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Abstract: The skeletal muscle of mammals is known to express myostatin (GDF-8) that acts as a potent negative regulator of skeletal muscle growth. However, the function of GDF-8 is not limited to skeletal muscle, because of its ubiquitous expression in fish. Here we investigated whether GDF-8 is expressed in various tissues including gonads during chicken embryogenesis. As revealed by RT-PCR and Western blotting, the transcript and protein for GDF-8 were detected in brain, eye, gizzard, muscle, heart, small gut, large gut, mesonephroi, testis and ovary of chicken embryos at E12, but not in liver. GDF-8 was constitutively expressed in testis and ovary as well as muscle at E6-E21, as demonstrated by in situ hybridization on section and whole-mount. Some cell population in testis, but not identified, highly expressed GDF-8. On the other hand, the medulla and germinal epithelium of ovary highly expressed it. Collectively, these results indicate that GDF-8 is ubiquitously expressed in various tissues of chicken embryos including testis and ovary through the stage of embryogenesis. [Copyright &y& Elsevier]

2-Methoxyestradiol (2-ME) has potent antiproliferative effects on cancer cells. Its utility alone or in combination with other therapies for treating prostate cancer, however, has not been fully explored. Androgen-dependent and independent human prostate cancer cells were examined in vivo for their response to combination therapy. Efficacy was assessed by terminal deoxynucleotide transferase--mediated dUTP nick-end labeling assay and measuring microvessel density (MVD) in excised tumors. Animals harboring hormone-dependent tumors treated with 2-ME alone, androgen deprivation therapy alone, or the combination of the two had a 3.1-fold, 5.3-fold, and 10.1-fold increase in apoptosis, respectively. For hormone-independent tumors, treatment with 2-ME resulted in a 2.43-fold increase in apoptosis and a 73% decrease in MVD. 2-ME was most effective against hormone-dependent tumors in vivo and combination therapy resulted in a significant increase in efficacy compared to no treatment controls and trended toward greater efficacy than either 2-ME or androgen deprivation alone. Combination therapy should be investigated further as an additional therapeutic option for early prostate cancer. [ABSTRACT FROM AUTHOR]

Pretreatment serum FGF-23 levels predict the efficacy of calcitriol therapy in dialysis patients.Background.The predictor for the result of calcitriol therapy would be useful in the clinical practice of secondary hyperparathyroidism. Fibroblast growth factor-23 (FGF-23) is a newly found circulating phosphaturic factor. Its circulating level is elevated in uremia.Methods.Dialysis patients with plasma intact parathyroid hormone (iPTH) levels greater than 300 pg/mL were included in the study. Calcitriol was intravenously injected three times a week. The patients whose plasma iPTH levels dropped below 300 pg/mL within 24 weeks were defined as those who had been successfully treated. A sandwich enzyme-linked immunosorbent assay (ELISA) system that detects human FGF-23 was applied.Results.Sixty-two patients were analyzed. The pretreatment FGF-23 levels were related to the iPTH levels, calcium× phosphate product levels, and history of active vitamin D therapy. The pretreatment FGF-23, iPTH, and calcium levels were lower in the patients who would be successfully treated with calcitriol. A logistic regression study revealed that the pretreatment iPTH and FGF-23 levels significantly affected the therapy results. Analyses using a receiver-operated curve revealed that FGF-23 was the best screening test for identifying patients with future refractory response to calcitriol therapy. The treatment would be successful in 88.2% of those with FGF-23≤9860 ng/L and iPTH≤591 pg/mL, while it would be successful in only 4.2% of those with FGF-23>9860 ng/L and iPTH>591 pg/mL.Conclusion.Pretreatment serum FGF-23 levels were a good indicator in predicting the response to calcitriol therapy. The measurement of serum FGF-23 levels, especially in combination with iPTH levels, is a promising laboratory examination for the clinical practice of secondary hyperparathyroidism. [ABSTRACT FROM AUTHOR]

