Inhibitory effects of adhesion oligopeptides on the invasion of squamous carcinoma cells with special reference to implication of αv integrins

We studied invasion-related adhesion events in vitro using three squamous carcinoma cell lines (HSC-3, poorly differentiated type; OSC-19, well-differentiated type; and KB cells, undifferentiated type). An in vitro invasion assay through matrigel in the transwell chamber revealed that HSC-3 cells were most invasive, OSC-19 cells moderately invasive and KB cells least invasive. Inhibition assay of invasion using synthetic peptides RGD, RGDV, RGDS, RGDT, IKVAV and YIGSR, showed that invasion of the three cell lines was significantly inhibited by RGDV. There were other peptides that inhibited invasion significantly including IKVAV for HSC-3, and RGDS and YIGSR for OSC-19. HSC-3 cells and OSC-19 cells adhered to fibronectin, laminin, vitronectin, and type IV collagen, and KB cells did not adhere to laminin but did to fibronectin, vitronectin and collagen type IV. Pretreatment of cells with RGDV peptide in the attachment assay reduced the ability of these cells to bind to vitronectin and fibronectin more efficiently than pretreatment with RGDS. Anti-v antibodies inhibited adhesion of HSC-3, OSC-19 and KB cells to vitronectin, but anti-1 antibodies did not inhibit adhesion. Immunofluorescent microscopic examinations showed that all cell lines were positive for anti-5 and anti-v antibodies, and only HSC-3 cells were positive for anti-3 antibody. 51 was not clearly demonstrated in any of the cell lines. RGDV was the most effective inhibitor of squamous cell carcinoma invasion among the synthetic oligopeptides used in this experiment, and it is suggested that it affects v3-and/or v5-mediated carcinoma cell invasion.Abbreviations BSA bovine serum albumin - MEM Eagle's minimal essential medium - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - PBS phosphatebuffered saline - FITC fluorescein isothiocyanate This work supported in part by a grant from the Osaka Cancer Research Foundation     

Prevalence of primary aldosteronism without hypertension in the general population: Results in Shika study

Objective: Recent studies have reported a high prevalence of primary aldosteronism (PA) among hypertensive patients. However, few data exist regarding the prevalence of PA in the general population. Therefore, we examined the prevalence of PA in the general population including normotensive subjects. Methods: Plasma renin activity (ng/mL/hr), plasma aldosterone concentration (pg/mL) and aldosterone renin ratio (ARR) were determined in 309 subjects aged >40 years in Horimatsu and Higashi-Matsuho district, Shika-machi, Ishikawa, Japan. Results: Among them, 195 subjects (78 males, mean age: 62 ± 11 years) did not take antihypertensive agents: 113 normotensive subjects and 82 hypertensive subjects. Under these conditions, 68 subjects (13 males, age 62 ± 10 years) had an ARR >200. In 14 subjects who underwent captopril suppression test, PA was documented in 5 subjects, yielding a minimum prevalence of 2.6% in total subjects (1.8% in normotensive subjects and 3.7% in hypertensive subjects). Interestingly, females subjects demonstrated significant differences in ARR between subjects with age <50 (172 ± 105) and those with age 51–60 (388 ± 531), although there were no differences in male subjects. Conclusions: These results demonstrate that PA including normotensive subjects exists more commonly than that expected in the general population. We suggest further investigation about the cause and progression of PA associated with sex and aging.     

Enzyme histochemistry is useful to assess viability of tumor tissue after microwave coagulation therapy (MCT): metastatic adenocarcinoma treated by lateral segmentectomy after MCT

We report on a case of metastatic adenocarcinoma of liver that was removed and examined histochemically after microwave coagulation therapy (MCT). The patient was a 65-year-old woman who had a metastatic tumor in the liver (S3) after high anterior resection due to a rectal adenocarcinoma and received MCT against the tumor. One month after MCT, multiple metastatic tumors were detected by abdominal computed tomography (CT) scan. As it was difficult to control them by MCT alone, we performed lateral segmentectomy. To assess the effects of microwave ablation on cellular viability of metastatic tumor, we used enzyme histochemistry for acid phosphatase (AcP), which is positive in macrophages infiltrating in the tumor. In a part of the ablated area of resected liver, there was remaining neoplastic tissue of which the morphology was maintained in H&E staining. This was found to be microwave-fixed non-viable tissue because no enzyme activity of AcP was detected in the infiltrating macrophages. This case report suggests that enzyme histochemistry was useful to assess the effect of MCT, enabling us to distinguish fixed cells from viable cells.     

