The Wnt signaling inhibitor dickkopf-1 is required for reentry into the cell cycle of human adult stem cells from bone marrow

Adult human mesenchymal stem cells from bone marrow stroma (hMSCs) differentiate into numerous mesenchymal tissue lineages and are attractive candidates for cell and gene therapy. When early passage hMSCs are plated or replated at low density, the cultures display a lag phase of 3-5 days, a phase of rapid exponential growth, and then enter a stationary phase without the cultures reaching confluence. We found that as the cultures leave the lag phase, they secrete high levels of dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt signaling pathway. The addition of recombinant Dkk-1 toward the end of the lag period increased proliferation and decreased the cellular concentration of beta-catenin. The addition of antibodies to Dkk-1 in the early log phase decreased proliferation. Also, expression of Dkk-1 in hMSCs decreased during cell cycle arrest induced by serum starvation. The results indicated that high levels of Dkk-1 allow the cells to reenter the cell cycle by inhibiting the canonical Wnt/beta-catenin signaling pathway. Since antibodies to Dkk-1 also increased the lag phase of an osteosarcoma line that expressed the gene, Dkk-1 may have a similar role in some other cell systems.     

Flow cytometric characterization of phenotype,DNA indices and p53 gene expression in 55 cases of acute leukemia

OBJECTIVE: To characterize the phenotype of acute leukemia cases using flow cytometry, to detect mixed lineage cases and to use DNA index determination, including S-phase fraction (SPF) and p53 detection, to find if there was any correlation of SPF and p53 expression with outcome. STUDY DESIGN: Fifty-five cases of acute leukemia were enrolled in this study. A complete hemogram and routine bone marrow examination, including cytochemistry, was done. Mycloperoxidase-negative cases were evaluated on a flow cytometer using monoclonal antibodies. DNA indices were determined by flow cytometry in all cases, and p53 was detected immunohistochemically using the alkaline phosphatase/antialkaline phosphatase technique. RESULTS: Acute myeloblastic leukemia (AML) was diagnosed in 32 cases; acute lymphoblastic leukemia (ALL) was diagnosed in 18 (14 B lineage and 4 T line age). Four cases showed mixed lineage leukemia, and undifferentiated acute leukemia was diagnosed in one case. The mean/range of SPF for these groups were 3.76/0.33-6.91, 6.25/0.15-21.4, 2.89/0.35-10.64, 2.60/0.72-6.94 and 7.34, respectively. Aneuploidy was detected in two cases of B-lineage ALL and tetraploidy in a case of AML-M7, while all others were diploid p53. Was detected in 6 of 55 cases (10.90%). Follow-up was available for 24 patients. Five patients relapsed, and four had B-cell type ALL and were diploid and expressed no p53 gene. SPF% did not show any correlation with outcome. CONCLUSION: These data suggest that within acute leukemia subtypes, there is a wide variation in SPF. SPF does not seem to correlate with outcome. Immunophenotyping is essential to determine the lineage in myeloperoxidase-negative cases. It is perhaps the only way to diagnose mixed lineage leukemia and aberrant expression of markers presently. The p53 gene was detected less frequently. However, more studies are required from different centers with longer follow-up to evaluate prognostic significance.     

Transcriptome mining, functional characterization, and phylogeny of a large terpene synthase gene family in spruce (Picea spp.)

Background  

In conifers, terpene synthases (TPSs) of the gymnosperm-specific TPS-d subfamily form a diverse array of mono-, sesqui-, and diterpenoid compounds, which are components of the oleoresin secretions and volatile emissions. These compounds contribute to defence against herbivores and pathogens and perhaps also protect against abiotic stress.     

Identification of phospholipase C gamma1 as a protein tyrosine phosphatase mu substrate that regulates cell migration

The receptor protein tyrosine phosphatase PTPµ has a cell‐adhesion molecule‐like extracellular segment and a catalytically active intracellular segment. This structure gives PTPµ the ability to transduce signals in response to cell–cell adhesion. Full‐length PTPµ is down‐regulated in glioma cells by proteolysis which is linked to increased migration of these cells in the brain. To gain insight into the substrates PTPµ may be dephosphorylating to suppress glioma cell migration, we used a substrate trapping method to identify PTPµ substrates in tumor cell lines. We identified both PKCδ and PLCγ1 as PTPµ substrates. As PLCγ1 activation is linked to increased invasion of cancer cells, we set out to determine whether PTPµ may be upstream of PLCγ1 in regulating glioma cell migration. We conducted brain slice assays using U87‐MG human glioma cells in which PTPµ expression was reduced by shRNA to induce migration. Treatment of the same cells with PTPµ shRNA and a PLCγ1 inhibitor prevented migration of the cells within the brain slice. These data suggest that PLCγ1 is downstream of PTPµ and that dephosphorylation of PLCγ1 is likely to be a major pathway through which PTPµ suppresses glioma cell migration. J. Cell. Biochem. 112: 39–48, 2011. © 2010 Wiley‐Liss, Inc.     

