Vascular endothelial growth factor (VEGF) and VEGF-receptor expression in placenta of hyperglycemic pregnant women

Hyperglycemia occurs in a variety of conditions such as overt diabetes, gestational diabetes and mild hyperglycemia, all of which are generally defined based on the oral glucose tolerance test and glucose profiles. Whereas diabetes has received considerable attention in recent decades, few studies have examined the mechanisms of mild hyperglycemia and its associated disturbances. Mild gestational hyperglycemia is associated with macrosomia and a high risk of perinatal mortality. Morphologically, the placenta of these women is characterized by an increase in the number of terminal villi and capillaries, presumably as part of a compensatory mechanism to maintain homeostasis at the maternal-fetal interface. In this study, we analised the expression of VEGF and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR) in placentas from mildly hyperglycemic women. This expression was compared with that of normoglycemic women and women with gestational and overt diabetes. Immunohistochemistry revealed strong staining for VEGF and VEGFR-2 in vascular and trophoblastic cells of mildly hyperglycemic women, whereas the staining for VEGFR-1 was discrete and limited to the trophoblast. The pattern of VEGF and VEGF-receptor reactivity in placentas from women with overt diabetes was similar to that of normoglycemic women. In women with gestational diabetes, strong staining for VEGFR-1 was observed in vascular and trophoblastic cells whereas VEGF and VEGFR-2 were detected only in the trophoblast. The expression of these proteins was confirmed by western blotting, which revealed the presence of an additional band of 75 kDa. In the decidual compartment, only extravillous trophoblast reacted with all antibodies. Morphological analysis revealed collagen deposition around large arteries in all groups with altered glycemia. These findings indicate a placental response to altered glycemia that could have important consequences for the fetus. The change in the placental VEGF/VEGFR expression ratio in mild hyperglycemia may favor angiogenesis in placental tissue and could explain the hypercapillarization of villi seen in this gestational disturbance. (C) 2010 Elsevier Ltd. All rights reserved.

Neurofilament heavy subunit in cerebrospinal fluid: A biomarker of amyotrophic lateral sclerosis?

The objectives of this study were to investigate the presence of the three neurofilament subunits, ubiquitin, proteasome and 3-nitrotyrosine, in CSF samples of ALS patients. CSF samples were obtained by lumbar puncture from 10 ALS patients and six controls. All samples were analysed by Western blotting. Results revealed that neurofilament heavy subunit was identified in 70% of ALS cases and we conclude that this subunit may be a promising biomarker for clinical diagnosis of ALS.

L-NAME Treatment Enhances Exercise-induced Content of Myocardial Heat Shock Protein 72 (Hsp72) in Rats

Background/Aim: Nitric oxide (NO) modulates the expression of the chaperone Hsp72 in the heart, and exercise stimulates both NO production and myocardial Hsp72 expression. The main purpose of the study was to investigate whether NO interferes with an exercise-induced myocardial Hsp72 expression. Methods: Male Wistar rats (70-100 days) were divided into control (C, n= 12), L-NAME-treated (L, n= 12), exercise (E, n= 13) and exercise plus L-NAME-treated (EL, n= 20) groups. L-NAME was given in drinking water (700 mg. L(-1)) and the exercise was performed on a treadmill (15-25 m.min(-1), 40-60 min. day(-1)) for seven days. Left ventricle (LV) protein Hsp content, NOS and phosphorylated-NOS (p-NOS) isoforms were measured using Western blotting. The activity of NOS was assayed in LV homogenates by the conversion of [(3)H] L-arginine to [(3)H] L-citrulline. Results: Hsp72 content was increased significantly (223%; p < 0.05) in the E group compared to the C group, but exercise alone did not alter the NOS content, p-NOS isoforms or NOS activity. Contrary to our expectation, L-NAME enhanced (p < 0.05) the exercise-induced Hsp72 content (EL vs. C, L and E groups = 1019%, 548% and 457%, respectively). Although the EL group had increased stimulatory p-eNOS(Ser1177) (over 200%) and decreased inhibitory p-nNOS(Ser852) (similar to 50%) compared to both the E and L groups (p < 0.05), NOS activity was similar in all groups. Conclusions: Our results suggest that exercise-induced cardiac Hsp72 expression does not depend on NO. Conversely, the in vivo L-NAME treatment enhances exercise-induced Hsp72 production. This effect may be due to an increase in cardiac stress. Copyright (C) 2011 S. Karger AG, Basel

An endogenous regulator of inflammation, resolvin E1, modulates osteoclast differentiation and bone resorption

