Retransformation of a revertant cell line with the adenovirus E1 oncogenes and vanadate

We have isolated a revertant cell line (G5) from an adenovirus transformed rat cell line (F4) which failed to express the integrated viral oncogenes. To determine whether the reversion mutation was acting in cis or trans the G5 cells were co-transfected with an E1 gene bearing expression plasmid and a neomycin phosphotransferase bearing plasmid. G418-resistant colonies were picked and shown to express the E1 proteins and to be tumorigenic. This re-transformation could be partially mimicked by treatment with vanadate, an inhibitor of phosphotyrosine phosphatases. These results show that the continued presence of the E1 proteins was required to maintain the transformed phenotype, and that the reversion mutation was a cis-acting event affecting directly the integrated E1 genes.     

Restoration of ultrastructural and biochemical changes in alloxan-induced diabetic rat sciatic nerve on treatment with Na3VO4 and Trigonella—a promising antidiabetic agent

Vanadium has been reported to have broad pharmacological activity both in vitro and in vivo. Vanadium compound, sodium orthovanadate, Na₃VO₄, is well known for its hypoglycaemic effects. However, Na₃VO₄ exerts these effects at relatively high doses (0.6 mg/ml) and exhibit several toxic effects. In the present study lower doses of Na₃VO₄ (0.2 mg/ml) are combined with Trigonella foenum graecum seed powder (TSP), another hypoglycaemic agent, to reduce its toxicity without compromising its antidiabetic potential. The efficacy of the lower doses of Na₃VO₄ has been investigated in restoring the altered glucose metabolism and histological structure in the sciatic nerves in 21 and 60 days alloxan diabetic rats. A portion of the glucose was found to be channelled from the normal glycolytic route to polyol pathway, evident by the reduced hexokinase activity and increased polyol pathway enzymes aldose reductase and sorbitol dehydrogenase activity causing accumulation of sorbitol and fructose in diabetic conditions. Ultrastructural observation of the sciatic nerve showed extensive demylination and axonal loss after eight weeks of diabetes induction. Blood glucose levels increased in diabetic rats were normalized with the lower dose of vanadium and Trigonella treatment. The treatment of the diabetic rats with vanadium and Trigonella prevented the activation of the polyol pathway and sugar accumulations. The sciatic nerves were also protected against the structural abnormalities found in diabetes with Trigonella foenum graecum as well as Na₃VO₄. Results suggest that lower doses of Na₃VO₄ may be used in combination with TSP as an efficient antidiabetic agent to effectively control the long-term complications of diabetes in tissues like peripheral nerve.     

Amelioration of altered antioxidant status and membrane linked functions by vanadium andTrigonella in alloxan diabetic rat brains

Trigonella foenum graecum seed powder (TSP) and sodium orthovanadate (SOV) have been reported to have antidiabetic effects. However, SOV exerts hypoglycemic effects at relatively high doses with several toxic effects. We used low doses of vanadate in combination with TSP and evaluated their antidiabetic effects on antioxidant enzymes and membrane-linked functions in diabetic rat brains. In rats, diabetes was induced by alloxan monohydrate (15 mg/100 g body wt.) and they were treated with 2 IU insulin, 0.6 mg/ml SOV, 5% TSP and a combination of 0.2 mg/ml SOV with 5% TSP for 21 days. Blood glucose levels, activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), Na⁺/K⁺ ATPase, membrane lipid peroxidation and fluidity were determined in different fractions of whole brain after 21 days of treatment. Diabetic rats showed high blood glucose (P < 0.001), decreased activities of SOD, catalase and Na⁺/K⁺ ATPase (P < 0.01,P < 0.001 andP < 0.01), increased levels of GPx and MDA (P < 0.01 andP < 0.001) and decreased membrane fluidity (P < 0.01). Treatment with different antidiabetic compounds restored the above-altered parameters. Combined dose ofTrigonella and vanadate was found to be the most effective treatment in normalizing these alterations. Lower doses of vanadate could be used in combination with TSP to effectively counter diabetic alterations without any toxic effects.     

