Ca2+/calmodulin-dependent protein kinase II and cytosolic phospholipase A2 contribute to mitogenic signaling in myeloblastic leukemia U-937 cells.

The signaling mechanisms downstream of growth factor-stimulated proliferation in myeloid leukemia cells have not yet been fully elucidated. Recent evidence suggests that alternate pathways to the mitogen-activated protein kinase cascade are required. We have previously shown that Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) activates cytosolic phospholipase A2 (cPLA2), which is involved in the proliferation of vascular smooth muscle cells. In the present study, the contribution of this pathway was investigated in the proliferation of U-937 myeloid leukemia cells. In U-937 cells, fetal bovine serum (FBS)-induced proliferation was attenuated by CaM kinase II inhibitor KN-93 but not by its inactive analog KN-92. Inhibitors of cPLA2 (methyl arachidonyl fluorophosphonate and arachidonyl trifluoromethyl ketone) also reduced proliferation of U-937 cells. FBS-induced proliferation was also attenuated by cotransfection with cPLA2 antisense oligonucleotides. These results suggest a role for CaM kinase II and cPLA2 in the proliferation of U-937 cells. FBS stimulated CaM kinase II and cPLA2 activities in a time-dependent manner. Moreover, FBS-stimulated phosphorylation and activation of cPLA2 activation was inhibited by KN-93. FBS-stimulated phosphorylation of CaM kinase II was blocked by KN-93 but not by cPLA2 inhibitors, suggesting that CaM kinase II activates cPLA2. The products of phospholipid hydrolysis produced by cPLA2, lysophosphatidylcholine but not arachidonic acid, increased [3H]thymidine incorporation in U-937 cells. These data suggest that exposure of U-937 cells to FBS promotes phosphorylation and activation of CaM kinase II, leading to stimulation of cPLA2 and generation of lysophosphatidylcholine and resultant proliferation of these cells.

[PI-3 kinase/AKT/PKB signal transduction pathway in kidneys mediates angiotensin II-induced hypertension]. / PPI-3 kinaza/AKT/PKB put transdukcije signala u bubregu posreduje razvoj angiotenzin II-inducirane hipertenzije.

INTRODUCTION: The activation of PI-3 kinase/AKT/PKB signal transduction pathway has implicated in the cell growth regulation and proliferation. AIM: To determine a role of PI-3 kinase/AKT/PKB signal transduction pathway in the kidney of Angiotensin II-induced hypertensive rats. METHODS: NZW (New Zealand White) rats have been infused by Angiotensin II using osmotic minipump for six days (n = 8). Control group was untreated rats (n = 6). PI-3 inase and AKT/PKB activities were measured in the presence or absence of different inhibitors. RESULTS: Angiotensin II elevated mean arterial blood pressure (MABP) to 182 +/- 2 mm Hg (p < 0.001) vs untreated control rats (95 +/- 3 mm Hg). Ras-GTPase and PI-3 Kinase activities were elevated in angiotensin II-treated group. Ras inhibitors FPT III and BMS/191563 attenuated MABP to 122 +/- 2 and 127 +/- 4 mm Hg (p < 0.05) and abolished Ras-GTPase and PI-3 Kinase activities. AKT/PKB activity followed the PI-3 kinase activity. CONCLUSION: PI-3 kinase/AKT/PKB signal transduction pathway in the kidney is activated and mediates Angiotesin II-induced hypertension.

[Disorders of diastolic function in patients on hemodialysis]. / Poremecaji dijastolne funkcije u pacijenata na hemodijalizi.

INTRODUCTION: Impaired cardiac function is frequently present in patients on maintenance haemodialysis. AIM: To assess the left ventricular diastolic function in patients on haemodialysis. MATERIAL AND METHODS: We used 2D and pulsed wave Doppler echocardiography to evaluate the left ventricular diastolic function in 40 patients on haemodialysis and compared those to healthy controls. RESULTS: Majority of Doppler parameters were changed in patients on haemodialysis. Diastolic dysfunction was present in 77.5% patients. In comparison to the healthy controls haemodialysis patients showed significant increase in peak velocity of late diastolic filling, (A wave, 76.82 +/- 23.76 cm/s vs. 58.46 +/- 9.65 cm/s p < 0.001) and reduction in the E/A ratio (1.00 +/- 0.26 vs. 1.26 +/- 0.31 p < 0.001). CONCLUSION: There was significant impairment in left ventricular diastolic function in patients on haemodialysis.

