Mesenchymal Stromal Cell Secretome Up-Regulates 47 kDa CXCR4 Expression,and Induce Invasiveness in Neuroblastoma Cell Lines

Neuroblastoma accounts for 15% of childhood cancer deaths and presents with metastatic disease of the bone and the bone marrow at diagnosis in 70% of the cases. Previous studies have shown that the Mesenchymal Stromal Cell (MSC) secretome, triggers metastases in several cancer types such as breast and prostate cancer, but the specific role of the MSC factors in neuroblastoma metastasis is unclear. To better understand the effect of MSC secretome on chemokine receptors in neuroblastoma, and its role in metastasis, we studied a panel of 20 neuroblastoma cell lines, and compared their invasive potential towards MSC-conditioned-RPMI (mRPMI) and their cytokine receptor expression profiles. Western blot analysis revealed the expression of multiple CXCR4 isoforms in neuroblastoma cells. Among the five major isoforms, the expression of the 47 kDa isoform showed significant correlation with high invasiveness. Pretreatment with mRPMI up-regulated the expression of the 47 kDa CXCR4 isoform and also increased MMP-9 secretion, expression of integrin α3 and integrin β1, and the invasive potential of the cell; while blocking CXCR4 either with AMD 3100, a CXCR4 antagonist, or with an anti-47 kDa CXCR4 neutralizing antibody decreased the secretion of MMP-9, the expression of integrin α3 and integrin β1, and the invasive potential of the cell. Pretreatment with mRPMI also protected the 47 kDa CXCR4 isoform from ubiquitination and subsequent degradation. Our data suggest a modulatory role of the MSC secretome on the expression of the 47 kDa CXCR4 isoform and invasion potential of the neuroblastoma cells to the bone marrow.     

Lack of an Efficient Endoplasmic Reticulum-localized Recycling System Protects Peroxiredoxin IV from Hyperoxidation

Typical 2-Cys peroxiredoxins are required to remove hydrogen peroxide from several different cellular compartments. Their activity can be regulated by hyperoxidation and consequent inactivation of the active-site peroxidatic cysteine. Here we developed a simple assay to quantify the hyperoxidation of peroxiredoxins. Hyperoxidation of peroxiredoxins can only occur efficiently in the presence of a recycling system, usually involving thioredoxin and thioredoxin reductase. We demonstrate that there is a marked difference in the sensitivity of the endoplasmic reticulum-localized peroxiredoxin to hyperoxidation compared with either the cytosolic or mitochondrial enzymes. Each enzyme is equally sensitive to hyperoxidation in the presence of a robust recycling system. Our results demonstrate that peroxiredoxin IV recycling in the endoplasmic reticulum is much less efficient than in the cytosol or mitochondria, leading to the protection of peroxiredoxin IV from hyperoxidation.     

Mosquito larvicidal activity of Aloe vera (Family: Liliaceae) leaf extract and Bacillus sphaericus,against Chikungunya vector,Aedes aegypti

The bio-efficacy of Aloe vera leaf extract and bacterial insecticide, Bacillus sphaericus larvicidal activity was assessed against the first to fourth instars larvae of Aedes aegypti, under the laboratory conditions. The plant material was shade dried at room temperature and powdered coarsely. A. vera and B. sphaericus show varied degrees of larvicidal activity against various instars larvae of A. aegypti. The LC₅₀ of A. vera against the first to fourth instars larvae were 162.74, 201.43, 253.30 and 300.05 ppm and the LC₉₀ 442.98, 518.86, 563.18 and 612.96 ppm, respectively. B. sphaericus against the first to fourth instars larvae the LC₅₀ values were 68.21, 79.13, 93.48, and 107.05 ppm and the LC₉₀ values 149.15, 164.67, 183.84, and 201.09 ppm, respectively. However, the combined treatment of A. vera + B. sphaericus (1:2) material shows highest larvicidal activity of the LC₅₀ values 54.80, 63.11, 74.66 and 95.10 ppm; The LC₉₀ values of 145.29, 160.14, 179.74 and 209.98 ppm, against A. aegypti in all the tested concentrations than the individuals and clearly established that there is a substantial amount of synergist act. The present investigation clearly exhibits that both A. vera and B. sphaericus materials could serve as a potential larvicidal agent. Since, A. aegypti is a container breeder vector mosquito this user and eco-friendly and low-cost vector control strategy could be a viable solution to the existing dengue disease burden. Therefore, this study provides first report on the mosquito larvicidal activity the combined effect of A. vera leaf extract and B. sphaericus against as target species of A. aegypti.     

Characterization of S1 nuclease. Involvement of carboxylate groups in metal binding.