Polarization spectroscopy in optical near-field measurements is an important method for evaluation of material properties and structures. A novel type of fiber probe has been developed with a sub-wavelength slit-shaped aperture as an optical device for polarization sensitive spectroscopic observations at the extreme nano-scale range. This probe features high throughput and sensitivity for a wide range of near-field sampling applications. The polarization degree is 10 times greater than that of conventional near-field probes. [Copyright &y& Elsevier]

In our search for a new agent, human neutrophil elastase (HNE) inhibitor, for the treatment of acute respiratory failure, we rationally designed and synthesized a series of peptide-based carboxylic acid-containing transition-state inhibitors. The presence of valyl moiety is found to be essential for potent in vitro inhibitory activity and also prevention of an undesirable toxicity. Of these, compound 9m has the most potent in vivo effect on HNE-induced lung hemorrhage in hamsters. [Copyright &y& Elsevier]

In higher vertebrates, cephalic neural crest cells (NCCs) form craniofacial skeleton by differentiating into chondrocytes and osteoblasts. A subpopulation of cephalic NCCs, cardiac NCCs (CNCCs), migrates to the heart. However, CNCCs mostly do not yield skeletogenic derivatives, and the molecular mechanisms of this fate restriction remain elusive. We identify a disintegrin and metalloprotease 19 (Adam19) as a position-specific fate regulator of NCCs. Adam19-depleted mice abnormally form NCC-derived cartilage in their hearts through the upregulation of Sox9 levels in CNCCs. Moreover, NCC-lineage-specific Sox9- overexpressing mice recapitulate CNCC chondrogenesis. In vitro experiments show that Adam19 mediates the cleavage of bone morphogenic protein (BMP) type I receptor Alk2 (Acvr1), whereas pharmacogenetic approaches reveal that Adam19 inhibits CNCC chondrogenesis by suppressing the BMP-Sox9 cascade, presumably through processing Alk2. These findings suggest a metalloprotease-dependent mechanism attenuating cellular responsiveness to BMP ligands, which is essential for both the positional restriction of NCC skeletogenesis and normal heart development. • Adam19 mutants exhibit neural crest-derived cardiac cartilage formation • High Sox9 expression in Adam19 −/− -CNCCs induces cardiac cartilage formation • Adam19 mediates proteolytic cleavage of BMP receptor Alk2 • Adam19 suppresses CNCC chondrogenesis by inhibiting the BMP-Sox9 signaling pathway Arai et al. show that metalloprotease Adam19 is essential for proper differentiation of cardiac neural crest cells (CNCCs) in murine embryogenesis. Adam19 suppresses the response of CNCCs to bone morphogenic protein by cleaving its receptor Alk2, thus restricting CNCC skeletogenic potential and preventing cartilage formation in the heart. [ABSTRACT FROM AUTHOR]