[Precarcinoma and early carcinomatous lesions in the uterine cervix and the use of oral contraceptives]. / Prekarcinomske i rane karcinomske lezije cerviksa uterusa i upotreba oralnih kontraceptiva.

Precancerous and early cancerous lesions of the cervix uteri (ASCUS, AGUS, LSIL and HSIL) are precursors of invasive cancer of the cervix uteri. By Papanicolaou test they are graded as Pap III and Pap IV. Different factors may increase a risk for those lesions as well as converting low stage lesion into higher one. Oral contraceptive use is one of the most potential risk factor for those lesions. The goal of this study was to examine a relationship between oral contraceptive use and precancerous and early cancerous lesion of the cervix lesion of the cervix uteri by using Papanicolaou test. After adjustment for other potential risk factors our results have shown: 1) there is high significant positive relationship between oral contraceptives use and precancerous and early cancerous lesions of the cervix uteri; 2) the users of oral contraceptives have shown Pap III and Pap IV smear grade five to ten years earlier than non-users; 3) long-term users have shown Pap III and Pap IV five years earlier than short-term users for middle age group (35-44 years); 4) the border between Pap III and Pap IV is shifted for five years toward earlier age.

[Catalytic properties of recombinant pancreatic ribonuclease A-K7H]. / Kataliticke osobine rekombinantne pankreasne ribonukleaze A-K7H.

Pancreatic ribonuclease A is an enzyme that binds up ribonucleic acid (RNA) along the multiple binding subsites that essentially recognize the negatively charged phosphates of the substrate. It is endoribonuclease that catalyse depolimerization of single-stranded RNA. This work gives additional support to the existence of the phosphate-binding site p2 and confirms the central role of Lys-7 in establishing and electrostatic interraction with a phosphate group of the substrate. In this work catalytic properties of recombinant ribonuclease K7H have been studied. This enzyme is a mutant enzyme which contains histidine instead of lysine in a position 7, amino-acid that participates in the main catalytic center of RNase A, named p1. It was obtained by site-directed mutagenesis. Kinetic parameters of K7H have determined with C > p i poli (C) as substrates at pH 5.5 i 7.5. Kinetic parameters of K7H for C > p and as a substrate at pH 5.5 have not altered, but at pH 7.5 were significantly increased. Value Km was also increased, that indicates decreasing of affinity. Increasing of catalysis was double. Results of kinetic parameters of K7H with poli (C) as a substrate in pH 5.5 have shown slight difference according to kinetic parameters of commercial RNase A with poli (C). Significant decreasing of values of all kinetic parameters for K7H were reaction at pH 7.5.

[Endonuclease activity of recombinant pancreatic nuclease (A-K7H)]. / Endonukleazna aktivnost rekombinantne pankreasne ribonukleaze A-K7H.

Pancreatic ribonuclease A (RNase A) is a endonuclease that catalyzes depolymerization of ribonucleic acid (RNA) releasing oligonucleotides. In the process of binding enzyme with substrate are involved several non-catalytic phosphate binding subsites, one of them is p2, additional to main catalytic site p1. RNaza A prefers binding and cleavage of longer substrate molecules, and 3',5'-phosphodiester bond should be some six-seven residues apart from the end of molecules of the chain of RNA. In this work is analysed endonuclease activity of recombinant pancreatic RNase A (K7H), that in position seven instead of a lysine there is a histidine, amino acid residue that participates in main catalytic site p1. Mutant enzyme is obtained by site-directed mutagenesis by Kunkel. Results of this investigation have shown that substitution of lysine by histidine in position seven of RNase A has produced total deletion of p2 subsite, and K7H has lost endonuclease activity, and has become exonuclease. These results confirm central role of Lys-7 in establishing p2 subsite and endonuclease activity of pancreatic RNase A.