Anti-chemorepulsive effects of vascular endothelial growth factor and placental growth factor-2 in dorsal root ganglion neurons are mediated via neuropilin-1 and cyclooxygenase-derived prostanoid production

Vascular endothelial growth factor (VEGF) displays neurotrophic and neuroprotective activities, but the mechanisms underlying these effects have not been defined. Neuropilin-1 (NP-1) is a receptor for VEGF165 and placental growth factor-2 (PlGF-2), but the role of NP-1 in VEGF-dependent neurotrophic actions is unclear. Dorsal root ganglion (DRG) neurons expressed high levels of NP-1 mRNA and protein, much lower levels of KDR, and no detectable Flt-1. VEGF165 and PlGF-2 promoted DRG growth cone formation with an effect similar to that of nerve growth factor, whereas the Flt-1-specific ligand, PlGF-1, and the KDR/Flt-4 ligand, VEGF-D, had no effect. The chemorepellent NP-1 ligand, semaphorin 3A, antagonized the response to VEGF and PlGF-2. The specific KDR inhibitor, SU5614, did not affect the anti-chemorepellent effects of VEGF and PlGF-2, whereas a novel, specific antagonist of VEGF binding to NP-1, called EG3287, prevented inhibition of growth cone collapse. VEGF stimulated prostacyclin and prostaglandin E2 production in DRG cultures that was blocked by inhibitors of cyclooxygenases; the anti-chemorepellent activities of VEGF and PlGF-2 were abrogated by cyclooxygenase inhibitors, and a variety of prostacyclin analogues and prostaglandins strikingly inhibited growth cone collapse. These findings support a specific role for NP-1 in mediating neurotrophic actions of VEGF family members and also identify a novel role for prostanoids in the inhibition of neuronal chemorepulsion.     

Association of VEGF genetic polymorphisms with prostate carcinoma risk and clinical outcome

OBJECTIVES: Vascular endothelial growth factor (VEGF) is a potent stimulus of angiogenesis that has an important role in many human malignancies including prostate carcinoma (PCa). We evaluated the role of the functional VEGF polymorphisms as genetic markers for PCa susceptibility and prognosis. METHODS: The study included 101 patients with PCa and [corrected] 100 age-matched healthy men. The VEGF genotypes -1154G>A were identified by allele-specific polymerase chain reaction (AS-PCR) and the genotypes -634G>C and 936C>T were identified by restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). RESULTS: A negative association was found between VEGF -1154AA genotype and PCa risk (OR=0.27; P=0.009). Furthermore, the presence of the VEGF -1154A allele appeared to be associated with a decreased [corrected] risk of higher tumor grade (OR=0.37; P=0.01). A significant increased risk of prostate cancer was associated with the VEGF -634 (GC+CC) combined genotype (OR=1.95; P=0.02). The VEGF -634C allele was associated with the aggressive phenotype of prostate cancer as defined by the high histological grade (OR=3.48; P=0.007). The VEGF -1154A/-634G haplotype was negatively associated with PCa risk (OR=0.48; P=0.005) and high tumor grade compared to low grade (OR=0.37; P=0.02). CONCLUSIONS: Genetic variations in the VEGF may predict not only PCa risk but also tumor aggressiveness.     

Fibrillation of α‐lactalbumin: Effect of crocin and safranal,two natural small molecules from Crocus sativus

Formation of toxic amyloid structures is believed to be associated with various late‐onset neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The fact that many proteins in addition to those that are associated with clinical conditions have the potential to form amyloid fibrils in vitro provides opportunities for studying the fundamentals of protein aggregation and amyloid formation in model systems. Accordingly, considerable interest and effort has been directed toward developing small molecules to inhibit the formation of fibrillar assemblies and their associated toxicities. In the present study, we investigated the inhibitory effect of crocin and safranal, two principal components of saffron, on fibrillation of apo‐α‐lactalbumin (a‐α‐LA), used as a model protein, under amyloidogenic conditions. In the absence of any ligand, formation of soluble oligomers became evident after 18 h of incubation, followed by subsequent appearance of mature fibrils. Upon incubation with crocin or safranal, while transition phase to monomeric beta structures was not significantly affected, formation of soluble oligomers and following fibrillar assemblies were inhibited. While both safranal and crocin had the ability to bind to hydrophobic patches provided in the intermediate structures, and thereby inhibit protein aggregation, crocin was found more effective, possibly due to its simultaneous hydrophobic and hydrophilic character. Cell viability assay indicated that crocin could diminish toxicity while safranal act in reverse order. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 854–865, 2010.     

