malaria risk in the highlands of western Kenya

Abstract: Epidemics of malaria in the East African highlands in the last 2 decades have often been associated with climate variability, particularly the El Nino-Southern Oscillation (ENSO). However, there are other factors associated with malaria risk and there is increased interest in the influences of the Indian Ocean Dipole (IOD), a climate mode of coupled ocean–atmosphere variability, on East African rainfall. This study explores the relationship between IOD and the number of malaria patients in 7 hospitals from 2 districts in the western Kenyan highlands, controlling for the effects of ENSO. We examined temporal patterns (1982–2001) in the number of malaria cases in relation to the dipole mode index (DMI), defined as the difference in sea surface temperature anomaly between the western (10°S-10°N, 50°-70°E) and eastern (10°S-0°, 90°-110°E) tropical Indian Ocean. We used Poisson regression models, adjusted for ENSO index Nino 3 region (NINO3), seasonal and interannual variations. The number of malaria patients per month increased by 3.4%–17.9% for each 0.1 increase above a DMI threshold (3–4 months lag). Malaria cases increased by 1.4%–10.7% per month, for each 10 mm increase in monthly rainfall (2–3 months lag). In 6 of 7 places, there was no evidence of an association between NINO3 and the number of malaria cases after adjusting for the effect of DMI. This study suggests that the number of malaria cases in the western Kenyan highlands increases with high DMI in the months preceding hospital visits.

Expression of autotaxin and acylglycerol kinase in prostate cancer: association with cancer development and progression.

Abstract: Lysophosphatidic acid (LPA) may enhance diverse biologic activities in prostate cancer. This study was conducted to analyze expression levels of LPA-producing enzymes, autotaxin (ATX) and acylglycerol kinase (AGK), in prostate cancer with relevance to clinicopathological parameters. Real-time RT-PCR and western blotting were performed for ATX and AGK in non-neoplastic prostate cells (PrECs and PrSCs) and prostate cancer cell-lines (DU-145, PC-3, LNCaP, and AILNCaP). Immunohistochemical analyses were conducted in tissue specimens of 132 localized prostate cancer patients who underwent radical prostatectomy between 2001 and 2007 (median observation period, 22 months). Both enzymes were negatively expressed in PrECs and PrSCs at mRNA and protein levels. ATX expression was higher than AGK in AILNCaP, DU-145, and PC-3 cell-lines, while AGK was mainly expressed in LNCaP cells. Immunohistochemically, ATX and AGK expressions were negative in non-neoplastic epithelia, while both were weakly expressed in the majority of high-grade intra-epithelial neoplasia (HG-PIN). In cancer foci, ATX and AGK expressions were strong in 49% and 62%, weak in 40% and 32%, and negative in 11% and 6%, respectively. Expressions of both enzymes were significantly correlated with primary Gleason grade of cancer foci (P < 0.0001) and capsular invasion (P = 0.03 and 0.003 respectively). ATX expression was significantly correlated with probability of prostate specific antigen (PSA)-failure after surgery (P < 0.0001). In conclusion, LPA-producing enzymes (ATX and AGK) were frequently expressed in prostate cancer cells and precancerous HG-PIN. In particular, high expression levels of ATX were associated with both malignant potentials and poor outcomes. (Cancer Sci 2009; 100: 1631–1638)

Clinical Outcomes of Stereotactic Body Radiotherapy for Small Lung Lesions Clinically Diagnosed as Primary Lung Cancer on Radiologic Examination

Abstract: Purpose Image-guided biopsy occasionally fails to diagnose small lung lesions, which are highly suggestive of primary lung cancer. The aim of the present study was to evaluate the outcome of stereotactic body radiotherapy (SBRT) for small lung lesions that were clinically diagnosed as primary lung cancer without pathologic confirmation. Methods and Materials A total of 115 patients were treated with SBRT in 12 institutions. Tumor size ranged from 5 to 45 mm in diameter, with a median of 20 mm. Results The 3-year and 5-year overall survival rates for patients with a tumor size ≤20 mm in diameter (n = 58) were both 89.8%, compared with 60.7% and 53.1% for patients with tumors >20 mm (n = 57) (p 20 mm. Among the patients with a tumor size ≤20 mm, Grade 2 pulmonary complications were observed in 2 (3.4%), but no Grade 3 to 5 toxicity was observed. In patients with a tumor size >20 mm, Grades 2, 3, and 5 toxicity were observed in 5 patients (8.8%), 3 patients (5.3%), and 1 patient (1.8%), respectively. Conclusion In patients with a tumor ≤20 mm in diameter, SBRT was reasonably safe in this retrospective study. The clinical implications of the high local control rate depend on the accuracy of clinical/radiologic diagnosis for small lung lesions and are to be carefully evaluated in a prospective study.