Background and purpose: The inflammation-resolving lipid mediator resolvin E1 (RvE1) effectively stops inflammation-induced bone loss in vivo in experimental periodontitis. It was of interest to determine whether RvE1 has direct actions on osteoclast (OC) development and bone resorption. Experimental approach: Primary OC cultures derived from mouse bone marrow were treated with RvE1 and analysed for OC differentiation, cell survival and bone substrate resorption. Receptor binding was measured using radiolabelled RvE1. Nuclear factor (NF)-kappa B activation and Akt phosphorylation were determined with western blotting. Lipid mediator production was assessed with liquid chromatography tandem mass spectrometry. Key results: OC growth and resorption pit formation were markedly decreased in the presence of RvE1. OC differentiation was inhibited by RvE1 as demonstrated by decreased number of multinuclear OC, a delay in the time course of OC development and attenuation of receptor activator of NF-kappa B ligand-induced nuclear translocation of the p50 subunit of NF-kappa B. OC survival and apoptosis were not altered by RvE1. Messenger RNA for both receptors of RvE1, ChemR23 and BLT(1) is expressed in OC cultures. Leukotriene B(4) (LTB(4)) competed with [(3)H] RvE1 binding on OC cell membrane preparations, and the LTB(4) antagonist U75302 prevented RvE1 inhibition of OC growth, indicating that BLT(1) mediates RvE1 actions on OC. Primary OC synthesized the RvE1 precursor 18R-hydroxy-eicosapentaenoic acid and LTB(4). Co-incubation of OC with peripheral blood neutrophils resulted in transcellular RvE1 biosynthesis. Conclusions and implications: These results indicate that RvE1 inhibits OC growth and bone resorption by interfering with OC differentiation. The bone-sparing actions of RvE1 are in addition to inflammation resolution, a direct action in bone remodelling.

Early Phase of Allergic Airway Inflammation in Diabetic Rats: Role of Insulin on the Signaling Pathways and Mediators

Background: Allergic lung inflammation is impaired in diabetic rats and is restored by insulin treatment. In the present study we investigated the effect of insulin on the signaling pathways triggered by allergic inflammation in the lung and the release of selected mediators. Methods: Diabetic male Wistar rats (alloxan, 42 mg/kg, i.v., 10 days) and matching controls were sensitized by s.c. injections of ovalbumin (OA) in aluminium hydroxide, 14 days before OA (1 mg/0.4 ml) or saline intratracheal challenge. A group of diabetic rats were treated with neutral protamine Hagedorn insulin (NPH, 4 IU, s.c.), 2 h before the OA challenge. Six hours after the challenge, bronchoalveolar lavage (BAL) was performed for mediator release and lung tissue was homogenized for Western blotting analysis of signaling pathways. Results: Relative to non-diabetic rats, the diabetic rats exhibited a significant reduction in OA-induced phosphorylation of the extracellular signal-regulated kinase (ERK, 59%), p38 (53%), protein kinase B (Akt, 46%), protein kinase C (PKC)-alpha (63%) and PKC-delta (38%) in lung homogenates following the antigen challenge. Activation of the NF-kappa B p65 subunit and phosphorylation of I kappa B alpha were almost suppressed in diabetic rats. Reduced expression of inducible nitric oxide synthase (iNOS, 32%) and cyclooxygenase-2 (COX-2, 46%) in the lung homogenates was also observed. The BAL concentration of prostaglandin (PG)-E(2), nitric oxide (NO) and interleukin (IL)-6 was reduced in diabetic rats (74%, 44% and 65%, respectively), whereas the cytokine-induced neutrophil chemoattractant (CINC)-2 concentration was not different from the control animals. Treatment of diabetic rats with insulin completely or partially restored all of these parameters. This protocol of insulin treatment only partially reduced the blood glucose levels. Conclusion: The data presented show that insulin regulates MAPK, PI3K, PKC and NF-kappa B pathways, the expression of the inducible enzymes iNOS and COX-2, and the levels of NO, PGE(2) and IL-6 in the early phase of allergic lung inflammation in diabetic rats. It is suggested that insulin is required for optimal transduction of the intracellular signals that follow allergic stimulation. Copyright (C) 2010 S. Karger AG, Basel

Complement 4 phenotypes and genotypes in Brazilian patients with classical 21-hydroxylase deficiency

The aim of this work was to analyse C4 genotypes, C4 protein levels, phenotypes and genotypes in patients with the classical form of 21-hydroxylase deficiency. Fifty-four patients from 46 families (36 female, 18 male; mean age 10.8 years) with different clinical manifestations (31 salt-wasting; 23 simple-virilizing) were studied. Taq I Southern blotting was used to perform molecular analysis of the C4/CYP21 gene cluster and the genotypes were defined according to gene organization within RCCX modules. Serum C4 isotypes were assayed by enzyme-linked immunosorbent assay. The results revealed 12 different haplotypes of the C4/CYP21 gene cluster. Total functional activity of the classical pathway (CH50) was reduced in individuals carrying different genotypes because of low C4 concentrations (43% of all patients) to complete or partial C4 allotype deficiency. Thirteen of 54 patients presented recurrent infections affecting the respiratory and/or the urinary tracts, none of them with severe infections. Low C4A or C4B correlated well with RCCX monomodular gene organization, but no association between C4 haplotypes and recurrent infections or autoimmunity was observed. Considering this redundant gene cluster, C4 seems to be a well-protected gene segment along the evolutionary process.

