Expression of the urokinase receptor regulates focal adhesion assembly and cell migration in adenoid cystic carcinoma cells

Adenoid cystic carcinoma (AdCC) cell lines (ACCS and ACCT) showed higher migration responses and adhesion to the extracellular matrix (ECM), especially types I and IV collagen, than did the oral squamous cell carcinoma (SCC) lines (NA and TF). The response to collagens was largely and exclusively inhibited by anti-alpha(2) integrin antibody. Moreover, AdCC cell lines expressed higher surface levels of urokinase-type plasminogen activator receptor (uPAR) than did SCC cell lines. When AdCC cells were plated on collagen, the surface level of uPAR was increased, and numerous focal adhesions consisting of uPAR, vinculin, and paxillin were assembled; whereas collagen-stimulated SCC cell counterparts or AdCC cells plated on other types of ECM, such as fibronectin, failed to assemble such definite focal adhesions. In order to elucidate the association of uPAR with collagen-induced events, an ACCS-AS cell line transfected with a vector expressing antisense uPAR RNA was established and shown to have reduced uPAR (about 10% that of parental ACCS at both the protein and mRNA levels). ACCS-AS showed a strong reduction of collagen-stimulated migration and focal adhesion assembly of alpha(2) integrin, vinculin, and paxillin. These findings suggest that AdCC has a proclivity for migrating to types I and IV collagens due to the overexpression of uPAR, which plays a key role in focal adhesion assembly and migration.     

Picrosides I and II,selective enhancers of the mitogen-activated protein kinase-dependent signaling pathway in the action of neuritogenic substances on PC12D cells

Picrosides I and II caused a concentration-dependent (> 0.1 microM) enhancement of basic fibroblast growth factor (bFGF, 2 ng/ml)-, staurosporine (10 nM)- and dibutyryl cyclic AMP (dbcAMP, 0.3 mM)-induced neurite outgrowth from PC12D cells. PD98059 (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, blocked the enhancement of bFGF (2 ng/ml)-, staurosporine (10 nM)- or dbcAMP (0.3 mM)-induced neurite outgrowth by picrosides, suggesting that picrosides activate MAP kinase-dependent signaling pathway. However, PD98059 did not affect the bFGF (2 ng/ml)-, staurosporine (10 nM)- and dbcAMP (0.3 mM)-induced neurite outgrowth in PC12D cells, indicating the existence of two components in neurite outgrowth induced by bFGF, staurosporine and dbcAMP, namely the MAP kinase-independent and the masked MAP kinase-dependent one. Furthermore, picrosides-induced enhancements of the bFGF-action were markedly inhibited by GF109203X (0.1 microM), a protein kinase C inhibitor. The expression of phosphorylated MAP kinase was markedly increased by bFGF (2 ng/ml) and dbcAMP (0.3 mM), whereas that was not enhanced by staurosporine (10 nM). Picrosides had no effect on the phosphorylation of MAP kinase induced by bFGF or dbcAMP and also unaffected it in the presence of staurosporine. These results suggest that picrosides I and II enhance bFGF-, staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying a down-stream step of MAP kinase in the intracellular MAP kinase-dependent signaling pathway. Picrosides I and II may become selective pharmacological tools for studying the MAP kinase-dependent signaling pathway in outgrowth of neurites induced by many kinds of neuritogenic substances including bFGF.     

