Ral Overactivation in Malignant Peripheral Nerve Sheath Tumors

Ras leads an important signaling pathway that is deregulated in neurofibromatosis type 1 and malignant peripheral nerve sheath tumor (MPNST). In this study, we show that overactivation of Ras and many of its downstream effectors occurred in only a fraction of MPNST cell lines. RalA, however, was overactivated in all MPNST cells and tumor samples compared to nontransformed Schwann cells. Silencing Ral or inhibiting it with a dominant-negative Ral (Ral S28N) caused a significant reduction in proliferation, invasiveness, and in vivo tumorigenicity of MPNST cells. Silencing Ral also reduced the expression of epithelial mesenchymal transition markers. Expression of the NF1-GTPase-related domain (NF1-GRD) diminished the levels of Ral activation, implicating a role for neurofibromin in regulating RalA activation. NF1-GRD treatment caused a significant decrease in proliferation, invasiveness, and cell cycle progression, but cell death increased. We propose Ral overactivation as a novel cell signaling abnormality in MPNST that leads to important biological outcomes with translational ramifications.The Ras family of guanine-nucleotide bound proteins exerts a fundamental role in cell biology and constitutes an important area of cancer research due to its significant involvement in the development and progression of malignancies (8, 10, 17, 18, 32). Ras-like (Ral) proteins are crucial members of this family and have been shown to play a pivotal role in human tumors (7, 28, 41, 66, 70). Because Ral guanine nucleotide exchange factors (Ral-GEFs) are direct effectors of Ras, the Ral signaling pathway has been traditionally considered a Ras-effector pathway. Activation of Ras (in resemblance to Ral) is regulated by two classes of proteins: Ras-GEFs (e.g., SOS) and Ras- GTPase activating proteins (Ras-GAPs such as neurofibromin). The latter induces hydrolysis of Ras from the active (GTP) form to the inactive (GDP) form (13). Ral-GEFs include two main groups: the proteins that are stimulated by Ras because of their carboxy-terminal Ras binding domain (RalGDS, RGL1, and RGL2) and the proteins that are activated by substrates of PI3K through a pleckstrin homology domain on their C-terminal (RALGPS1 and RALGPS2) (19). Although highly similar to Ras, Ral proteins (RalA and RalB) involve a series of distinctly different effectors that influence gene expression and translation through interaction with ZO-1-associated nucleic acid binding protein (ZONAB) and RalA binding protein 1 (RalBP1) (11, 23, 33). RalB directly interacts with the SEC5 subunit of exocyst to facilitate the host defense response (48, 58).In addition to overactivation of GEFs, inactivation of GAPs is another mechanism for overactivation of GTP-bound proteins. The lack of neurofibromin (encoded by NF1 on human chromosome 17q11.2), a Ras-GAP protein, is the main molecular event in neurofibromatosis type 1 (NF-1), an autosomal-dominant human genetic disease occurring in approximately 1 in 2,500 to 3,500 births (22, 27, 42). One of the main tumor-causing effects of inactivating mutations in the tumor suppressor NF1 gene is postulated to be the subsequent activation of Ras (3, 29, 53, 57, 69). With two main functional domains, SEC14 and Ras-GAP, neurofibromin is best known for its Ras-GAP function. Although the yeast SEC14p is shown to be involved in regulating intracellular proteins and lipid trafficking, the function of its homologous domain in neurofibromin is unknown (49, 62). Although neurofibromas are the most common tumors in NF-1, 10% of patients with plexiform develop malignant peripheral nerve sheath tumors (MPNSTs), which are typically high grade and often fatal (21, 34, 65).The molecular events involved in the malignant transformation of benign neurofibromas to MPNST are poorly defined. Usually arising in the third through sixth decades of life, these tumors are composed of tightly packed hyperchromatic spindle-shaped cells with frequent mitotic figures. Inactivation of both copies of the NF1 gene has been demonstrated in benign human neurofibromas and shown to cause tumors in murine models (56). Loss of heterozygosity of NF1 and p53 has frequently been observed in human MPNST (35, 47, 54). Recombinant mouse strains (NP mice), which harbor inactivated Nf1 and p53 alleles (cis-Nf1^+/−:p53^+/−), demonstrate the cumulative effects of loss of both Nf1 and p53 genes in the etiology of MPNST (14, 68).In the present study, we show that while both Ras activation and activation of a series of its downstream effector pathways are observed in a fraction of MPNST cells, RalA is activated globally in all studied mouse and human MPNST cells and tumor samples. Our results also explain the involvement of this signaling molecule in a series of key biological functions of MPNST cells, as shown in a variety of in vitro assays and an in vivo model of MPNST. Such information may play a role in designing novel therapies for treatment of MPNST or other tumors with overactivation of the Ral pathway.     

