Inhibition of growth of rat hepatoma by local injection of liposomes containing recombinant interleukin-2

Human recombinant interleukin-2 (IL-2) was entrapped in liposome, consisting of egg phosphatidylcholine (PC) and cholesterol.The peri-tumor injections of IL-2 liposome inhibited significantly the growth of solid tumor and prolonged the survival time of rats with solid tumors which were induced by a subcutaneous (s.c.) inoculation of AH-66 cells.Immunohistochemical staining of peritoneal exudate cells and tumor tissues revealed a marked accumulation of activated macrophages in and around the tumor tissues induced by the local injections of IL-2 liposome.     

Effect of prostaglandin E1 on renin and aldosterone in hypertensive patients.

The effect of prostaglandin E1 (PGE1) on plasma renin activity (PRA) and plasma aldosterone concentration (PAC) was studied in the hypertensive subjects treated with or without 75 mg indomethacin or 60 mg propranolol for a week. Subsequent to the treatment with indomethacin for a week, PRA and PAC levels were decreased as compared to the control, without changes in the blood pressure and heart rate. During the infusion of PGE1, the blood pressure was decreased and the pulse rate was increased. PRA and PAC levels were also elevated. These changes of parameters were not different between the control and the indomethacin-treated subjects. PRA and PAC were suppressed after the treatment with propranolol. With the infusion of PGE1, the level of PRA was not significantly elevated, while, PAC was significantly increased by the infusion of 100 ng/Kg/min of PGE1. During the infusion of PGE1, the blood pressure was decreased while the pulse rate was increased in the subjects treated with propranolol. However, the elevation of the pulse rate was less remarkable than the control. These data indicate that PGE1 have important roles in the regulation of the release of renin and aldosterone. These findings also suggest that PGE1 may act to stimulate the secretion of aldosterone in man.     

Prion protein with Y145STOP mutation induces mitochondria-mediated apoptosis and PrP-containing deposits in vitro

A pathogenic truncation of an amber mutation at codon 145 (Y145STOP) in Gerstmann-Straussler-Scheinker disease (GSS) was investigated through the real-time imaging in living cells, by utilizing GFP-PrP constructs. GFP-PrP(1-144) exhibited an aberrant localization to mitochondria in mouse neuroblastoma neuro2a (N2a) and HpL3-4 cells, a hippocampal cell line established from prnp gene-ablated mice, whereas full-length GFP-PrP did not. The aberrant mitochondrial localization was also confirmed by Western blot analysis. Since GFP-PrP(1-121), as previously reported, and full-length GFP-PrP do not exhibit such mitochondrial localization, the mitochondrial localization of GFP-PrP(1-144) requires not only PrP residues 121-144 (in human sequence) but also COOH-terminal truncation in the current experimental condition. Subsequently, the GFP-PrP(1-144) induced a change in the mitochondrial innermembrane potential (DeltaPsi(m)), release of cytochrome c from the intermembrane space into the cytosol, and DNA fragmentation in these cells. Non-fluorescent PrP(1-144) also induced the DNA fragmentation in N2a and HpL3-4 cells after the proteasomal inhibition. These data may provide clues as to the molecular mechanism of the neurotoxic property of Y145STOP mutation. Furthermore, immunoelectron microscopy revealed numerous electron-dense deposits in mitochondria clusters of GFP-PrP(1-144)-transfected N2a cells, whereas no deposit was detected in the cells transfected with full-length GFP-PrP. Co-localization of GFP/PrP-immunogold particles with porin-immunogold particles as a mitochondrial marker was observed in such electron-dense vesicular foci, resembling those found in autophagic vacuoles forming secondary lysosomes. Whether such electron-dense deposits may serve as a seed for the growth of amyloid plaques, a characteristic feature of GSS with Y145STOP, awaits further investigations.     

Catalase regulates cell growth in HL60 human promyelocytic cells: evidence for growth regulation by H(2)O(2)

