Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice

The structure and function of blood vessels adapt to environmental changes such as physical development and exercise. This phenomenon is based on the ability of the endothelial cells to sense and respond to blood flow; however, the underlying mechanisms remain unclear. Here we show that the ATP-gated P2X4 ion channel, expressed on endothelial cells and encoded by P2rx4 in mice, has a key role in the response of endothelial cells to changes in blood flow. P2rx4(-/-) mice do not have normal endothelial cell responses to flow, such as influx of Ca(2+) and subsequent production of the potent vasodilator nitric oxide (NO). Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2rx4(-/-) mice. Furthermore, P2rx4(-/-) mice have higher blood pressure and excrete smaller amounts of NO products in their urine than do wild-type mice. Moreover, no adaptive vascular remodeling, that is, a decrease in vessel size in response to a chronic decrease in blood flow, was observed in P2rx4(-/-) mice. Thus, endothelial P2X4 channels are crucial to flow-sensitive mechanisms that regulate blood pressure and vascular remodeling.     

Trypsin-catalyzed peptide synthesis withm-guanidinophenyl andm-(guanidinomethyl)phenyl esters as acyl donor component

Summary Two series of inverse substrates,m-guanidinophenyl andm-(guanidinomethyl)phenyl esters derived fromN-(tert-butyloxycarbonyl)amino acid, were prepared as an acyl donor component for trypsin-catalyzed peptide synthesis. The kinetic behavior of these esters toward tryptic hydrolysis was analyzed. They were found to couple with an acyl acceptor such asl-alaninep-nitroanilide to produce dipeptide in the presence of trypsin.Streptomyces griseus trypsin was a more efficient catalyst than the bovine trypsin. Within the enzymatic peptide coupling methods, this approach was shown to be advantageous, since the resulting peptides are resistant to the enzymatic hydrolysis.Abbreviations Boc tert-butyloxycarbonyl - Aib -aminoisobutyric acid - DMSO dimethylsulfoxide - Tris tris(hydroxymethyl)aminomethane - MOPS 3-morpholino-l-prop anesulfonate - G guanidinophenyl - GM (guanidinomethyl)phenyl - pNA p-nitroanilide     

Sphingosine 1-phosphate transactivates c-Met as well as epidermal growth factor receptor (EGFR) in human gastric cancer cells

Receptor tyrosine kinases (RTKs) are transactivated by the stimulation of G protein-coupled receptors (GPCRs). Sphingosine 1-phosphate (S1P), a ligand of GPCR, is known as a tumor-promoting lipid, but its signaling pathways are not fully understood. We here demonstrated that S1P induces rapid and transient tyrosine phosphorylation of epidermal growth factor receptor (EGFR) and c-Met in gastric cancer cells, both of which have been proposed as prognostic markers of gastric cancers. The pathway of S1P-induced c-Met transactivation is Gi-independent and matrix metalloproteinase-independent, which differs from that of EGFR transactivation. Our results indicate that S1P acts upstream of various RTKs and thus may act as a potent stimulator of gastric cancer.     

Effect on endothelial cell gene expression of shear stress, oxygen concentration, and low-density lipoprotein as studied by a novel flow cell culture system

A new cell culture system has been developed that reflects the vascular microenvironment. By means of this system the cultured cells are exposed not only to shear stress by the circulating culture medium, but also to an oxygen concentration gradient and certain critical blood components such as low-density lipoprotein (LDL) and monocytes. DNA microarray analysis was performed for human umbilical vein endothelial cells cultured in this system in the absence and presence of laminar flow at a low shear stress, 0.2 dyn/cm(2). In addition to shear stress, either an oxygen concentration gradient, or LDL (1 mg/ml), or both were applied. Many Nrf-2-regulating genes, such as heme oxygenase 1, NAD(P)H quinone oxidoreductase 1, solute carrier family 7 No. 11, and glutamate-cysteine ligase modifier subunit, were induced by laminar flow at very low shear stress regardless of the additional conditions. Certain genes were specifically affected by exposure to the oxygen gradient and/or LDL under shear stress, but the degree was very low. These results suggest that shear stress is the most critical factor affecting gene expression in endothelial cells and that Nrf-2-regulating proteins may contribute to protecting endothelial cells against other vascular stress. This system should provide highly relevant and useful information about both vascular physiology and pathology, in the latter on such urgent matters as the specific steps involved in atherogenesis.     

