Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus

Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 ??mol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.     

Serotonin mediates cross-modal reorganization of cortical circuits

Loss of one type of sensory input can cause improved functionality of other sensory systems. Whereas this form of plasticity, cross-modal plasticity, is well established, the molecular and cellular mechanisms underlying it are still unclear. Here, we show that visual deprivation (VD) increases extracellular serotonin in the juvenile rat barrel cortex. This increase in serotonin levels facilitates synaptic strengthening at layer 4 to layer 2/3 synapses within the barrel cortex. Upon VD, whisker experience leads to trafficking of the AMPA-type glutamate receptors (AMPARs) into these synapses through the activation of ERK and increased phosphorylation of AMPAR subunit GluR1 at the juvenile age when natural whisker experience no longer induces synaptic GluR1 delivery. VD thereby leads to sharpening of the functional whisker-barrel map at layer 2/3. Thus, sensory deprivation of one modality leads to serotonin release in remaining modalities, facilitates GluR1-dependent synaptic strengthening, and refines cortical organization.     

Induction of Cancer Stem Cell Properties in Colon Cancer Cells by Defined Factors

Cancer stem cells (CSCs) are considered to be responsible for the dismal prognosis of cancer patients. However, little is known about the molecular mechanisms underlying the acquisition and maintenance of CSC properties in cancer cells because of their rarity in clinical samples. We herein induced CSC properties in cancer cells using defined factors. We retrovirally introduced a set of defined factors (OCT3/4, SOX2 and KLF4) into human colon cancer cells, followed by culture with conventional serum-containing medium, not human embryonic stem cell medium. We then evaluated the CSC properties in the cells. The colon cancer cells transduced with the three factors showed significantly enhanced CSC properties in terms of the marker gene expression, sphere formation, chemoresistance and tumorigenicity. We designated the cells with CSC properties induced by the factors, a subset of the transduced cells, as induced CSCs (iCSCs). Moreover, we established a novel technology to isolate and collect the iCSCs based on the differences in the degree of the dye-effluxing activity enhancement. The xenografts derived from our iCSCs were not teratomas. Notably, in contrast to the tumors from the parental cancer cells, the iCSC-based tumors mimicked actual human colon cancer tissues in terms of their immunohistological findings, which showed colonic lineage differentiation. In addition, we confirmed that the phenotypes of our iCSCs were reproducible in serial transplantation experiments. By introducing defined factors, we generated iCSCs with lineage specificity directly from cancer cells, not via an induced pluripotent stem cell state. The novel method enables us to obtain abundant materials of CSCs that not only have enhanced tumorigenicity, but also the ability to differentiate to recapitulate a specific type of cancer tissues. Our method can be of great value to fully understand CSCs and develop new therapies targeting CSCs.     

Overexpression of a minimal domain of calpastatin suppresses IL-6 production and Th17 development via reduced NF-κB and increased STAT5 signals

Calpain, a calcium-dependent cysteine protease, is reportedly involved in the pathophysiology of autoimmune diseases such as rheumatoid arthritis (RA). In addition, autoantibodies against calpastatin, a natural and specific inhibitor of calpain, are widely observed in RA. We previously reported that E-64-d, a membrane-permeable cysteine protease inhibitor, is effective in treating experimental arthritis. However, the exact role of the calpastatin-calpain balance in primary inflammatory cells remains unclear. Here we investigated the effect of calpain-specific inhibition by overexpressing a minimal functional domain of calpastatin in primary helper T (Th) cells, primary fibroblasts from RA patients, and fibroblast cell lines. We found that the calpastatin-calpain balance varied during Th1, Th2, and Th17 development, and that overexpression of a minimal domain of calpastatin (by retroviral gene transduction) or the inhibition of calpain by E-64-d suppressed the production of IL-6 and IL-17 by Th cells and the production of IL-6 by fibroblasts. These suppressions were associated with reductions in RORγt expression and STAT3 phosphorylation. Furthermore, inhibiting calpain by silencing its small regulatory subunit (CPNS) suppressed Th17 development. We also confirmed that overexpressing a minimal domain of calpastatin suppressed IL-6 by reducing NF-κB signaling via the stabilization of IκBα, without affecting the upstream signal. Moreover, our findings indicated that calpastatin overexpression suppressed IL-17 production by Th cells by up-regulating the STAT5 signal. Finally, overexpression of a minimal domain of calpastatin suppressed IL-6 production efficiently in primary fibroblasts derived from the RA synovium. These findings suggest that inhibiting calpain by overexpressing a minimal domain of calpastatin could coordinately suppress proinflammatory activities, not only those of Th cells but also of synovial fibroblasts. Thus, this strategy may prove viable as a candidate treatment for inflammatory diseases such as RA.     