Characteristics of in vivo murine erythropoietic response to sodium orthovanadate

Current knowledge about the effects of vanadium compounds on erythropoiesis is still reduced and even contradictory. The aim of this work was to evaluate the in vivo effects of a single dose of sodium orthovanadate (OV, 33 mg/kg i.p.) on CF-1 mice in a time course study (0-8 days). Murine erythropoiesis was assessed through a combinatory of experimental approaches. Classical peripheral and bone marrow (BM) hematological parameters were determined. Erythroid maturation in blood stream and hemopoietic tissues (59Fe uptake assays), BM erythroid progenitor frequency (clonogenic assays) and erythroid crucial protein expressions for commitment and survival: GATA-1, erythropoietin receptor (Epo-R) and Bcl-xL (immunoblottings) were evaluated. Neither BM cellularities nor BM viabilities changed noticeably during the study. Peripheral reticulocytes showed a biphasic increment on days 2 and 8 post-OV. hematocrits enhanced transiently between days 2 and 4. 59Fe uptake percentages enhanced in peripheral blood nearly two-fold over control values between 4 and 8 days (p<0.01) without changes in BM and spleen. Additionally, mature erythroid BM compartments: polychromatophilic erythroblasts and orthochromatic normoblasts increased by the eighth day. BFU-E colonies remained near basal values during the whole experience, whilst CFU-E colonies raised 60% over control at 8 days post-OV (p<0.05). GATA-1 and Epo-R were significantly over-expressed from the third until the end of the experimental protocol (p<0.01). Surprisingly, Bcl-xL showed a constitutive expression pattern without changes during the experience. Experimental data let us suggest that OV does not to cause bone marrow cytotoxicity and that it accelerates maturation of BM committed erythroid precursors. Moreover, there are significant correlations among erythroid-related protein expressions: GATA-1 and Epo-R and the frequency of CFU-E. In addition, Bcl-xL expression invariance during the time course study would indicate that the stimulatory effect of OV treatment on erythropoiesis was mainly exerted on the maturation of red cell precursors rather than on the antiapoptosis of erythroid terminal progenitors.     

TREATMENT OF RAT PROXIMAL AND DISTAL COLONIC CELLS WITH SODIUM ORTHOVANADATE ENHANCES THEIR ADHESION AND SURVIVAL IN PRIMARY CULTURE

We have studied the effect of sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, on primary cultures of colonocytes and stromal cells. Everted proximal and distal colonic tissue of adult rats were disintegrated by a collagenase/dispase solution for 60 min at 37°C to prepare viable gland fragments and isolated cells. Cell preparations were inoculated onto plastic substratum or cytodex-3 microcarriers in a defined maintenance medium or in 1% fetal calf serum media. Incorporation of sodium orthovanadate (≥50 μm) in these media constantly enhanced the survival (cell enumeration and trypan blue exclusionP<0.05) and the adhesion (up to four-fold by crystal violet staining,P<0.01) of colonocytes (characterized by cytokeratin-18, transforming growth factor-α or alkaline phosphatase expression) and stromal cells. Removal of sodium orthovanadate from culture media restored cellular death processes. Incorporation of 10 mmn-butyric acid did not promote cell adhesion and survival except for distal cells exposed to 2 mm sodium orthovanadate. Besides studies in the regulation of anoikis in primary culture, the model will help to assay the influences of dietary and growth factors on the biology of non-cancerous colonic cells.     

Selective blockade of axonogenesis in cultured hippocampal neurons by the tyrosine phosphatase inhibitor orthovanadate

Protein tyrosine phosphorylation has been implicated in several aspects of neurite outgrowth regulation. To address specific roles in early neuronal morphogenesis, hippocampal neurons in culture were treated with the tyrosine phosphatase inhibitor orthovanadate. This treatment completely suppressed axon formation, yet enhanced formation of minor neurites. The inhibition of axonogenesis was dose dependent and occurred in parallel with a marked increase in cellular phosphotyrosine immunoreactivity, which was especially concentrated within neuritic growth cones and showed partial colocalization with f-actin. Both the blockade of axonogenesis and the elevation of phosphotyrosine were completely reversible. An additional and unexpected effect of orthovanadate was the appearance of many binucleate neurons. Immunoblotting experiments using a phosphotyrosine-specific antibody revealed an orthovanadate-induced reversible hyperphosphorylation of several protein bands, especially of two at 115 and 125 kD. These data suggest a potentially important role for tyrosine phosphatases and their phosphoprotein substrates in axonogenesis. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 17–28, 1998     

Akt inhibits MLK3/JNK3 signaling by inactivating Rac1: a protective mechanism against ischemic brain injury