Reactive oxygen species (ROS) including hydrogen peroxide (H(2)O(2)) are generated constitutively in mammalian cells. Because of its relatively long life and high permeability across membranes, H(2)O(2) is thought to be an important second messenger. Generation of H(2)O(2) is increased in response to external insults, including radiation. Catalase is located at the peroxisome and scavenges H(2)O(2). In this study, we investigated the role of catalase in cell growth using the H(2)O(2)-resistant variant HP100-1 of human promyelocytic HL60 cells. HP100-1 cells had an almost 10-fold higher activity of catalase than HL60 cells without differences in levels of glutathione peroxidase, manganese superoxide dismutase (MnSOD), and copper-zinc SOD (CuZnSOD). HP100-1 cells had higher proliferative activity than HL60 cells. Treatment with catalase or the introduction of catalase cDNA into HL60 cells stimulated cell growth. Exposure of HP100-1 cells to a catalase inhibitor resulted in suppression of cell growth with concomitant increased levels of intracellular H(2)O(2). Moreover, exogenously added H(2)O(2) or depletion of glutathione suppressed cell growth in HL60 cells. Extracellular signal regulated kinase 1/2 (ERK1/2) was constitutively phosphorylated in HP100-1 cells but not in HL60 cells. Inhibition of the ERK1/2 pathway suppressed the growth of HP100-1 cells, but inhibition of p38 mitogen-activated protein kinase (p38MAPK) did not affect growth. Moreover, inhibition of catalase blocked the phosphorylation of ERK1/2 but not of p38MAPK in HP100-1 cells. Thus our results suggest that catalase activates the growth of HL60 cells through dismutation of H(2)O(2), leading to activation of the ERK1/2 pathway; H(2)O(2) is an important regulator of growth in HL60 cells.     

More than a 100-fold increase in immunoblot signals of laser-microdissected inclusion bodies with an excessive aggregation property by oligomeric actin interacting protein 2/D-lactate dehydrogenase protein 2

We established a histobiochemical approach targeting micron-order inclusion bodies possessing extensive aggregation properties in situ by using a nonchemical denaturant (oligomeric actin interacting protein 2/d-lactate dehydrogenase protein 2 [Aip2p/Dld2p]) with the combinatorial method of laser-microdissection and immunoblot analysis. As a model, pick bodies were chosen and laser-microdissected from three different brain regions of two patients with Pick's disease. Initially, 500 to 2000 pick bodies were applied onto SDS-PAGE gels after boiling in Laemmli's sample buffer according to established immunoblotting procedures; however, only faint signals were obtained. Following negative results with chemical denaturants or detergent, including 6 M guanidine hydrochloride, 8 M urea, and 2% SDS, the laser-microdissected pick bodies were pretreated with oligomeric Aip2p/Dld2p, which possesses robust protein unfolding activity under biological conditions. Strikingly, only one pick body was sufficient to illustrate an immunoblot signal, indicating that pretreatment with oligomeric Aip2p/Dld2p enhanced the immunoblot sensitivity by more than 100-fold. Pretreatment with oligomeric Aip2p/Dld2p also allowed us to quantify the total protein content of pick bodies. Thus, use of oligomeric Aip2p/Dld2p significantly contributed toward the acquisition of information pertaining to the molecular profile of proteins possessing an extensive aggregation property, particularly in small amounts.     

Identification of angiotensin I in a cyclostome, Lampetra fluviatilis

Angiotensin I (ANG I) was isolated from incubates of plasma and kidney extracts of the river lamprey, Lampetra fluviatilis, using eel vasopressor activity as an assay during purification. Its sequence was Asn-Arg-Val-Tyr-Val-His-Pro-Phe-Thr-Leu as determined by the sequence analysis and mass spectrometry. The sequence was confirmed by identity of the elution profile with the synthetic peptide in two different reverse-phase columns of high-performance liquid chromatography. Lamprey ANG I produced dorsal-aortic pressor responses in L. fluviatilis but the rise was very small in comparison to that produced by angiotensin II. Angiotensin III produced an even bigger increase. It was not possible to demonstrate a difference in response to Asn(1) (lamprey) ANG I and Asp(1) (human) ANG I. The present study directly demonstrated the presence and biological activity of the renin-angiotensin system in the most primitive extant vertebrates, the cyclostomes. Thus the renin-angiotensin system is a phylogenetically old hormonal system that is present throughout the vertebrates.     

MSF, a novel cytoplasmic chaperone which functions in precursor targeting to mitochondria.

Mitochondrial import stimulation factor (MSF) unfolds wheat germ lysate synthesized aggregated mitochondrial precursor proteins and stimulates their mitochondrial import in an ATP dependent manner. Here we analysed the function of MSF mainly by utilizing chemically pure adrenodoxin precursor (pAd). MSF bound to the unfolded pAd and prevented it from losing import competence and also restored the import competence of the aggregated pAd dependent on ATP hydrolysis. The import incompetent aggregated mitochondrial precursors induced the ATPase activity of MSF and the activity was strongly inhibited by isolated mitochondrial outer membrane (OM) but not by trypsin treated outer membrane (tOM). The precursor induced ATPase activity of N-ethylmaleimide (NEM)-treated MSF was not inhibited by OM. In this context, the MSF-precursor complex specifically bound to OM and binding was abolished both by the treatment of OM with trypsin and by the treatment of MSF with NEM. These results show that MSF is a novel cytoplasmic chaperone protein with a mitochondrial precursor-targeting function.