Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment

Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.     

Targeting lysophosphatidic acid signaling retards culture-associated senescence of human marrow stromal cells

Marrow stromal cells (MSCs) isolated from mesenchymal tissues can propagate in vitro to some extent and differentiate into various tissue lineages to be used for cell-based therapies. Cellular senescence, which occurs readily in continual MSC culture, leads to loss of these characteristic properties, representing one of the major limitations to achieving the potential of MSCs. In this study, we investigated the effect of lysophosphatidic acid (LPA), a ubiquitous metabolite in membrane phospholipid synthesis, on the senescence program of human MSCs. We show that MSCs preferentially express the LPA receptor subtype 1, and an abrogation of the receptor engagement with the antagonistic compound Ki16425 attenuates senescence induction in continually propagated human MSCs. This anti-aging effect of Ki16425 results in extended rounds of cellular proliferation, increased clonogenic potential, and retained plasticity for osteogenic and adipogenic differentiation. Expressions of p16(Ink4a), Rb, p53, and p21(Cip1), which have been associated with cellular senescence, were all reduced in human MSCs by the pharmacological inhibition of LPA signaling. Disruption of this signaling pathway was accompanied by morphological changes such as cell thinning and elongation as well as actin filament deformation through decreased phosphorylation of focal adhesion kinase. Prevention of LPA receptor engagement also promoted ubiquitination-mediated c-Myc elimination in MSCs, and consequently the entry into a quiescent state, G(0) phase, of the cell cycle. Collectively, these results highlight the potential of pharmacological intervention against LPA signaling for blunting senescence-associated loss of function characteristic of human MSCs.     

Neoplastic transformation and induction of H+,K+-adenosine triphosphatase by N-methyl-N′-nitro-N-nitrosoguanidine in the gastric epithelial RGM-1 cell line

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces gastric cancer in animal models. We established an MNNG-induced mutant of the rat murine RGM-1 gastric epithelial cell line, which we named RGK-1, that could be used as an in vitro model of gastric cancer. This cell line showed signs of neoplasia and transformation, in that it lost contact inhibition and formed tumors in nude mice. The mutant cells also expressed parietal cell-specific H⁺,K⁺-adenosine triphosphatase (H⁺,K⁺-ATPase), which parent RGM-1 did not. The results suggested that parent RGM-1 cells were gastric progenitor cells. This mutant RGK-1 cell line will contribute to future investigation on gastric carcinogenesis and to the development of other pathophysiologic fields.     

Effects of Choto-san on hemorheological factors and vascular function in stroke-prone spontaneously hypertensive rats

Choto-san is a formula used for the treatment of headache and vertigo. Recently it has often also been used for hypertension and dementia. One of the mechanisms involved is thought to be the improvement of blood circulation, but the details are still unclear. In this study, the effect of Chotosan was studied on nitric oxide (NO) function, hemorheological factors and endothelial function in stroke-prone spontaneously hypertensive rats (SHR-SP). Rats were given Choto-san in drinking water for eight weeks. Body weight, blood pressure, serum NO2-/NO3-, lipid peroxides, blood viscosity, erythrocyte deformability and endothelium-dependent/-independent relaxation were measured. The results indicated that Choto-san caused a decrease in blood pressure and an increase in erythrocyte deformability and NO function. Blood viscosity was not changed. Furthermore, endothelium-dependent relaxation by acetylcholine was significantly increased as compared to control. In this study, it was supposed that Choto-san had a protective effect on the endothelium. SHR-SP is a useful model for human brain stroke, and Choto-san showed a protective effect against cerebral vascular injury in the susceptible rat.     

Inhibitory effects of Choto-san (Diao-teng-san), and hooks and stems of Uncaria sinensis on free radical-induced lysis of rat red blood cells.

The present study is designed to test our hypothesis that the ingestion of Uncaria sinensis (US), the main medicinal plant of Choto-san (Diao-teng-san, CS), would protect red blood cell (RBC) membrane from free radical-induced oxidation if polyphenolics in US could be absorbed and circulated in blood. When incubated with RBC suspension, Choto-san extract (CSE) and Uncaria sinensis extract (USE) exhibited strong protection for RBC membrane against hemolysis induced by 2,2-azo-bis (2-amidinopropane) dihydrochloride (AAPH), an azo free-radical initiator. The inhibitory effect was dose-dependent at concentrations of 50 to 1000 microg/mL. Ingestion of 200 mg of USE was associated with a significant decrease in susceptibility of RBC to hemolysis in rats. Furthermore, caffeic acid, an antioxidative hydroxycinnamic acid, was identified in rat plasma after administration of URE.     

Study on the inhibitory effects of Korean medicinal plants and their main compounds on the 1,1-diphenyl-2-picrylhydrazyl radical

A 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-generating system was used to evaluate the antioxidant properties of Korean medicinal plants that have been used widely as folk medicines for several disorders, as well as compounds isolated from them. Among the Rosaceae, Rosa rugosa and Rosa davurica showed strong DPPH radical-scavenging activity. The most effective medicinal plant from families other than Rosaceae was Cedrela sinensis, followed in order by Nelumbo nucifera, Eucommia ulmoides, Zanthoxylum piperitum, Cudrania tricuspidata and Houttuynia cordata. These results serve as a good index of the free radical-scavenging activities of Korean medicinal plants. Furthermore, the polyphenols isolated from these plants, procyanidin B-3, (+)-catechin, gallic acid, methyl gallate, quercetin, quercetin-3-O-beta-D-glucoside, quercetin-3-O-beta-galactoside, quercetin-3-O-rutinose and kaempferol, exerted strong DPPH radical-scavenging activity. These results suggest that the Korean medicinal plants and the polyphenols isolated from them that exhibited effective radical-scavenging activity may be promising agents for scavenging free radicals and treating diseases associated with excess free radicals.     

Tenascin: cDNA cloning and induction by TGF-beta.

cDNA clones coding for tenascin, an extracellular matrix glycoprotein with a restricted tissue distribution, were isolated from a chicken fibroblast cDNA expression library using a specific tenascin antiserum. Antibodies eluted from the cDNA-encoded fusion proteins reacted exclusively with tenascin. Limited trypsin treatment of purified tenascin resulted in a peptide which confirmed the deduced protein sequences. The largest clone encoding 632 amino acids showed a cysteine-rich region containing 13 consecutive epidermal growth factor-like repeats of unusual uniformity. Northern blot analysis revealed 8- to 9-kb messages. Tenascin is shown to be induced in vitro by fetal calf serum as well as by transforming growth factor beta (TGF-beta). A 4-fold increase in tenascin secretion by chick embryo fibroblasts was seen after TGF-beta treatment. The induction of tenascin protein synthesis was preceded by an increase of tenascin mRNA as determined by Northern blot analysis. The induction of tenascin was compared with fibronectin. The accumulation of the two extracellular matrix proteins in the medium was differentially affected by fetal calf serum and TGF-beta and the increase was in both cases higher for tenascin.