Background Recently, increased plasma mid-regional pro-adrenomedullin (MR-proADM) concentrations have been demonstrated in patients with chronic renal failure. In this study, we attempted to identify significant clinical factors associated with MR-proADM concentration in stable kidney transplant recipients. Methods Forty-seven Japanese kidney transplant recipients who underwent transplantation > 180 days prior to the study were analyzed. To facilitate comparability of anti-hypertensive regimens across recipients taking different drugs, we calculated the treatment intensity score of anti-hypertensive drugs in each recipient. Morning blood samples were collected and plasma MR-proADM concentrations were measured using an enzyme immunoassay. Results Multiple regression analysis identified treatment intensity score for anti-hypertensive drugs, serum albumin, creatinine clearance and use of lipid-lowering agents as significant independent factors associated with plasma MR-proADM concentration. Adjusted coefficient of determination for this model was 0.46. Conclusion Apart from indicating lowered renal function, plasma MR-proADM concentration may be a useful biomarker for metabolic disorders, especially hypertension and hyperlipidemia, in stable kidney transplant patients. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: Tumor hypoxia is a common feature of any cancer, including prostate cancer (CaP), and associated with tumor cell aggressiveness. Although some reports are available on acute hypoxia-response in CaP cells aggressiveness, little is known about chronic hypoxia-response. We investigated the effects of chronic hypoxia on human CaP cells. Materials and methods: The human androgen-dependent CaP cell line LNCaP was cultured under normoxia (21% O2), acute hypoxia (1% O2), or chronic hypoxia (1% O2 for over 6 months). The cell growth, cell cycle and cell behavior of these cells were analyzed by cell count, flow cytometric analysis and in vitro cell migration and invasion assay, respectively. The expression of matrix metalloproteinases and intracellular signaling pathways were tested by real time reverse transcriptase-polymerase chain reaction and Western blotting. Results: Chronic hypoxia-conditioned LNCaP cells grew in an androgen-independent manner with acceleration of G1 to S phase cell cycle progression. Chronic hypoxia, but not acute hypoxia, accelerated cell migration and invasion. The expressions of matrix metalloproteinase-7, -9, -14, and -15 were significantly up-regulated in LNCaP cells under chronic hypoxia, but not under acute hypoxia. In addition, PI3K/Akt, JAK/STAT, and HIF-1 pathways were activated in chronic hypoxia-conditioned LNCaP cells. Conclusions: These results suggested that chronic hypoxia plays an important role in enhancement of malignant potential during androgen-independent CaP progression. [Copyright &y& Elsevier]

MUC20, a novel mucin protein highly expressed in kidney, was isolated as an up-regulated gene in renal tissues of patients with IgA nephropathy (IgAN) ( J Biol Chem 279:1968, 2004). Functional analyses of MUC20 have demonstrated its role as a negative regulator of hepatocyte growth factor (HGF)–induced Grb2-Ras pathway. The C-terminus of MUC20 associates with the multifunctional docking site of Met, preventing Grb2 recruitment to Met and thus attenuating HGF-induced proliferation and matrix metalloproteinase expression ( Mol Cell Biol 24:7456, 2004). In addition, it has been suggested that the oligomerization of MUC20, caused by its overproduction or some other unknown factor(s), which leads to this association with Met . Interestingly, in human MUC20, the repeat numbers of the extracellular tandem domain, which may have an influence on the oligomerization, showed a divergence, with two to six repeat types in several human cell lines. In this study, to clarify the role of MUC20 in human kidney diseases, we analyzed the expression of MUC20 in kidney tissues of patients with IgAN by in situ hybridization. We also investigated the possible association of the tandem repeat polymorphism of MUC20 with renal survival in 236 patients with histologically proven IgAN. In normal kidneys, the expression of MUC20 was confined to the distal tubules, where Met was colocalized by immunohsitochemistry. In addition, glomerular podocytes and the parietal epithelial cells lining Bowman's capsule demonstrated positive expression of MUC20. In renal tissues of IgAN, up-regulation of MUC20 expression in proximal tubules, as well as in distal tubules, was observed. By Kaplan-Meier analysis, the prognosis of IgAN patients with five or six of tandem repeat of MUC20 ( N= 130) was significantly better than those without ( N= 106, log-rank, χ2= 10.51) ( P= 0.0012). The tandem repeat polymorphism of MUC20 was an independent risk factor for the progression of renal dysfunction even after adjusting for other clinical risk factors, including hypertension, urinary protein excretion of more than 1.0 g/day, and no administration of renin angiotensin system inhibitors. This study supports the role of MUC20 in regulating the Met signaling cascade, which is implicated not only in renal development and maintenance of kidney functions but also in tubular repair and regeneration under pathological conditions in human glomerulonephritis. The tandem repeat polymorphism in MUC20, which may directly affect its oligomerization and binding to Met, is associated with the renal prognosis of IgAN. Factors that regulate the function of MUC20 may be useful therapeutic agents for progression of renal injury. [ABSTRACT FROM AUTHOR]

