Cross Species Genomic Analysis Identifies a Mouse Model as Undifferentiated Pleomorphic Sarcoma/Malignant Fibrous Histiocytoma

Undifferentiated pleomorphic sarcoma/Malignant Fibrous Histiocytoma (MFH) is one of the most common subtypes of human soft tissue sarcoma. Using cross species genomic analysis, we define a geneset from the LSL-Kras^G12D; Trp53^Flox/Flox mouse model of soft tissue sarcoma that is highly enriched in human MFH. With this mouse geneset as a filter, we identify expression of the RAS target FOXM1 in human MFH. Expression of Foxm1 is elevated in mouse sarcomas that metastasize to the lung and tissue microarray analysis of human MFH correlates overexpression of FOXM1 with metastasis. These results suggest that genomic alterations present in human MFH are conserved in the LSL-Kras^G12D; p53^Flox/Flox mouse model of soft tissue sarcoma and demonstrate the utility of this pre-clinical model.     

uPAR expression under hypoxic conditions depends on iNOS modulated ERK phosphorylation in the MDA-MB-231 breast carcinoma cell line

Urokinase plasminogen activator receptor （uPAR） plays a major role in cancer-invasion and metastasis and uPAR expression is correlated with a poor prognosis in various cancer types. Moreover, the expression of uPAR is increased under hypoxic conditions. Nitric oxide （NO） and its metabolites produced by inducible nitric oxide synthase （iNOS） are important products ofhypoxic stress, and NO may activate or modulate extracellular signal regulated kinase （ERK）. Here, we evaluated uPA, uPAR, and activated ERK levels under hypoxic conditions, and the modulatory effects of iNOS and NO in the MDA-MB-231 human breast cancer cell line. Cells were incubated in a hypoxic or normoxic incubator and treated with PD98059 （a MEK 1/2 inhibitor, which abrogates ERK phosphorylation） and aminoguanidine （a selective iNOS inhibitor）, uPAR expression, ERK phosphorylation, and uPA activity were found to be increased under hypoxic conditions. Moreover, when cells were treated with PD98059 under hypoxic conditions, uPAR was downregulated, whereas aminoguanidine markedly increased ERK phosphorylation in a dose dependent manner. Furthermore, aminoguanidine increased uPAR expression and prevented the inhibition of uPAR expression by PD98059. These results demonstrated that uPAR is induced by hypoxia and that increased uPAR expression is mediated by ERK phosphorylation, which in turn is modulated by iNOS/NO in MDA-MB-231 cells. We conclude that iNOS/NO downregulates the expression of uPAR under hypoxic conditions via ERK pathway modulation.     

Nanoporous protein matrix made of amyloid fibrils of β2‐microglobulin

Amyloid fibrils are considered as novel nanomaterials because of their nanoscale width, a regular constituting structure of cross β‐sheet conformation, and considerable mechanical strength. By using an amyloidogenic protein of β₂‐microglobulin (β₂M) related to dialysis‐related amyloidosis, nanoporous protein matrix has been prepared. The β₂M granules made of around 15 monomers showed an average size of 23.1 nm. They formed worm‐like fibrils at pH 7.4 in 20 mM sodium phosphate containing 0.15 M NaCl following vigorous nondirectional shaking incubation, in which they became laterally associated and interwound to generate the porous amyloid fibrillar matrix with an average pore size of 30–50 nm. This nanoporous protein matrix was demonstrated to be selectively disintegrated by reducing agents, such as tris‐(2‐carboxyethyl) phosphine. High surface area with nanopores on the surface has been suggested to make the matrix of β₂M amyloid fibrils particularly suitable for applications in the area of nanobiotechnology including drug delivery and tissue engineering. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3μM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1μM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7? distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.     

The small GTPase Rac1 is involved in the maintenance of stemness and malignancies in glioma stem-like cells

A subpopulation of cancer cells with stem cell properties is responsible for tumor formation, maintenance, and malignant progression; however, the molecular mechanisms underlying the maintenance of cancer stem-like cell properties have remained unclear. Here, we show that the Rho family GTPase Rac1 is involved in the glioma stem-like cell (GSLC) maintenance and tumorigenicity in human glioma. The Rac1-Pak signaling was markedly activated in GSLCs. Knockdown of Rac1 caused reduction of expression of GSLC markers, self-renewal-related proteins and neurosphere formation. Moreover, down-regulation of Rac1 suppressed the migration, invasion, and malignant transformation in GSLCs. Furthermore, inhibition of Rac1 enhanced radiation sensitivity of GSLCs. These results indicate that the small GTPase Rac1 is involved in the maintenance of stemness and malignancies in GSLCs.     

Genome sequence of Lactobacillus johnsonii PF01, isolated from piglet feces

Lactobacillus johnsonii PF01, an autochthonous bacterium of the gastrointestinal tract, was isolated from a fecal sample from a piglet. The strain adhered specifically to the duodenal and jejunal epithelial cells of the piglet and had high bile resistance activity. Here we report the genomic sequence of L. johnsonii PF01.     

Simultaneous measurements of cytoplasmic viscosity and intracellular vesicle sizes for live human brain cancer cells

Intracellular vesicles, comprised of protein clusters, were individually tracked inside human brain cancer cells and characterized to simultaneously determine the average vesicle size and effective cytoplasmic viscosity. The cells were transfected with a TGF‐β superfamily gene, non‐steroidal anti‐inflammatory drug‐Activated Gene‐1 (NAG‐1) tagged with green fluorescent proteins (GFPs). Using total internal reflection fluorescent microscopy (TIRFM) the individual movements of the vesicles were categorized into either Brownian, caged, or directional type motion. In the near‐field region confined by the evanescent wave field of TIRFM, the hindrance of these vesicles was created by interactions with the glass coverslip and/or sub‐cellular structures. Measured particle motions were compared with theoretical predictions of hindered motion to estimate the unknown size and viscosity parameters using a nonlinear regression technique. For the tested human brain cancer cells, the average vesicle size and effective intracellular fluid viscosity were calculated to be 496 nm and 0.068 Pa s, respectively. This finding suggests that most of the hindrance experienced by vesicles can be due to non‐hydrodynamic interactions with microtubules and other intracellular structures. It should be also noted that this method provides a way to examine changes in vesicle size due to outside stimulus such as drug interaction, cytotoxicity, etc., unlike standard measurement techniques which require fixing the cells themselves. Biotechnol. Bioeng. 2011;108: 2504–2508. © 2011 Wiley Periodicals, Inc.