Modification of the carboxylate groups of purified S1 nuclease resulted in a loss of its single-stranded DNAase, RNAase and phosphomonoesterase activities. The inactivation was due to the removal of zinc atoms from the enzyme and this in turn was dependent on the degree of modification. While the removal of one zinc atom resulted in the partial inactivation of the enzyme, removal of the remaining zinc atoms resulted in the complete inactivation of the enzyme. Similar results were obtained when the purified enzyme was incubated with various concentrations of the metal chelator, EDTA. The EDTA-(1 mM)-treated enzyme, depleted of one zinc atom, showing 40-45% residual activity, when incubated with 1 mM Zn2+ or 1 mM Co2+, regained a significant amount of its initial activity towards all the substrates. However, Woodward's-Reagent-K-modified enzyme depleted of one zinc atom and having the same level of activity (40-45%) could not regain its activity, indicating that the carboxylate groups are involved in the metal binding. Data obtained with carboxylate-group modification, EDTA-treatment, reconstitution with metal ions, zinc estimation and CD analysis of the enzyme suggests that, out of three zinc atoms present in S1 nuclease, zinc I is easily replaceable and is probably involved in the catalytic activity while zinc II and zinc III are involved in maintaining the enzyme structure.     

Distinct functions of maternal and somatic Pat1 protein paralogs

We previously identified Xenopus Pat1a (P100) as a member of the maternal CPEB RNP complex, whose components resemble those of P-(rocessing) bodies, and which is implicated in translational control in Xenopus oocytes. Database searches have identified Pat1a proteins in other vertebrates, as well as paralogous Pat1b proteins. Here we characterize Pat1 proteins, which have no readily discernable sequence features, in Xenopus oocytes, eggs, and early embryos and in human tissue culture cells. xPat1a and 1b have essentially mutually exclusive expression patterns in oogenesis and embryogenesis. xPat1a is degraded during meiotic maturation, via PEST-like regions, while xPat1b mRNA is translationally activated at GVBD by cytoplasmic polyadenylation. Pat1 proteins bind RNA in vitro, via a central domain, with a preference for G-rich sequences, including the NRAS 5′ UTR G-quadruplex-forming sequence. When tethered to reporter mRNA, both Pat proteins repress translation in oocytes. Indeed, both epitope-tagged proteins interact with the same components of the CPEB RNP complex, including CPEB, Xp54, eIF4E1b, Rap55B, and ePAB. However, examining endogenous protein interactions, we find that in oocytes only xPat1a is a bona fide component of the CPEB RNP, and that xPat1b resides in a separate large complex. In tissue culture cells, hPat1b localizes to P-bodies, while mPat1a-GFP is either found weakly in P-bodies or disperses P-bodies in a dominant-negative fashion. Altogether we conclude that Pat1a and Pat1b proteins have distinct functions, mediated in separate complexes. Pat1a is a translational repressor in oocytes in a CPEB-containing complex, and Pat1b is a component of P-bodies in somatic cells.     

Protection of crystal-induced polymorphonuclear leukocyte membranolysis by phosphocitrate

The protection afforded by phosphocitrate, a phosphorylated polycarboxylic acid, against crystal-induced membrane damage to polymorphonuclear leukocytes was studied in vitro. Membranolysis was assessed by nitro blue tetrazolium salt reduction, lactate dehydrogenase release, and scanning electron microscopy. Phosphocitrate protected strongly against hydroxyapatite crystal-induced damage, an action attributable to crystal surface binding of phosphocitrate rather than to the membrane. The ability of phosphocitrate to prevent hydroxyapatite crystallization, together with its membrane protective effect against preformed crystals, would suggest that the compound might have a useful future role against crystal-induced arthropathies.     

Responses of a sterile red fungus to soil types,wheat varieties and the presence of certain isolates ofStreptomyces

The biological activities of a sterile red fungus (SRF) capable of plant growth promotion and suppression of take-all disease were investigated in soils collected from Lancelin, Newdegate and Mt. Barker regions of Western Australia. Further, the effects of three wheat cultivars and the presence of two isolates ofStreptomyces on the biological activities of the SRF were tested using the Lancelin soil. The biological activities of the SRF were greatest in the Lancelin and Newdegate (wheat field) soils and with the wheat cultivar Gutha. In in vitro studies the soil streptomycetes tested showed either a significant increase in the exudate production by the SRF, which had antifungal and growth promoting properties, or an inhibition of growth of the fungus. Streptomycete A63 which stimulated the exudate production by the SRF in vitro, however, did not enhance disease protection in vivo. On the other hand, protection from root rot by the SRF in vivo was reduced in the presence of the streptomycete isolate Ax which is capable of inhibiting the growth of the SRF in vitro.