Redox regulation of CLIC1 by cysteine residues associated with the putative channel pore

Chloride intracellular channels (CLICs) are putative pore-forming glutathione-S-transferase homologs that are thought to insert into cell membranes directly from the cytosol. We incorporated soluble, recombinant human CLIC1 into planar lipid bilayers to investigate the associated ion channels, and noted that channel assembly (unlike membrane insertion) required a specific lipid mixture. The channels formed by reduced CLIC1 were similar to those previously recorded from cells and "tip-dip" bilayers, and specific anti-CLIC1 antibodies inhibited them. However, the amplitudes of the filtered single-channel currents were strictly regulated by the redox potential on the "extracellular" (or "luminal") side of the membrane, with minimal currents under strongly oxidizing conditions. We carried out covalent functional modification and site-directed mutagenesis of this controversial ion channel to test the idea that cysteine 24 is a critical redox-sensitive residue located on the extracellular (or luminal) side of membrane CLIC1 subunits, in a cysteine-proline motif close to the putative channel pore. Our findings support a simple structural hypothesis to explain how CLIC1 oligomers form pores in membranes, and suggest that native channels may be regulated by a novel mechanism involving the formation and reduction of intersubunit disulphide bonds.     

Molecular modulation of autophagy: New venture to target resistant cancer stem cells

Autophagy is a highly regulated catabolic process in which superfluous,damaged organelles and other cytoplasmic constituents are delivered to the lysosome for clearance and the generation of macromolecule substrates during basal or stressed conditions. Autophagy is a bimodal process with a context dependent role in the initiation and the development of cancers. For instance,autophagy provides an adaptive response to cancer stem cells to survive metabolic stresses, by influencing disease propagation via modulation of essential signaling pathways or by promoting resistance to chemotherapeutics. Autophagy has been implicated in a cross talk with apoptosis. Understanding the complex interactions provides an opportunity to improve cancer therapy and the clinical outcome for the cancer patients. In this review, we provide a comprehensive view on the current knowledge on autophagy and its role in cancer cells with a particular focus on cancer stem cell homeostasis.     

Protective Effect of Lycopene on Oxidative Stress and Cognitive Decline in Rotenone Induced Model of Parkinson’s Disease

Evidence from clinical and experimental studies indicate that oxidative stress is involved in pathogenesis of Parkinson’s disease. The present study was designed to investigate the neuroprotective potential of lycopene on oxidative stress and neurobehavioral abnormalities in rotenone induced PD. Rats were treated with rotenone (3 mg/kg body weight, intraperitoneally) for 30 days. NADH dehydrogenase a marker of rotenone action was observed to be significantly inhibited (35%) in striatum of treated animals. However, lycopene administration (10 mg/kg, orally) to the rotenone treated animals for 30 days increased the activity by 39% when compared to rotenone treated animals. Rotenone administration increased the MDA levels (75.15%) in striatum, whereas, lycopene administration to rotenone treated animals decreased the levels by 24.33%. Along with this, significant decrease in GSH levels (42.69%) was observed in rotenone treated animals. Lycopene supplementation on the other hand, increased the levels of GSH by 75.35% when compared with rotenone treated group. The activity of SOD was inhibited by 69% in rotenone treated animals and on lycopene supplementation; the activity increased by 12% when compared to controls. This was accompanied by cognitive and motor deficits in rotenone administered animals, which were reversed on lycopene treatment. Lycopene treatment also prevented release of cytochrome c from mitochondria. Collectively, these observations suggest that lycopene supplementation along with rotenone for 30 days prevented rotenone-induced alterations in antioxidants along with the prevention of rotenone induced oxidative stress and neurobehavioral deficits. The results provide an evidence for beneficial effect of lycopene supplementation in rotenone-induced PD and suggest therapeutic potential in neurodegenerative diseases involving accentuated oxidative stress.     

Effect of carbon,nitrogen sources and inducers on ligninolytic enzyme production by Morchella crassipes

The effect of different carbon, nitrogen sources and inducers on growth and ligninolytic activity by Morel mushroom Morchella crassipes was investigated. The maximum growth was observed in mineral salts broth containing glucose as the carbon source and sodium nitrate as the nitrogen source. Among the inducers, chemical inducers inhibited the growth whereas in natural substrates, growth was not affected much. Manganese peroxidase and lignin peroxidase activity were not detected in the medium with different carbon and nitrogen sources, whereas laccase activity varied depending on carbon source (0.7–3.48 U/ml). Among the inducers, natural inducers resulted in an increase in the enzyme activities. Maximum laccase activity was observed in rice straw (12. 6 U/ml) followed by ABTS (11.6 U/ml); Manganese peroxidase activity was maximum in rice straw (14.32 U/l) wheat straw (12.16 U/l) and phenol red (15 U/l) as the inducers, whereas for Lignin peroxidase activity, rice straw (22 U/l), wheat straw (16 U/l) and veratrylalcohol (20 U/l) served as the best inducers.