Adrenergic regulation of clock gene expression in mouse liver

A main oscillator in the suprachiasmatic nucleus (SCN) conveys circadian information to the peripheral clock systems for the regulation of fundamental physiological functions. Although polysynaptic autonomic neural pathways between the SCN and the liver were observed in rats, whether activation of the sympathetic nervous system entrains clock gene expression in the liver has yet to be understood. To assess sympathetic innervation from the SCN to liver tissue, we investigated whether injection of adrenaline/noradrenaline (epinephrine/norepinephrine) or sympathetic nerve stimulation could induce mPer gene expression in mouse liver. Acute administration of adrenaline or noradrenaline increased mPer1 but not mPer2 expression in the liver of mice in vivo and in hepatic slices in vitro. Electrical stimulation of the sympathetic nerves or adrenaline injection caused an elevation of bioluminescence in the liver area of transgenic mice carrying mPer1 promoter-luciferase. Under a light-dark cycle, destruction of the SCN flattened the daily rhythms of not only mPer1, mPer2, and mBmal1 genes but also noradrenaline content in the liver. Daily injection of adrenaline, administered at a fixed time for 6 days, recovered oscillations of mPer2 and mBmal1 gene expression in the liver of mice with SCN lesion on day 7. Sympathetic nerve denervation by 6-hydroxydopamine flattened the daily rhythm of mPer1 and mPer2 gene expression. Thus, on the basis of the present results, activation of the sympathetic nerves through noradrenaline and/or adrenaline release was a factor controlling the peripheral clock.     

Thrombopoietin-producing hepatocellular carcinoma

A 66-year-old man with hepatocellular carcinoma (HCC) showed marked thrombocytosis (110.7 x 10(4)/microl). Bone marrow (BM) aspirates demonstrated an increase of mature megakaryocytes (MgK). The serum thrombopoietin (TPO) level was increased to about 100-fold that of the normal level in the terminal stage. However, the platelet count gradually decreased to 13.5 x 10(4)/microl. The autopsy specimen revealed normoplastic BM with decreased MgK, mainly consisting of the immature type, and it was negative for tumor cells. Liver specimen showed markedly fatty metamorphosis. Immunohistochemical staining of TPO demonstrated that hepatocytes were weakly stained and HCC cells strongly stained, suggesting TPO-producing HCC.     

A glutamate residue at the C terminus regulates activity of inward rectifier K+ channels: implication for Andersen's syndrome

G protein-coupled inward rectifiers (GIRKs) are activated directly by G protein betagamma subunits, whereas classical inward rectifiers (IRKs) are constitutively active. We found that a glutamate residue of GIRK2 (E315), located on a hydrophobic domain of the C terminus, is crucial for the channel activation. This glutamate (or aspartate) residue is conserved in all members of the Kir family. Substitution of alanine for the glutamate on GIRK1, GIRK2, and IRK2, expressed in HEK293 cells, greatly reduced the whole-cell currents. The whole-cell current of GIRK channels with a constitutively active gate, GIRK2(V188A), [Yi, B. A., Lin, Y. F., Jan, Y. N. & Jan, L. Y. (2001) Neuron 29, 657-667] was also reduced by the same glutamate mutation. Mean open time and conductance of single channels in GIRK2 and IRK2 were not affected by the mutation, indicating that the reduced whole-cell current resulted from a lowered probability of channel activation. The mutated GIRK and IRK showed normal trafficking to the cell membrane. The mutated GIRK2 retained the ability to interact with G protein betagamma subunits, and it showed almost the same inwardly rectifying property as the wild type. The mutated GIRK1 and GIRK2 retained ion selectivity to K(+) ions. This glutamate residue corresponds to one of the residues causing Andersen's syndrome [Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., et al. (2001) Cell 105, 511-519]. Our interpretation is that this region of the glutamate residue is crucial in relaying the activating message from the ligand sensor region to the gate.     