A fluorescence-based assay for indoleamine 2,3-dioxygenase

A rapid and sensitive fluorescence-based bioassay for determination of indoleamine 2,3-dioxygenase (IDO) activity has been developed. This assay relies on the quantification of the amount of kynurenine produced in the assay medium by fluorescence and complements the standard absorbance and high-performance liquid chromatography (HPLC) assay methods. The fluorescence method has limits of detection similar to those of the standard assay methods. Measured activities of IDO, including in the presence of tryptophan-based inhibitors, were in statistical agreement with the absorbance and HPLC assay methods. The fluorescence-based assay was also suitable for assessment of IDO inhibition by compounds that are incompatible with the absorbance method.     

Somatic mutations contribute to genotypic diversity in sterile and fertile populations of the threatened shrub, Grevillea rhizomatosa (Proteaceae)

Background and Aims

Grevillea rhizomatosa is a spreading shrub which exhibits multiple breeding strategies within a narrow area in the fire-prone heathlands of eastern Australia. Reproductive strategies include self-compatibility, self-incompatibility and clonality (with and without sterility). The close proximity of contrasting breeding systems provides an opportunity to explore the evolution of sterility and to compare and contrast the origins of genotypic diversity (recombinant or somatic) against degrees of sexual expression.

Methods

ISSR markers for 120 band positions (putative loci) were used to compare genetic diversity among five populations at a macro-scale of 5 m between samples (n = 244 shrubs), and at a micro-scale of nearest neighbours for all plants in five 25-m² quadrats with contrasting fertilities (n = 162 shrubs). Nearest-neighbour sampling included several clusters of connected ramets. Matrix incompatibility (MIC) analyses were used to evaluate the relative contribution of recombination and somatic mutation to genotype diversity.

Key Results

High levels of genotypic diversity were found in all populations regardless of fertilities (fertile populations, G/N ≥ 0·94; sterile populations, G/N ≥ 0·97) and most sterile populations had a unique genetic profile. Somatic mutations were detected along connected ramets in ten out of 42 ramet clusters. MIC analyses showed that somatic mutations have contributed to diversity in all populations and particularly so in sterile populations.

Conclusions

Somatic mutations contribute significantly to gene diversity in sterile populations of Grevillea rhizomatosa, the accumulation of which is the likely cause of male and female sterility. High levels of genetic diversity therefore may not always be synonymous with sexual fitness and genetic health. We hypothesize that frequent fires drive selection for clonal reproduction, at the cost of flowering such that sexual functions are not maintained through selection, and the build-up of somatic mutations in meristems results in high genotype diversity at the cost of pollen and ovule fertilities.     

Impacts of very warm temperature on egg production rates of three Acartiidae (Crustacea,Copepoda) in a Northern African lagoon

The daily Egg Production Rate (EPR) of the three Acartiidae copepods namely Acartia clausi, Paracartia latisetosa and Paracartia grani in the North Lagoon of Tunis was compared to temperature and food availability every season from March, 2003 to February, 2004 corresponding to the major seasonal peaks of their abundance and over a 20-days period. The daily EPR was evaluated by assuming a direct correlation with chlorophyll a, particulate organic carbon (POC), and some easily extractable macromolecular compounds from the seston, such as proteins, carbohydrates and lipids. The results showed significant differences in EPR between seasons and species. Temperature and salinity negatively affected the fecundity of A. clausi and positively that of P. latisetosa and P. grani. The A. clausi EPR was supported by chlorophyll a and sestonic proteins while that of P. latisetosa and P. grani correlated with POC suggesting that these two taxa feed on microzooplankton and detritus more than on phytoplankton. Overall, EPR data showed potential population recruitment evidently the highest in P. latisetosa in summer, P. grani in autumn and A. clausi in winter. Our results indicate that temperature and food quality are the determining factors of both abundance and reproduction of the copepods under study.     

Factor h and properdin recognize different epitopes on renal tubular epithelial heparan sulfate