Angelica keiskei extract improves insulin resistance and hypertriglyceridemia in rats fed a high-fructose drink

Angelica keiskei is a traditional herb peculiar to Japan and abundantly contains vitamins, dietary fiber and such polyphenols as chalcone. We investigated in the present study the effect of A. keiskei on insulin resistance and hypertriglyceridemia in fructose-drinking rats as a model for the metabolic syndrome. Male Wistar rats were given a 15% fructose solution as drinking water for 11 weeks. Fructose significantly increased the levels of serum insulin and triglyceride (TG) compared with the control level. Treatment with an ethanol extract of A. keiskei (AE) significantly reduced the levels of blood glucose (-16.5%), serum insulin (-47.3%), HOMA-R (-56.4%) and TG (-24.2%). A hepatic gene analysis showed that fructose reduced the expression of the genes related to fatty acid β-oxidation and high-density lipoprotein (HDL) production. Treatment with AE enhanced the expression of the acyl-CoA oxidase 1 (ACO1), medium-chain acyl-CoA dehydrogenase (MCAD), ATP-binding membrane cassette transporter A1 (ABCA1) and apolipoprotein A1 (Apo-A1) genes. These results suggest that AE improved the insulin resistance and hypertriglyceridemia of the fructose-drinking rats.     

Engineered <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> as an endotoxin-free platform strain for lactate-based polyester production

The first biosynthetic system for lactate (LA)-based polyesters was previously created in recombinant Escherichia coli (Taguchi et al. 2008). Here, we have begun efforts to upgrade the prototype polymer production system to a practical stage by using metabolically engineered Gram-positive bacterium Corynebacterium glutamicum as an endotoxin-free platform. We designed metabolic pathways in C. glutamicum to generate monomer substrates, lactyl-CoA (LA-CoA), and 3-hydroxybutyryl-CoA (3HB-CoA), for the copolymerization catalyzed by the LA-polymerizing enzyme (LPE). LA-CoA was synthesized by D-lactate dehydrogenase and propionyl-CoA transferase, while 3HB-CoA was supplied by β-ketothiolase (PhaA) and NADPH-dependent acetoacetyl-CoA reductase (PhaB). The functional expression of these enzymes led to a production of P(LA-co-3HB) with high LA fractions (96.8 mol%). The omission of PhaA and PhaB from this pathway led to a further increase in LA fraction up to 99.3 mol%. The newly engineered C. glutamicum potentially serves as a food-grade and biomedically applicable platform for the production of poly(lactic acid)-like polyester.     

Heat-shock proteins and nucleolar hypertrophy in the liver of rat infused with methionine-free total parenteral nutrition

Infusing a methionine-free solution into rats for 7 days resulted in a marked enlargement of liver nucleoli. By the analysis of two-dimensional gel electrophoresis, a spot 'a' (76 kDa, pI 5.3) stained with Coomassie blue was observed to accumulate highly in liver cytosol from rat infused with methionine-free solution. Metabolically labeling experiments with [35S]methionine showed that 'a' was more heavily labeled in primary hepatocytes of rats infused with methionine-free solution than in those of control rat. To ascertain whether 'a' is one of stress proteins, primary hepatocyte cultures were incubated at 42 degrees C for 2 h. 'a' (76 kDa, pI 5.3) was slightly induced in control hepatocytes but not appreciably in hepatocytes from the treated rat. In contrast, two other spots 'b' (74 kDa, pI 5.6) and 'c' (74 kDa, pI 5.3) were highly induced at 42 degrees C in hepatocytes from control and treated rats. The antibody against the consensus sequence peptide of hsp70 family reacted with 'a' (76 kDa, pI 5.3) as well as 'b' and 'c'. Immunoblot analysis revealed that 'a' accumulates highly in hepatocytes of treated rats. These results indicate that infusion of methionine-free solution into rats induces one member of the hsp70 family in hepatocytes.