Pulmonary artery obstruction caused by thoracic aortic dissection: a case with unique pathological findings.

Pulmonary artery obstruction is a rare complication of acute thoracic aortic dissection. A 74-year-old woman was admitted to hospital with respiratory distress. Computed tomography scan showed right pulmonary artery occlusion and a thoracic aortic dissection of the Stanford A type. Lung perfusion scan revealed a defect in the entire right lung field. These findings mimicked acute pulmonary thromboembolism accompanying aortic dissection. On the other hand, pulmonary angiography revealed a round smooth defect of the right pulmonary artery, indicating an extrinsic compression. The patient finally died of cardiac tamponade. Autopsy disclosed that the right pulmonary artery was compressed by a hematoma in the adventitial space around the pulmonary artery. Such a mechanism of pulmonary artery obstruction caused by acute aortic dissection is unique, and is distinct from that caused by chronic non-dissecting aortic aneurysms, which themselves compress the pulmonary arteries. This complication inevitably follows aortic rupture; therefore, emergency operation to repair the dissected aorta must be performed to avoid the following catastrophic event.     

Estimated Daily Intake and Seasonal Food Sources of Quercetin in Japan

Quercetin is a promising food component, which can prevent lifestyle related diseases. To understand the dietary intake of quercetin in the subjects of a population-based cohort study and in the Japanese population, we first determined the quercetin content in foods available in the market during June and July in or near a town in Hokkaido, Japan. Red leaf lettuce, asparagus, and onions contained high amounts of quercetin derivatives. We then estimated the daily quercetin intake by 570 residents aged 20–92 years old in the town using a food frequency questionnaire (FFQ). The average and median quercetin intakes were 16.2 and 15.5 mg day⁻¹, respectively. The quercetin intakes by men were lower than those by women; the quercetin intakes showed a low correlation with age in both men and women. The estimated quercetin intake was similar during summer and winter. Quercetin was mainly ingested from onions and green tea, both in summer and in winter. Vegetables, such as asparagus, green pepper, tomatoes, and red leaf lettuce, were good sources of quercetin in summer. Our results will help to elucidate the association between quercetin intake and risks of lifestyle-related diseases by further prospective cohort study and establish healthy dietary requirements with the consumption of more physiologically useful components from foods.     

Glycated high-density lipoprotein regulates reactive oxygen species and reactive nitrogen species in endothelial cells

Nonenzymatic glycosylation of plasma proteins may contribute to the excess risk of developing atherosclerosis in patients with diabetes mellitus. Although it is believed that high-density lipoprotein (HDL) is glycosylated at an increased level in diabetic individuals, little is known about a possible linkage between glycated HDL and endothelial dysfunction in diabetes. To clarify whether glucose-modified HDL affects the function of endothelial cells, we first examined herein the level of H(2)O(2) generation from cultured human aortic endothelial cells (HAECs) exposed to a glycated oxidized HDL (gly-ox-HDL) prepared in vitro. Incubation for 48 hours with 100 microg/mL of gly-ox-HDL induced significant release of H(2)O(2) from cells and gly-ox-HDL-induced H(2)O(2) formation was inhibited in the presence of diphenyleneiodonium, an inhibitor of NADPH oxidase. In addition, stimulation of HAECs with gly-ox-HDL for 48 hours elicited a marked downregulation of catalase and Cu(2+), Zn(2+)-superoxide dismutase (CuZn-SOD), suggesting H(2)O(2) formation by gly-ox-HDL to be due to a disturbance involving oxidant and antioxidant enzymes in the cells. Treatment of HAECs with gly-ox-HDL attenuated the expression of endothelial nitric oxide synthase (eNOS), but not inducible nitric oxide synthase (iNOS), and this was followed by decreased production of nitric oxide (NO) by the cells. Furthermore, in vitro experiments with glycated HDL (gly-HDL) in the presence of 2 mmol/L EDTA and Cu(2+)-oxidized HDL suggested the effect of gly-HDL on endothelial function to be possibly potentiated by additional oxidative modification. Taking all of the above findings together, gly-ox-HDL may lead to the deterioration of vascular function through altered production of reactive oxygen species and reactive nitrogen species in endothelial cells.