Determination of urinary 12(S)-hydroxyeicosatetraenoic acid by liquid chromatography-tandem mass spectrometry with column-switching technique: sex difference in healthy volunteers and patients with diabetes mellitus

We developed a determination method for human urinary 12-hydroxyeicosatetraenoic acid (12-HETE) using LC-MS-MS. This method, which includes simple extraction and detection in the SRM mode, allows precise and accurate determination of 12-HETE. There was a significant sex difference in urinary 12-HETE levels. Chiral analysis of 12-HETE using LC-MS-MS with column-switching technique revealed that the major enantiomer was 12(S)-HETE. Furthermore, the urinary level in patients with diabetes mellitus (DM) was analyzed. The present in vivo findings indicate that there could be difference in production of 12(S)-HETE between genders and 12(S)-HETE may play a role in the pathogenesis of DM.     

Generation of polymorphic markers tightly linked to the thymus enlargement loci by phenotype-directed representational difference analysis

To obtain genetic markers linked to a specific genetic locus, genomic subtraction with a DNA pool of backcross or F₂ intercross animals with a specific genotype at the locus is known to be effective. To determine whether the pooling strategy is also effective for isolation of genetic markers linked to a quantitative phenotype that can potentially be controlled by multiple genetic loci, we tested the ability of representational difference analysis (RDA) to isolate genetic markers linked to the thymus enlargement observed in the BUF/Mna (BUF) rat. This is known to be controlled by single major and minor genes, Ten1 and Ten2, on Chromosomes (Chrs) 1 and 13, respectively, both of which have dose effects on the normal WKY/Ncj (WKY) allele. DNA from an inbred WKY rat was used as the tester, and the driver was prepared from a DNA pool of 12 (WKY × BUF)F₁× BUF backcross rats with high thymus ratios (thymus weight/body weight), expected to have dominance of the BUF allele in the responsible loci. By two RDA series with the restriction enzymes BglII and BamHI, respectively, 28 polymorphic markers were isolated, and 8 of them were shown to be linked to Ten1, and one to Ten2. One of the 8 markers linked to Ten1 demonstrated no recombination in 18 rats with high thymus ratios. RDA with a DNA pool based on a quantitative phenotype (phenotype-directed RDA) can thus be considered an efficient approach for direct isolation of polymorphic markers linked to a quantitative trait. Received: 15 April 1997 / Accepted: 8 May 1998     

Effects of acidic pH on the formation of vinblastine-induced paracrystals in Chinese hamster ovary cells

Summary— The pH-related change in morphology of vinblastine (VLB)-induced paracrystals formed in Chinese hamster ovary (CHO) cells was examined immunohistochemically in order to determine both the mechanism of tubulin crystallization and the influence of acidic pHs on cytoskeletal microtubules. Lowering the extracellular pH (pHe) rapidly reduced the intracellular pH (pHi) in CHO cells. Lowering the pHi to near the neutral range significantly accelerated the growth of VLB-induced paracrystals, compared to that of paracrystals formed at a physiological pHe. However, further cytoplasmic acidification caused by the addition of sodium azide into the culture medium induced the disappearance of typical paracrystals and the appearance of a highly organized meshwork of tubulin appearing as short, thick filaments at the light microscopic level. Treatments using different concentrations of VLB at different pHe's showed that low pHi's (6.7 and 6.3) suppressed paracrystal-formation at lower concentrations of VLB (5×10^?6 M and 10^?5 M). At higher concentrations of VLB (5×10^?5 M and 10^?4 M), only short filaments were formed at pHi 6. 3. Electron microscopy revealed that the filaments had a ladder-like structure probably consisting of a stacked series of fused rings. This indicates that paracrystals may be modified by extremely low pH. These results show that paracrystals are unstable in living cells and that their formation is regulated by environmental pH.