Three Raman probes of sphingomyelin (SM) were synthesized and evaluated for their applicability to imaging experiments. One probe containing a hydroxymethyl-1,3-butadiyne moiety in the polar head group showed strong scattering. The solid-state 2 H NMR spectra of this probe in oriented bilayer membrane revealed excellent compatibility with natural SM in phase behavior since the probe undergoes phase separation to form raft-like liquid ordered (L o ) domains in the raft-mimicking mixed bilayers. [ABSTRACT FROM AUTHOR]

Skeletal muscle regeneration requires processes different from developmental myogenesis. One important difference is a requirement of inflammatory reactions prior to regenerative myogenesis, by which injured muscle fibers must be eliminated to make new myotubes. In this study, we show that efficient elimination of injured muscle fibers during regeneration requires ADAM8, a member of a d isintegrin a nd m etalloprotease (ADAM) family. Skeletal muscle of dystrophin-null mice, an animal model for Duchenne Muscular Dystrophy, deteriorates by the lack of ADAM8, which is characterized by increased area of muscle degeneration and increased number of necrotic and calcified muscle fibers. Adam8 is highly expressed in neutrophils. Upon cardiotoxin-induced skeletal muscle injury, neutrophils invade into muscle fibers through the basement membrane and form large clusters in wild type, but not in ADAM8-deficient mice, although neutrophils of the latter infiltrate into interstitial tissues similarly to those of wild type mice. Neutrophils lose their adhesiveness to blood vessels after infiltration, which includes an ectodomain shedding of P-Selectin Glycoprotein Ligand-1 (PSGL-1) on their surface. Expression of PSGL-1 on the surface of neutrophils remains higher in ADAM8-deficient than in wild type mice. These results suggest that ADAM8 mediates an enhanced invasiveness of neutrophils into injured muscle fibers by the removal of their adhesiveness to blood vessels after infiltration into interstitial tissues. [ABSTRACT FROM AUTHOR]

Lipid organization has been at the center of research on lipid rafts. Dioleoylphosphatidylcholine (DOPC) is a typical unsaturated lipid. Very few studies have reported its thermodynamics in raft-like membranes. Herein, we have developed a highly efficient synthetic method for [C6- 2 H 2 ] oleic acid, and newly synthesized [C6- 2 H 2 ] DOPC. In raft-like oriented bilayers, [C6- 2 H 2 ] DOPC shows clear phase separation and characteristic phase behavior at various temperature. It has been successfully utilized for the comparison of membrane properties between sphingomyelin (SM) and dihydrosphingomyelin (DHSM) membranes. [ABSTRACT FROM AUTHOR]

Abstract: The role of heart-type fatty acid-binding protein (FABP3) in human physiology as an intracellular carrier of fatty acids (FAs) has been well-documented. In this study, we aimed to develop an analytical method to study real-time interaction kinetics between FABP3 immobilized on the sensor surface and unsaturated C18 FAs using surface plasmon resonance (SPR). To establish the conditions for SPR experiments, we used an FABP3-selective inhibitor 4-(2-(1-(4-bromophenyl)-5-phenyl-1H-pyrazol-3-yl)-phenoxy)-butyric acid. The affinity index thus obtained was comparable to that reported previously, further supporting the usefulness of the SPR-based approach for evaluating interactions between FABPs and hydrophobic ligands. A pseudo-first-order affinity of FABP3 to K+ petroselinate (C18:1 Δ6 cis), K+ elaidate (C18:1 Δ9 trans), and K+ oleate (C18:1 Δ9 cis) was characterized by the dissociation constant (K d) near micromolar ranges, whereas K+ linoleate (C18:2 Δ9,12 cis/cis) and K+ α-linolenate (C18:3 Δ9,12,15 cis/cis/cis) showed a higher affinity to FABP3 with K d around 1×10−6 M. Interactions between FAPB3 and C18 FAs incorporated in large unilamellar vesicles consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and FAs (5:1 molar ratio) were also analysed. Control DMPC liposomes without FA showed only marginal binding to FABP3 immobilized on a sensor chip while liposome-incorporated FA revealed significant responses in sensorgrams, demonstrating that the affinity of FAs to FABP3 could be evaluated by using the liposome-incorporated analytes. Significant affinity to FABP3 was observed for monounsaturated fatty acids (K d in the range of 1×10−7 M). These experiments demonstrated that highly hydrophobic compounds in a liposome-incorporated form could be subjected to SPR experiments for kinetic analysis. [Copyright &y& Elsevier]