The overall goal of this study was to determine the molecular basis by which mixed-lineage kinase 3 (MLK3) kinase and its signaling pathways are negatively regulated by the pro-survival Akt pathway in cerebral ischemia. We demonstrated that tyrosine phosphorylation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) underlies the increased Akt-Ser473 phosphorylation by orthovanadate. Co-immunoprecipitation analysis revealed that endogenous Akt physically interacts with Rac1 in the hippocampal CA1 region, and this interaction is promoted on tyrosine phosphatase inhibition. The elevated Akt activation can deactivate MLK3 by phosphorylation at the Ser71 residue of Rac1, a small Rho family of guanidine triphosphatases required for MLK3 autophosphorylation. Subsequently, inhibition of c-Jun N-terminal kinase 3 (JNK3) results in decreased serine phosphorylation of 14-3-3, a cytoplasmic anchor of Bax, and prevents ischemia-induced mitochondrial translocation of Bax, release of cytochrome c and activation of caspase 3. At the same time, the expression of Fas-ligand decreases in the CA1 region after inhibition of c-Jun activation. The neuroprotective effect of Akt activation is significant in the CA1 region after global cerebral ischemia. Our results suggest that the activation of the pro-apoptotic MLK3/JNK3 cascade induced by ischemic stress can be suppressed through activation of the anti-apoptotic phosphatidylinositol 3-kinase/Akt pathway, which provides a direct link between Akt and the family of stress-activated kinases.     

Modulation of glucose transporter (GLUT4) by vanadate and Trigonella in alloxan-diabetic rats

Oral administration of vanadate is an effective treatment for diabetes in animal models. However, vanadate exerts these effects at high doses and several toxic effects are produced. Low doses of vanadate are relatively safe but are unable to elicit any antidiabetic effect. The present study explored the prospect of using low doses of vanadate in combination with Trigonella seed powder (TSP) to evaluate their antidiabetic effect in alloxan-diabetic rats. Alloxan-diabetic rats were treated with insulin, vanadate, TSP and vanadate and TSP in combination for 3 weeks. The effect of these antidiabetic compounds was examined on general physiological parameters and distribution of glucose transporter (GLUT4) in skeletal muscle by immunoblotting and immunohistochemistry. Treatment of alloxan-diabetic rats with insulin, vanadate, TSP and vanadate in combination with TSP revived normoglycemia and restored the disturbances in the distribution of GLUT4 in skeletal muscle. TSP treatment was only partially effective in the restoration of diabetic alterations. The treatment of diabetic rats with combined doses of vanadate and TSP was most effective in the normalization of plasma glucose levels and correction of altered GLUT4 distribution.     

Structure and photoluminescent properties of green‐emitting terbium‐doped GdV1−xPxO4 phosphor prepared by solution combustion method

Terbium‐doped gadolinium orthovanadate (GdVO₄:Tb³⁺), orthophosphate monohydrate (GdPO₄·H₂O:Tb³⁺) and orthovanadate–phosphate (GdV,PO₄:Tb³⁺) powder phosphors were synthesized using a solution combustion method. X‐Ray diffraction analysis confirmed the formation of crystalline GdVO₄, GdPO₄·H₂O and GdV,PO₄. Scanning electron microscopy images showed that the powder was composed of an agglomeration of particles of different shapes, ranging from spherical to oval to wire‐like structures. The chemical elements present were confirmed by energy dispersive spectroscopy, and the stretching mode frequencies were determined by Fourier transform infrared spectroscopy. UV–visible spectroscopy spectra showed a strong absorption band with a maximum at 200 nm assigned to the absorption of VO₄^3? and minor excitation bands assigned to f → f transitions of Tb³⁺. Four characteristic emission peaks were observed at 491, 546, 588 and 623 nm, and are attributed to ⁵D₄ → ⁷F_j (j = 6, 5, 4 and 3). The photoluminescent prominent green emission peak (⁵D₄ → ⁷F₅) was centred at 546 nm. The structure and possible mechanism of light emission from GdV_1?xP_xO₄:% Tb³⁺ are discussed. Copyright © 2016 John Wiley & Sons, Ltd.     

Regulation of anthocyanin and lignin synthesis in Petunia hybrida cell suspensions

In Petunia hybrida cv. Violet 30 cell suspensions the phenylpropanoid pathway can be induced to produce lignin and anthocyanins. Orthovanadate addition leads to lignin accumulation, subculturing the cells using small inoculum sizes (<2 g fresh weight l^-1) gives rise to both anthocyanin and lignin production. Orthovanadate has a negative effect on cell growth. By replacing the medium, one day after orthovanadate addition, by medium without elicitor, we were able to restore growth without disturbing the lignin accumulation. The activity of phenylalanine ammonia-lyase (PAL) increased immediately after orthovanadate addition; this increase stopped upon medium replacement without affecting the lignin production. Reduction of the NAA concentration from 2 mg l^-1 to 0.1 mg l^-1, subsequent to the elicitation by orthovanadate or dilution stress, gave rise to a further increase in the production of lignin and anthocyanins respectively. Decreasing the NAA concentration without a prior elicitation, didn't have any effect on either PAL activity or product formation.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BSA bovine serum albumine - FW fresh weight - NAA naphthaleneacetic acid - PAL phenylalanine ammonia-lyase - PPP phenyl propanoid pathway