Prevention and Reversal of Obesity and Glucose Intolerance in Mice by DHA Derivatives

The n‐3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exert hypolipidemic effects and prevent development of obesity and insulin resistance in animals fed high‐fat diets. We sought to determine the efficacy of α‐substituted DHA derivatives as lipid‐lowering, antiobesity, and antidiabetic agents. C57BL/6 mice were given a corn oil‐based high‐fat (35% weight/weight) diet (cHF), or cHF with 1.5% of lipids replaced with α‐methyl DHA ethyl ester (Substance 1), α‐ethyl DHA ethyl ester (Substance 2), α,α‐di‐methyl DHA ethyl ester (Substance 3), or α‐thioethyl DHA ethyl ester (Substance 4) for 4 months. Plasma markers of glucose and lipid metabolism, glucose tolerance, morphology, tissue lipid content, and gene regulation were characterized. The cHF induced obesity, hyperlipidemia, impairment of glucose homeostasis, and adipose tissue inflammation. Except for Substance 3, all other substances prevented weight gain and Substance 2 exerted the strongest effect (63% of cHF‐controls). Glucose intolerance was significantly prevented (～67% of cHF) by both Substance 1 and Substance 2. Moreover, Substance 2 lowered fasting glycemia, plasma insulin, triacylglycerols, and nonesterified fatty acids (73, 9, 47, and 81% of cHF‐controls, respectively). Substance 2 reduced accumulation of lipids in liver and skeletal muscle, as well as adipose tissue inflammation associated with obesity. Substance 2 also induced weight loss in dietary obese mice. In contrast to DHA administered either alone or as a component of the EPA/DHA concentrate (replacing 15% of dietary lipids), Substance 2 also reversed established glucose intolerance in obese mice. Thus, Substance 2 represents a novel compound with a promising potential in the treatment of obesity and associated metabolic disturbances.     

ATP-type DNA ligase requires other proteins for its activity in vitro and its operon components for radiation resistance in Deinococcus radiodurans in vivo

A multiprotein DNA processing complex isolated from Deinococcus radiodurans contains the DNA repair protein PprA, an ATP-type DNA repair ligase (LigB) encoded by the drB0100 gene, and protein kinase activity. An ATP-dependent DNA end-joining activity was detected in the complex. To elucidate the function of the drB0100 gene, we generated the deletion mutant for the DR_B0100 ORF. The mutant exhibited a nearly 2-log cycle reduction in growth rate when exposed to a 10,000 Gray dose of γ-radiation, and a significant loss in mitomycin C and methylmethane sulphonate tolerance as compared with wild type. Functional complementation of these phenotypes required the wild-type copy of drB0100 along with other genes such as drb0099 and drb0098, organized downstream in the operon. The in vitro DNA ligase activity of LigB was stimulated severalfold by PprA in the presence of the recombinant DRB0098 protein. However, this activity did not improve when PprA was substituted with purified DRB0099 protein or when DRB0098 protein was substituted with the DRB0099 protein in the presence of PprA in solution. These results suggest that PprA and DRB0098 protein are required for LigB function. Furthermore, they also suggest that the LigB operon components contribute to radiation resistance and double-strand break (DSB) repair in D. radiodurans.     

Phenylacetic acid induced organogenesis in cultured leaf segments of Dianthus chinensis

Summary Shoot regeneration was achieved from leaf derived callus of Dianthus chinensis using Phenylacetic acid (PAA). Callus from basal leaf segments, raised on Murashige and Skoog's (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 1-Naphthaleneacetic acid (NAA) in combination with 6-benzylamino purine (BAP), was subcultured on medium supplemented with BAP in combination with 2,4-D, NAA or PAA. Shoots were induced only when leaf derived callus was subcultured on medium containing BAP (2.0, 5.0 mg/l) in combination with PAA (0.5, 1.0 mg/l). No shoot regeneration was observed when 2,4-D, NAA or BAP were used in the medium either singly or in different combinations. These results demonstrate that PAA in combination with BAP was essential to trigger shoot regeneration from cultured leaf callus of D. chinensis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DPX dibutylphthalate xylol - MS Murashige and Skoog (1962) basal medium - NAA 1-Naphthaleneacetic acid - PAA Phenylacetic acid     

Redistribution of subcellular calcium in rat liver on administration of vanadate

Summary Mitochondria isolated from the livers of rats administered with sodium meta-, ortho-, or polyvanadate, but not vanadyl sulphate, exhibited enhanced Ca²⁺ — stimulated respiration and uptake of calcium. These effects were shown also by mitochondria isolated from livers perfused with polyvanadate. The concentration of acid-soluble calcium decreased significantly in the mitochondrial fraction on vanadate treatment, while that in the cytosol showed a corresponding increase. Phenoxybenzamine, an antagonist to a-adrenergic receptors, effectively inhibited vanadate-induced Ca²⁺ mobilization, but surgical sympathectomy was without effect. This is the first demonstration of vanadate mimicking -adrenergic agonists in vivo.     