During proteinuria, renal tubular epithelial cells become exposed to ultrafiltrate-derived serum proteins, including complement factors. Recently, we showed that properdin binds to tubular heparan sulfates (HS). We now document that factor H also binds to tubular HS, although to a different epitope than properdin. Factor H was present on the urinary side of renal tubular cells in proteinuric, but not in normal renal tissues and colocalized with properdin in proteinuric kidneys. Factor H dose-dependently bound to proximal tubular epithelial cells (PTEC) in vitro. Preincubation of factor H with exogenous heparin and pretreatment of PTECs with heparitinase abolished the binding to PTECs. Surface plasmon resonance experiments showed high affinity of factor H for heparin and HS (K(D) values of 32 and 93 nm, respectively). Using a library of HS-like polysaccharides, we showed that chain length and high sulfation density are the most important determinants for glycosaminoglycan-factor H interaction and clearly differ from properdin-heparinoid interaction. Coincubation of properdin and factor H did not hamper HS/heparin binding of one another, indicating recognition of different nonoverlapping epitopes on HS/heparin by factor H and properdin. Finally we showed that certain low anticoagulant heparinoids can inhibit properdin binding to tubular HS, with a minor effect on factor H binding to tubular HS. As a result, these heparinoids can control the alternative complement pathway. In conclusion, factor H and properdin interact with different HS epitopes of PTECs. These interactions can be manipulated with some low anticoagulant heparinoids, which can be important for preventing complement-derived tubular injury in proteinuric renal diseases.     

Extracellular 14-3-3 from human lung epithelial cells enhances MMP-1 expression

Airway remodelling in asthma involves various mediators modulating the production/breakdown of collagen by lung fibroblasts. Matrix metalloproteinase-1 (MMP-1) plays an important role in collagen breakdown. We recently showed that epithelial cell-derived extracellular form of 14-3-3σ is an important inducer of MMP-1 expression in skin fibroblasts. Thus, we hypothesized that 14-3-3 proteins are important regulators of MMP-1 expression in the respiratory airway. We examined the presence of extracellular 14-3-3 proteins in conditioned media obtained from primary lung epithelial cells, A549 and HS24 cells, and their effect on MMP-1 expression by lung fibroblasts (IMR-90). In addition, we evaluated IMR-90 response to 14-3-3 proteins in the presence of transforming growth factor-β(1) (TGF-β(1)), a cytokine known to decrease MMP-1 expression by fibroblasts. Extracellular 14-3-3α/β, but not -σ, is released by the human-derived lung epithelial cell lines, A549 and HS24. Unlike dermal fibroblasts, IMR-90 cells do not produce MMP-1 in response to 14-3-3σ. Conversely, MMP-1 production was induced following treatment of IMR-90 with recombinant or lung epithelial cell-derived 14-3-3α/β. These findings were also confirmed using primary human bronchial epithelial cells and lung fibroblasts obtained from non-asthmatic patients. The MMP-1-inducing effect of 14-3-3α/β on IMR-90 was not inhibited by TGF-β(1). Lung epithelial cell-derived 14-3-3α/β has a potent MMP-1-inducing effect on airway fibroblasts. Modulation of MMP-1 by 14-3-3α/β, may be important in the alteration of collagenase production associated with airway remodelling in obstructive lung diseases. Our data indicate that 14-3-3 proteins may be potential targets for future therapeutic strategies aimed at modulating tissue remodelling in asthma.     

Flavonoid Baicalein Modulates H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Mitogen-Activated Protein Kinases Activation and Cell Death in SK-N-MC Cells

It is believed that ROS-induced oxidative stress triggers numerous signaling pathways which are involved in neurodegenerative diseases, including Alzheimer’s disease. To find the effective drugs for neurodegenerative diseases, the deep delve into molecular mechanisms underlie these diseases is necessary. In the current study, we investigated the effects of flavonoid baicalein on H₂O₂-induced oxidative stress and cell death in SK-N-MC cells. Our results revealed that the treatment of SK-N-MC cells with H₂O₂ led to a decrease in cell viability through phosphorylation and activation of extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs) pathways followed by increase in Bax/Bcl2 ratio and initiation of caspase-dependent apoptotic pathways. In addition, our results showed that the exposure of SK-N-MC cells to H₂O₂ ended up in reduction of glutathione (GSH) levels of SK-N-MC cells via JNK/ERK-mediated down-regulation of γ-glutamyl-cysteine synthetase (γ-GCS) expression. Our results demonstrated that flavonoid baicalein protected against H₂O₂-induced cell death by inhibition of JNK/ERK pathways activation and other key molecules in apoptotic pathways, including blockage of Bax and caspase-9 activation, induction of Bcl-2 expression and prevention of cell death. Baicalein supported intracellular defense mechanisms through maintaining GSH levels in SK-N-MC cells by the removal of inhibition effects of JNK/ERK pathways from γ-GCS expression. In addition, baicalein attenuated lipid and protein peroxidation and intracellular reactive oxygen species in SK-N-MC cells. In accordance with these observations, baicalein can be a promising candidate in antioxidant therapy and designing of natural-based drug for ROS-induced neurodegenerative disorders.