Abstract: Objectives: We had previously reported that chronic hypoxia induces androgen-independent growth in the human prostate cancer cell line LNCaP. In this study, we have identified a key molecule, the Vav3 oncogene, and investigated the effects of Vav3 overexpression on cancer cell growth and malignant behavior and the possible apoptosis-inducing effect of Vav3 expression knockdown by small interfering ribonucleic acid (siRNA) in LNCaP cells under chronic hypoxia (LNCaP/CH). Methods and materials: Hypoxia-inducible oncogenes were identified by complementary deoxyribonucleic acid (cDNA) microarray and Ingenuity Pathway Analysis in order to investigate gene ontology and functional pathways and networks. siRNA was used to knockdown the Vav3 target gene and analyze the effects on proliferation, invasion, migration, and apoptosis of LNCaP/CH cells. Vav3 cDNA was transfected into LNCaP cells under normoxia (LNCaP/N) to establish Vav3-overexpressing clonal cell lines, whose proliferation, invasion, and migration was then examined. Immunoblot analysis was used to investigate the activation of Akt, a Vav3 downstream target molecule. Results: cDNA microarray analysis and Ingenuity Pathway Analysis identified Vav3 as a hypoxia-inducible oncogene that was highly associated with malignant behavior. Vav3 messenger RNA and protein expression in LNCaP/CH cells were higher than in LNCaP/N and LNCaP cells cultured under acute hypoxia (LNCaP/AH). The growth rate of LNCaP/CH cells was lower than that of LNCaP/N cells but higher than that of LNCaP/AH cells. LNCaP/CH cells showed higher invasion and migration than LNCaP/N and LNCaP/AH cells. Interrupting Vav3 expression strongly suppressed the proliferation, invasion, and migration of LNCaP/CH cells. Furthermore, siRNA led to apoptosis with increased caspase-3 and cleaved poly (adenosine diphosphate-ribose) polymerase activation in LNCaP/CH cells. Stable Vav3 overexpression in LNCaP cells promoted cell proliferation, invasion, and migration with Akt activation. Conclusions: Our results demonstrate that Vav3 plays a crucial role in prostate cancer growth and malignant behavior, thus revealing a novel potential therapeutic target. [Copyright &y& Elsevier]

Highlights: [•] Kidney transplant recipients at least 90 days after transplantation were studied. [•] MR-proADM level correlates significantly with creatinine clearance. [•] MR-proADM level correlates significantly with treatment intensity score of antihypertensives. [•] MR-proADM level is an independent factor associated with treatment intensity score. [•] MR-proADM level may be useful for evaluating resistance to antihypertensive therapy. [ABSTRACT FROM AUTHOR]

Abstract: We have successfully synthesized chiral β-amino trifluoromethyl alcohol (2S,3S)-7a, which is a key intermediate in the production of AE-3763, by stereoselective reduction of N-Cbz-protected 5-hydroxy-5-(trifluoromethyl)-1,3-oxazolidine 4 prepared from L-valine in 3 steps followed by alkaline hydrolysis. This new method can be applied to the industrial-scale synthesis of AE-3763. [ABSTRACT FROM AUTHOR]