Ischemic preconditioning and morphine attenuate myocardial apoptosis and infarction after ischemia-reperfusion in rabbits: role of delta-opioid receptor

We examined whether ischemic preconditioning (IPC) attenuates ischemia-reperfusion injury, in part, by decreasing apoptosis and whether the delta-opioid receptor (DOR) plays a pivotal role in the regulation of apoptosis. Rabbits were subjected to 30-min coronary artery occlusion (CAO) and 180 min of reperfusion. IPC was elicited with four cycles of 5-min ischemia and 10-min reperfusion before CAO. Morphine (0.3 mg/kg iv) was given 15 min before CAO. Naloxone (Nal; 10 mg/kg iv) and naltrindole (Nti; 10 mg/kg iv), the respective nonselective and selective DOR antagonists were given 10 min before either morphine or IPC. Infarct size (%risk area) was reduced from 46 +/- 3.8 in control to 11.6 +/- 1.0 in IPC and 19.5 +/- 3.8 in the morphine group (means +/- SE; P < 0.001 vs. control). Nal blocked the protective effects of IPC and morphine, as shown by the increase in infarct size to 38.6 +/- 7.2 and 44.5 +/- 1.8, respectively. Similarly, Nti blocked IPC and morphine-induced protection. The percentage of apoptotic cells (revealed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay) decreased in IPC (3.6 +/- 1.9) and morphine groups (5.2 +/- 1.2) compared with control group (12.4 +/- 1.6; P < 0.001). Nti pretreatment increased apoptotic cells 11.2 +/- 2.2% in IPC and 12.1 +/- 0.8% in morphine groups. Nal failed to block inhibition of apoptosis in the IPC group (% of cells: 5.7 +/- 1.3 vs. 3.6 +/- 1.9 in IPC alone; P > 0.05). These results were also confirmed by nucleosomal DNA laddering pattern. We conclude that IPC reduces lethal injury, in part, by decreasing apoptosis after ischemia-reperfusion and activation of the DOR may play a crucial role in IPC or morphine-induced myocardial protection.     

Rho-kinase phosphorylates eNOS at threonine 495 in endothelial cells

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO), which is involved in various physiological functions of the cardiovascular system. eNOS is activated by dephosphorylation at Thr495 and phosphorylation at Ser1177. Inhibition of Rho-kinase, an effector of the small GTPase RhoA, leads to activation of Akt/PKB, which phosphorylates eNOS at Ser1177 and thereby promotes NO production. However, little is known about the effects of Rho-kinase on phosphorylation of Thr495. We here found that the constitutively active form of Rho-kinase phosphorylated eNOS at Thr495 in vitro. Expression of the constitutively active form of RhoA or Rho-kinase increased this phosphorylation in COS-7 cells. Addition of thrombin to cultured human umbilical vein endothelial cells induced phosphorylation of eNOS at Thr495. Treatment with Y27632, a Rho-kinase inhibitor, suppressed thrombin-induced phosphorylation at Thr495. These results indicate that Rho-kinase can directly phosphorylate eNOS at Thr495 to suppress NO production in endothelium.     

Identification and origin of the germline stem cells as revealed by the expression of nanos-related gene in planarians

The planarian's remarkable regenerative ability is thought to be supported by the stem cells (neoblasts) found throughout its body. Here we report the identification of a subpopulation of neoblasts, which was revealed by the expression of the nanos-related gene of the planarian Dugesia japonica, termed Djnos. Djnos-expressing cells in the asexual planarian were distributed to the prospective ovary or testes forming region in the sexual planarian. During sexualization, Djnos-expressing cells produce germ cells, suggesting that in the asexual state these cells were kept as germline stem cells for the oogonia and spermatogonia. Interestingly, the germline stem cells were indistinguishable from the neoblasts by morphology and X-ray sensitivity and did not seem to contribute to the regeneration at all. Germline stem cells initially appear in the growing infant planarian, suggesting that germline stem cells are separated from somatic stem cells in the planarian. Thus, planarian neoblasts can be classified into two groups; somatic stem cells for regeneration and tissue renewal, and germline stem cells for production of germ cells during sexualization. However, Djnos-positive cells appeared in the newly formed trunk region from the head piece, suggesting that somatic stem cells can convert to germline stem cells.     

Synthesis of double-labeled lactosylceramide

The total stereo-controlled synthesis of lactosylceramide and introduction of two kinds of isotopes,³H and¹⁴C, into synthetic lactosylceramide are described.