Andrzej Klein

The ability of HDL to inhibit VCAM-1 expression and oxidized LDL uptake is impaired in renal patients.

OBJECTIVES This study examines the ability of HDL from hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients to suppress the expression of adhesion molecules in endothelial cells (ICAM-1, VCAM-1) and in monocytes (LFA-1, VLA-4) and to inhibit the uptake of oxidized LDL by macrophages. DESIGN AND METHODS Gene expression and the uptake of oxidized LDL were determined in 12 HD patients, 12 CAPD patients and 14 healthy volunteers. RESULTS HDL from renal patients were less effective than control lipoproteins in reducing VCAM-1 expression. HDL from CAPD patients inhibited LFA-1 expression to the highest extent. The ability of HDL from renal patients to reduce oxidized LDL uptake was lower compared to control group. CONCLUSIONS Decreased ability of HDL to suppress expression of VCAM-1 in endothelial cells and the uptake of oxidized LDL by macrophages can be one of the risk factors for atherosclerosis development in patients with renal failure.

The effect of tyrosine kinase inhibitors, tyrphostins: AG1024 and SU1498, on autocrine growth of prostate cancer cells (DU145).

It is well established that autocrine growth of human prostate cancer cell line DU145 is dependent on TGF (EGF)/EGFR loop. However, the participation of several other growth factors in proliferation of DU145 cells has been also proposed. We employed two selective tyrosine kinase inhibitors (tyrphostins): AG1024 (an IGFIR inhibitor) and SU1498 (a VEGFR2 inhibitor) for growth regulation of DU145 cells, cultured in chemically defined DMEM/F12 medium. Both the tested compounds inhibited autocrine growth of DU145 cells at similar concentration values (IC50 approximately 2.5 microM). The tyrphostins arrested cell growth of DU145 in G1 phase, similarly as inhibitors of EGFR. However, in contrast to selective inhibitors of EGFR, neither AG1024, nor SU1498 (at concentration < or =10 microM) decreased the viability of the investigated cells. These results strongly suggest that autocrine growth of DU145 cells is stimulated by, at least, three autocrine loops: TGFalpha(EGF)/EGFR, IGFII/IGFIr and VEGF/VEGFR2(VEGFR1). These data support the hypothesis of multi-loops growth regulation of metastatic prostate cancer cell lines.

The effect of tyrphostins AG494 and AG1478 on the autocrine growth regulation of A549 and DU145 cells

We employed two selective EGFR tyrosine kinase inhibitors: AG494 (reversible) and AG1478 (irreversible) for growth regulation of human lung (A549) and prostate (DU145) cancer cell lines, cultured in chemically defined DMEM/F12 medium. Both tested tyrphostins significantly inhibited autocrine growth of the investigated cell lines. The action of AG494 was dose dependent, and at highest concentrations led to complete inhibition of growth. AG1478 seemed to be more effective at lower concentrations, but was unable to completely inhibit growth of A549 cells. Inhibition of EGFR kinase activity by AG494 in contrast to AG1478 had no effect on the activity of ERK in both cell lines. Both EGFRs inhibitors induced apoptosis of the investigated lung and prostate cancer cell lines, but the proapoptotic effect of the investigated tyrphostins was greater in A549 than in DU145 cells. The tyrphostins arrested cell growth of DU145 and A549 cells in the G1 phase, similarly to other known inhibitors of EGFR. The influence of AG494 and AG1478 on the activity of two signaling proteins (AKT and ERK) was dependent upon the kind of investigated cells. In the case of DU145 cells, there was an evident decline in enzymatic activity of both kinases (stronger for AG1478), while in A549, only AG1478 effectively inhibited the phosphorylation of Akt. Tyrphostins AG494 and AG1478 are ATP-competitors and are supposed to have a similar mechanism of action, but our results suggest that this is not quite true.

Isolation of middle-sized non-diffusible sugar-peptide (nsp) fraction from calf liver. Its aggregation properties and similarity to the corresponding fraction obtained from bovine blood plasma

Abstract 1. 1. The fraction of middle-sized non-diffusible sugar-peptide components may be obtained by the two-step dialysis method from homogenate of calf liver blooded by perfusion with 0.9% NaCl. 2. 2. The constituents of the fraction show great ability to aggregate. Thin-layer electrophoresis resolves the fraction into seven ninhydrin-positive and one ninhydrin-negative bands each of which appears to be an aggregate formed of the same constituents. 3. 3. Elution profiles in ion exchange chromatography on Hamilton 77756 resin and nitration curves on Bio-Gel P-6 in the presence of SDS and 2-mercaptoethanol show the similarity of this fraction to that from bovine plasma obtained both by means of Sephadex G-25 filtration followed by dialysis against H 2 O and by the two-step dialysis method.

Isolation of the homogeneous constituents from non-diffusible sugar-peptide (nsp) fraction originating from bovine plasma—i. method of disruption of a sugar-peptide aggregate

1. High molecular weight sugar-peptide aggregate contains 84--92% of sugar-peptide compounds of the whole non-diffusible sugar-peptide (NSP) fraction obtained from bovine blood plasma by the two-step dialysis method. 2. The aggregate can be resolved into various numbers, dependent on conditions, of electrophoretic homogeneous bands, which appeared to be heterogeneous in thin-layer chromatography on MN-Cellulose. 3. Electrophoresis in 5 M guanidine-hydrochloride allows the disruption of the aggregate and isolation of seven chromatographic and electrophoretic homogeneous constituents with different molecular weights and chemical characters.

Isolation of the homogeneous constituents from non-diffusible sugar-peptide (nsp) fraction originating from bovine plasma—II. the influence on cell growth of bhk-21, bhk/rsv and pr-rsh cells In vitro

1. The sugar-peptide aggregate isolated from the NSP fraction of bovine blood, added in concentration 20 microgram/ml, decrease of [3H]thymidine incorporation and cell proliferation of BHK-21 and BHK/RSV cells in vitro to approx. 25% of untreated control. 2. Two of four peptide constituents of the aggregate show distinct and extremely different effects on cell growth in vitro. The effects of peptide D mol. wt 5000-6000 is characteristic for peptide growth factors, while the peptide G mol. wt 1200-1800 decreases rapidly the growth of all investigated kinds of cells to 8-16% relative to untreated control. 3. The NSP fraction contains also two non-peptide constituents, which inhibit the growth of Rous sarcoma virus transformed cells to a statistically significant degree and have no effect on the growth of BHK-21 cells.

Iron inhibits respiratory burst of peritoneal phagocytes in vitro.

Objective. This study examines the effects of iron ions Fe3+ on the respiratory burst of phagocytes isolated from peritoneal effluents of continuous ambulatory peritoneal dialysis (CAPD) patients, as an in vitro model of iron overload in end-stage renal disease (ESRD). Material and Methods. Respiratory burst of peritoneal phagocytes was measured by chemiluminescence method. Results. At the highest used concentration of iron ions Fe3+ (100 μM), free radicals production by peritoneal phagocytes was reduced by 90% compared to control. Conclusions. Iron overload may increase the risk of infectious complications in ESRD patients.