Causes of large-scale landslides in the Lesser Himalaya of central Nepal

Abstract: Geologically and tectonically active Himalayan Range is characterized by highly elevated mountains and deep river valleys. Because of steep mountain slopes, and dynamic geological conditions, large-scale landslides are very common in Lesser and Higher Himalayan zones of Nepal Himalaya. Slopes along the major highways of central Nepal namely Prithvi Highway, Narayangadh-Mugling Road and Tribhuvan Highway are considered in this study of large-scale landslides. Geologically, the highways in consideration pass through crushed and jointed Kathmandu Nappe affected by numerous faults and folds. The relict large-scale landslides have been contributing to debris flows and slides along the highways. Most of the slope failures are mainly bechanced in geological formations consisting phyllite, schist and gneiss. Laboratory test on the soil samples collected from the failure zones and field investigation suggested significant hydrothermal alteration in the area. The substantial hydrothermal alteration in the Lesser Himalaya during advancement of the Main Central Thrust (MCT) and thereby clay mineralization in sliding zones of large-scale landslide are the main causes of large-scale landslides in the highways of central Nepal. This research also suggests that large-scale landslides are the major cause of slope failure during monsoon in the Lesser Himalaya of Nepal. Similarly, hydrothermal alteration is also significant in failure zone of the large-scale landslides. For the sustainable road maintenance in Nepal, it is of utmost importance to study the nature of sliding zones of large-scale landslides along the highways and their role to cause debris flows and slides during monsoon period.

Regulation of megalin expression in cultured proximal tubule cells by angiotensin II type 1A receptor- and insulin-mediated signaling cross talk.

Abstract: Impairment of proximal tubular endocytosis of glomerular-filtered proteins including albumin results in the development of proteinuria/albuminuria in patients with chronic kidney disease. However, the mechanisms regulating the proximal tubular function are largely unknown. This study aimed to investigate the role of angiotensin II type 1A receptor (AT(1A)R)- and insulin-mediated signaling pathways in regulating the expression of megalin, a multiligand endocytic receptor in proximal tubule cells (PTCs). Opossum kidney PTC-derived OK cells that stably express rat AT(1A)R but are deficient in endogenous angiotensin II receptors (AT(1A)R-OK cells) were used for this study. Treatment of the cells with angiotensin II suppressed mRNA and protein expression of megalin at 3- and 24-h incubation time points, respectively. Cellular uptake and degradation of albumin and receptor-associated protein, megalin's endocytic ligands were suppressed 24 h after angiotensin II treatment. The AT(1A)R-mediated decrease in megalin expression was partially prevented by ERK inhibitors. Insulin competed with the AT(1A)R-mediated ERK activation and decrease in megalin expression. Inhibitors of phosphatidylinositol 3-kinase (PI3K), a major component of insulin signaling, also suppressed megalin expression, and activation of the insulin receptor substrate (IRS)/PI3K system was prevented by angiotensin II. Collectively the AT(1A)R-mediated ERK signaling is involved in suppressing megalin expression in the OK cell line, and insulin competes with this pathway. Conversely, the insulin-IRS/PI3K signaling, with which angiotensin II competes, tends to stimulate megalin expression. In conclusion, there is AT(1A)R- and insulin-mediated competitive signaling cross talk to regulate megalin expression in cultured PTCs.

Angiotensin II Type 1 Receptor Blockers Reduce Urinary Angiotensinogen Excretion and the Levels of Urinary Markers of Oxidative Stress and Inflammation in Patients with Type 2 Diabetic Nephropathy.

Abstract: Objective: To demonstrate that the administration of an angiotensin (Ang) II type 1 receptor (AT1R) blocker (ARB) inhibits the vicious cycle of high glucose (HG)-reactive oxygen species (ROS)-angiotensinogen (AGT)-Ang II-AT1R-ROS by suppressing ROSs and inflammation, thus ameliorating diabetic nephropathy (DN). Research Design and Methods: Thirteen hypertensive DN patients were administered ARBs, and the following parameters were evaluated before and 16 weeks after the treatment: urinary AGT (UAGT), albumin (albumin-creatinine ratio: ACR), 8-hydroxyde- oxyguanosine (8-OHdG), 8-epi-prostaglandin F2α (8-epi-PGF2α), monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6, and IL-10. Results: ARB treatment reduced the blood pressure and urinary levels of AGT, ACR, 8-OHdG, 8-epi-PGF2α, MCP-1, and IL-6 but increased the urinary levels of IL-10. The reduction rate of UAGT correlated with the reduction rate of blood pressure; the reduction rates of the urinary ACR, 8-OHdG, 8-epi-PGF2α, MCP-1, and IL-6 levels; and the increase rate of the urinary IL-10 levels. Moreover, subjects who had high UAGT values at baseline exhibited higher reduction rates of urinary albumin excretion. Conclusions: ARB-induced blockade of the abovementioned vicious cycle contributes to the renoprotective effects of ARBs in DN. The urinary levels of AGT could represent a predictive factor for reduced ACR in patients receiving ARB treatment.