Vitellin-binding proteins in the nematode Oscheius tipulae (Nematoda, Rhabditida)

We describe the first application of a non-radioactive ligand-blotting technique to the characterization of proteins interacting with nematode vitellins. Chromatographically purified vitellins from the free-living nematode Oscheius tipulae were labeled with fluorescein in vitro. Ligand-blotting assays with horseradish peroxidase-conjugated anti-fluorescein antibodies showed that labeled vitellins reacted specifically with a polypeptide of approximately 100 kDa, which we named P100. This polypeptide is a specific worm`s vitellin-binding protein that is present only in adult worms. Blots containing purified O. tipulae vitellin preparations showed no detectable signal in the 100 kDa region, ruling out any possibility of yolk polypeptides self-assembling under the conditions used in our assay. Experiments done in the presence of alpha-methyl mannoside ruled out the possibility of vitellins binding to P100 through mannose residues. Triton X-114 fractionation of whole worm extracts showed that P100 is either a membrane protein or has highly hydrophobic regions. (C) 2008 Elsevier Inc. All rights reserved.

Exercise training enhances rat pancreatic islets anaplerotic enzymes content despite reduced insulin secretion

Endurance exercise has been shown to reduce pancreatic islets glucose-stimulated insulin secretion (GSIS). Anaplerotic/cataplerotic pathways are directly related to GSIS signaling. However, the effect of endurance training upon pancreatic islets anaplerotic enzymes is still unknown. In this sense, we tested the hypothesis that endurance exercise decreases GSIS by reducing anaplerotic/cataplerotic enzymes content. Male Wistar rats were randomly assigned to one of the four experimental groups as follows: control sedentary group (CTL), trained 1 day per week (TRE1x), trained 3 days per week (TRE3x) and trained 5 days per week (TRE5x) and submitted to an 8 weeks endurance-training protocol. After the training protocol, pancreatic islets were isolated and incubated with basal (2.8 mM) and stimulating (16.7 mM) glucose concentrations for GSIS measurement by radioimmunoassay. In addition, pyruvate carboxylase (PYC), pyruvate dehydrogenase (PDH), pyruvate dehydrogenase kinase 4 (PDK4), ATP-citrate lyase (ACL) and glutamate dehydrogenase (GDH) content were quantified by western blotting. Our data showed that 8 weeks of chronic endurance exercise reduced GSIS by 50% in a dose-response manner according to weekly exercise frequency. PYC showed significant twofold increase in TRE3x. PYC enhancement was even higher in TRE5x (p < 0.0001). PDH and PDK4 reached significant 25 and 50% enhancement, respectively compared with CTL. ACL and GDH also reported significant 50 and 75% increase, respectively. The absence of exercise-induced correlations among GSIS and anaplerotic/cataplerotic enzymes suggests that exercise may control insulin release by activating other signaling pathways. The observed anaplerotic and cataplerotic enzymes enhancement might be related to beta-cell surviving rather than insulin secretion.

Preliminary report: Leucine supplementation enhances glutamate dehydrogenase expression and restores glucose-induced insulin secretion in protein-malnourished rats

Low-protein diet impairs insulin secretion in response to nutrients and may induce several metabolic disorders including diabetes, obesity, and cardiovascular disease. In the present study, the influence of leucine supplementation on glutamate dehydrogenase (GDH) expression and glucose-induced insulin secretion (GIIS) was investigated in malnourished rats. Four groups were fed with different diets for 12 weeks: a normal-protein diet (17%) without or with leucine supplementation or a low (6%)-protein diet without (LP) or with leucine supplementation (LPL). Leucine (1.5%) was supplied in the drinking water. Western blotting analysis revealed reduced GIN! expression in LP, whereas LPL displayed improved GDH expression, similar to control. The GHS and leucinc-induced insulin release were also enhanced in LPL compared with LP and similar to those observed in rats fed a normal-protein diet without leucine supplementation. In addition, GDH allosteric activators produced an increased insulin secretion in LPL. These findings indicate that leucine supplementation was able to increase GDH expression leading to Cl IS restoration, probably by improved leucine metabolic pathways. (C) 2010 Elsevier Inc. All rights reserved.