Long pepper (Piper longum) derived carbon dots as fluorescent sensing probe for sensitive detection of Sudan I

In this piece of work, microwave-assisted conversion of a natural precursor in to high-valued nano-scale material was carried out by a completely greener method. The fluorescent carbon dots prepared, designated as long pepper derived carbon dots (LPCDs), have been thoroughly characterized to explore the physical and chemical properties. The system exhibits excitation dependent emission behavior and from the optimal studies the excitation and emission wavelength of the system was found to be 330 nm and 455 nm respectively. On account of the superior fluorescent behavior of the LPCDs, it was successfully employed as a fluorescent sensing probe to detect Sudan I with good level of selectivity and sensitivity. This carcinogenic dye extensively used as food adulterant can impart several health issues. Food product safety is of high concern, therefore a simple facile and economical analytical method was proposed based on the fluorescence of LPCDs for this dye detection with satisfactory statistical parameters. A linear relationship was maintained in the range of 0 to 27.27 μM Sudan I with limit of detection of 0.92 μM. The quenching mechanism was studied and finally attributed to Förster resonance energy transfer (FRET) mechanism. In addition, the probe was effectively implemented for Sudan I detection in commercial chili powder samples with good level of recovery parameters.     

Embryogenesis in suspension cultures of Datura innoxia Mill.

In vitro plant regeneration via embryogenesis was obtained in suspension cultures of Datura innoxia Mill. Embryogenesis was induced in suspension cultures raised from callus of androgenetic origin, using LS liquid medium supplemented with 0.22 mg/l 2,4-D. The total number of embryos formed was variable over time in culture. Embryos differentiated and matured in the liquid medium itself as also evidenced by histological observation. Embryos germinated to form plantlets on semisolid MS medium without growth substances. The regenerated plants had haploid number of chromosomes.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn Kinetin - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962)     

Microbial biodiversity and in situ bioremediation of endosulfan contaminated soil

Molecular characterization based on 16s rDNA gene sequence analysis of bacterial colonies isolated from endosulfan contaminated soil showed the presence of Ochrobacterum sp, Burkholderia sp, Pseudomonas alcaligenes, Pseudomonas sp and Arthrobacter sp which degraded 57–90% of α-endosulfan and 74–94% of β-endosulfan after 7days. Whole cells of Pseudomonas sp and Pseudomonas alcaligenes showed 94 and 89% uptake of α-isomer and 86 and 89% of β-endosulfan respectively in 120 min. In Pseudomonas sp, endosulfan sulfate was the major metabolite detected during the degradation of α-isomer, with minor amount of endosulfan diol while in Pseudomonas alcaligenes endosulfan diol was the only product during α-endosulfan degradation. Whole cells of Pseudomonas sp also utilized 83% of endosulfan sulfate in 120 min. In situ applications of the defined consortium consisting of Pseudomonas alcaligenes and Pseudomonas sp (1:1) in plots contaminated with endosulfan showed that 80% of α-endosulfan and 65% of β-endosulfan was degraded after 12 weeks of incubation. Endosulfan sulfate formed during endosulfan degradation was subsequently degraded to unknown metabolites. ERIC-PCR analysis indicated 80% survival of introduced population of Pseudomonas alcaligenes and Pseudomonas sp in treated plots.     

Metabolic flux maps comparing the effect of temperature on protein and oil biosynthesis in developing soybean cotyledons

Metabolic flux maps developed from ¹³C metabolic flux analysis (¹³C MFA) are effective tools for assessing the response of biological systems to genetic or environmental perturbations, and for identifying possible metabolic engineering targets. Experimental treatments were designed to distinguish between temperature effects prior to, and during incubation in vitro , on primary metabolism in developing soybeans. Biomass accumulation increased with temperature as did carbon partitioning into lipids. The flux through the plastidic oxidative pentose phosphate pathway (pgl^P) relative to sucrose intake remained fairly constant [∼56% (±24%)] when cotyledons were transferred from an optimum growth temperature to varying temperatures in in vitro culture, signifying a rigid node under these conditions. However, pgl^P flux ranged from 57 to 77% of sucrose intake when growth temperature in planta varied and were cultured in vitro at the same temperature (as the plant), indicating a flexible node for this case. The carbon flux through the anaplerotic reactions catalysed by plastidic malic enzyme (me^P), cytosolic phosphoenolpyruvate (PEP) carboxylase and the malate (Mal) transporter from the cytosol to mitochondrion varied dramatically with temperature and had a direct influence on the carbon partitioning into protein and oil from the plastidic pyruvate (Pyr) pool. These results of the in vitro culture indicate that temperature during early stages of development has a dominant effect on establishing capacity for flux through certain components of central carbon metabolism.