Abstract: A series of peptide-based transition-state human neutrophil elastase (HNE) inhibitors with N-terminal acidic moieties were synthesized and their inhibitory activity against HNE was evaluated both in vitro and in vivo. Our results show that compounds containing cyclic amide bridged acidic moieties at the N-terminal have not only improved water solubility but also high in vivo potency. Among these compounds, AE-3763 showed remarkable efficacy in hamster models of elastase-induced lung hemorrhage and lipopolysaccharide (LPS)-induced lung injury as well as in a mouse model of LPS/galactosamine-induced acute multiple organ dysfunctions. The water solubility of AE-3763 (>1000mg/ml in H2O) was also far superior to that of any of the other compounds synthesized. Thus, it is believed that AE-3763 would be useful for treatment of HNE-associated respiratory disorders, such as acute respiratory distress syndrome (ARDS), acute lung injury (ALI), and acute exacerbation of chronic obstructive pulmonary disease (COPD). [Copyright &y& Elsevier]

Bezafibrate suppresses rat antiglomerular basement membrane crescentic glomerulonephritis. Background. The immunoregulatory activity of ligands for peroxisome proliferator-activated receptors (PPARs) has been recently paid attention. The regulatory effect of bezafibrate (BZF), a ligand for PPARα on glomerulonephritis was investigated using a rat anti-glomerular basement membrane (GBM) glomerulonephritis model. Methods. The effect on development of anti-GBM glomerulonephritis was examined by treatment with BZF from day−7 to day 7 after intravenous injection of rabbit anti-GBM serum into Wistar Kyoto (WKY) rats. The therapeutic efficacy after onset of the glomerulonephritis was also checked by treatment with BZF from day 3 to 7. On day 7, the condition was evaluated histologically. The expression of a tissue injury molecule, macrophage metalloesterase (MME), was measured by Northern blot analysis. The suppressive effect on immune cells was assessed by proliferation assay with mitogen-stimulated rat spleen cells. Results. Histopathologic changes induced by anti-GBM in rats treated with BZF (day−7 to day 7) were markedly suppressed in a dose-dependent fashion. Infiltration of ED-1+ macrophages in glomeruli, proteinuria, and mRNA expression of MME in kidneys were diminished in parallel with histologic improvement. Moreover, the disease activity was also attenuated even by the treatment after onset of the glomerulonephritis (day 3 to 7). The mitogen-induced proliferation of spleen cells was down-regulated at concentrations of BZF, which were equivalent to those in sera of BZF-treated rats. Conclusion. BZF markedly suppresses the activity of rat anti-GBM crescentic glomerulonephritis. Fibrates might serve as a therapeutic option for crescentic glomerulonephritis. [ABSTRACT FROM AUTHOR]

Noninvasive imaging of differences between the molecular properties of cancer and normal tissue has the potential to enhance the detection of tumors. Because overexpression of endogenous transferrin receptor (TfR) has been qualitatively described for various cancers and is presumably due to malignant transformation of cells, TfR may represent a suitable target for application of molecular imaging technologies to increase detection of smaller tumors. In the work reported here, investigation into the biology of this receptor using electron microscopy has demonstrated that iron oxide particles targeted to TfR are internalized and accumulate in lysosomal vesicles within cells. Biochemical analysis of the interaction of imaging probes with cells overexpressing the TfR demonstrated that the extent of accumulation, and therefore probe efficacy, is dependent on the nature of the chemical cross-link between transferrin and the iron oxide particle. These data were utilized to design and synthesize an improved imaging probe. Experiments demonstrate that the novel magnetic resonance imaging (MRI) probe is sensitive enough to detect small differences in endogenous TfR expression in human cancer cell lines. Quantitative measurement of TfR overexpression in a panel of 27 human breast cancer patients demonstrated that 74% of patient cancer tissues overexpressed the TfR and that the sensitivity of the new imaging agent was suitable to detect TfR overexpression in greater than 40% of these cases. Based on a biochemical and cell biological approach, these studies have resulted in the synthesis and development of an improved MRI probe with the best in vitro and in vivo imaging properties reported to date. [ABSTRACT FROM AUTHOR]

Highlights: [•] Plasma MR-proADM level decreased significantly after kidney transplantation. [•] Plasma MR-proADM level correlates significantly with creatinine clearance. [•] Kidney transplantation may lead to a decrease in plasma MR-proADM level. [•] Plasma MR-proADM level may depend largely on renal function. [Copyright &y& Elsevier]