Gamma-linolenic acid inhibits both tumour cell cycle progression and angiogenesis in the orthotopic C6 glioma model through changes in VEGF, Flt1, ERK1/2, MMP2, cyclin D1, pRb, p53 and p27 protein expression

Background: Gamma-linolenic acid is a known inhibitor of tumour cell proliferation and migration in both in vitro and in vivo conditions. The aim of the present study was to determine the mechanisms by which gamma-linolenic acid (GLA) osmotic pump infusion alters glioma cell proliferation, and whether it affects cell cycle control and angiogenesis in the C6 glioma in vivo. Methods: Established C6 rat gliomas were treated for 14 days with 5 mM GLA in CSF or CSF alone. Tumour size was estimated, microvessel density (MVD) counted and protein and mRNA expression measured by immunohistochemistry, western blotting and RT-PCR. Results: GLA caused a significant decrease in tumour size (75 +/- 8.8%) and reduced MVD by 44 +/- 5.4%. These changes were associated with reduced expression of vascular endothelial growth factor (VEGF) (71 +/- 16%) and the VEGF receptor Flt1 (57 +/- 5.8%) but not Flk1. Expression of ERK1/2 was also reduced by 27 +/- 7.7% and 31 +/- 8.7% respectively. mRNA expression of matrix metalloproteinase-2 (MMP2) was reduced by 35 +/- 6.8% and zymography showed MMP2 proteolytic activity was reduced by 32 +/- 8.5%. GLA altered the expression of several proteins involved in cell cycle control. pRb protein expression was decreased (62 +/- 18%) while E2F1 remained unchanged. Cyclin D1 protein expression was increased by 42 +/- 12% in the presence of GLA. The cyclin dependent kinase inhibitors p21 and p27 responded differently to GLA, p27 expression was increased (27 +/- 7.3%) while p21 remained unchanged. The expression of p53 was increased (44 +/- 16%) by GLA. Finally, the BrdU incorporation studies found a significant inhibition (32 +/- 11%) of BrdU incorporation into the tumour in vivo. Conclusion: Overall the findings reported in the present study lend further support to the potential of GLA as an inhibitor of glioma cell proliferation in vivo and show it has direct effects upon cell cycle control and angiogenesis. These effects involve changes in protein expression of VEGF, Flt1, ERK1, ERK2, MMP2, Cyclin D1, pRb, p53 and p27. Combination therapy using drugs with other, complementary targets and GLA could lead to gains in treatment efficacy in this notoriously difficult to treat tumour.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel alpha-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1). Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein`s biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1), showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of alpha-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian alpha-amylase activity in vitro.

Expression, Purification, Bioactivity, and Partial Characterization of a Recombinant Human Bone Morphogenetic Protein-7 Produced in Human 293T Cells

Bone morphogenetic protein-7 (BMP-7) is a secreted multifunctional growth factor of the TGF-beta superfamily, which is predominantly known for its osteoinductive properties and emerging potential for treatment of kidney diseases. The mature 34-38 kDa disulfide-linked homodimer protein plays a key role in the differentiation of mesenchymal cells into bone and cartilage. In this study, the full-length sequence of hBMP-7 was amplified and, then, cloned, expressed, and purified from the conditioned medium of 293T cells stably transfected with a lentiviral vector. The mature protein dimer form was properly secreted and recognized by anti-BMP-7 antibodies, and the protein was shown to be glycosilated by treatment with exoglycosidase, followed by western blotting. Moreover, the activity of the purified protein was demonstrated both in vitro, by alkaline phosphatase activity in C2C12 cells, and in vivo by induction of ectopic bone formation in Balb/c Nude mice after 21 days, respectively. This recombinant protein platform may be very useful for expression of different human cytokines and other proteins for medical applications.

Genomic organization and transcription analysis of the 195-bp satellite DNA in Trypanosoma cruzi

The 195-bp satellite DNA is the most abundant Trypanosoma cruzi repetitive sequence. Here we show by RNA blotting and RT-PCR that 195 SAT is intensely transcribed. We observed a positive correlation between the level of satellite RNA and the abundance of the satellite copies in the genome of T cruzi strains and that the satellite expression is not developmentally regulated. By analyzing CL Brener individual reads, we estimated that 195 SAT corresponds to approximately 5% of the CL Brener genome. 195 SAT elements were found in only 37 annotated contigs, indicating that a large number of satellite copies were not incorporated into the assembled data. The assembled satellite units are distributed in non-syntenic regions with Trypanosoma brucei and Leishmania major genomes, enriched with surface proteins, retroelements, RHS and hypothetical proteins. Satellite repeats were not observed in annotated subtelomeric regions. We report that 12 satellite sequences are truncated by the retroelement VIPER. (C) 2008 Elsevier B.V. All rights reserved.