Ruud D. Fontijn

Rosuvastatin Reduces Atherosclerosis Development Beyond and Independent of Its Plasma Cholesterol–Lowering Effect in APOE*3-Leiden Transgenic Mice Evidence for Antiinflammatory Effects of Rosuvastatin

Background—Statins can exert anti-inflammatory antiatherosclerotic effects through an anti-inflammatory action, independent of lowering cholesterol. We addressed the question whether the anti-inflammatory activities of statins can reduce atherosclerosis beyond the reduction achieved by cholesterol lowering per se. Methods and Results—Two groups of 20 female APOE*3-Leiden mice received either a high-cholesterol diet (HC) or a high-cholesterol diet supplemented with 0.005% (wt/wt) rosuvastatin (HC+R). The HC diet alone resulted in a plasma cholesterol concentration of 18.9±1.4 mmol/L, and administration of rosuvastatin lowered plasma cholesterol to 14.1±0.7 mmol/L. In a separate low-cholesterol (LC) control group, the dietary cholesterol intake was reduced, which resulted in plasma cholesterol levels that were comparable to the HC+R group (13.4±0.8 mmol/L). Atherosclerosis in the aortic root area was quantified after 24 weeks. As compared with the HC group, the LC group had a 62% (P <0.001) reduction in cross-sectional lesion area. When compared with the LC group, the HC+R group showed a further decrease in cross-sectional lesion area (80%, P <0.001), size of individual lesions (63%, P <0.05), lesion number (58%, P <0.001), monocyte adherence (24%, P <0.05), and macrophage-containing area (60%, P <0.001). Furthermore, rosuvastatin specifically suppressed the expression of the inflammation parameters MCP-1 and TNF-&agr; in the vessel wall and lowered plasma concentrations of serum amyloid A and fibrinogen, independent of its cholesterol-lowering effect. Conclusions—Rosuvastatin reduces atherosclerosis beyond and independent of the reduction achieved by cholesterol lowering alone. This additional beneficial effect of rosuvastatin may be explained, at least partly, by its anti-inflammatory activity.

The human kinesin-like protein RB6K is under tight cell cycle control and is essential for cytokinesis

ABSTRACT Several members of the kinesin superfamily are known to play a prominent role in the motor-driven transport processes that occur in mitotic cells. Here we describe a new mitotic human kinesin-like protein, RB6K (Rabkinesin 6), distantly related to MKLP-1. Expression of RB6K is regulated during the cell cycle at both the mRNA and protein level and, similar to cyclin B, shows a maximum during M phase. Isolation of the RB6K promoter allowed identification of a CDE-CHR element and promoter activity was shown to be maximal during M phase. Immunofluorescence microscopy using antibodies raised against RB6K showed a weak signal in interphase Golgi but a 10-fold higher signal in prophase nuclei. During M phase, the newly synthesized RB6K does not colocalise with Rab6. In later stages of mitosis RB6K localized to the spindle midzone and appeared on the midbodies during cytokinesis. The functional significance of this localization during M phase was revealed by antibody microinjection studies which resulted exclusively in binucleate cells, showing a complete failure of cytokinesis. These results substantiate a crucial role for RB6K in late anaphase B and/or cytokinesis, clearly distinct from the role of MKLP-1.

Monovalent nickel in hydrogenase from Chromatium vinosum: Light sensitivity and evidence for direct interaction with hydrogen

Redox titrations with hydrogenase from Chromatium vinosum show that its nickel ion can exist in 3, possibly 4, different redox states: the 3 +, 2 +, 1 + and possibly a zero valent state. The 1 + state is unstable: oxidation to Ni(II) occurs unless H2 gas is present. The Ni(I) coordination, but not that of Ni(III), is highly light sensitive. A photoreaction occurs on illumination. It is irreversible below 77 K, but reversible at 200 K. The rate of this photodissociation reaction in 2H2O is nearly 6‐times slower than in H2O, indicating the breakage of a nickel‐hydrogen bond. This forms the first evidence for an H atom in the direct coordination sphere of Ni in hydrogenase and for the involvement of this metal in the reaction with hydrogen.

Biogenesis of von Willebrand factor-containing organelles in heterologous transfected CV-1 cells.

Von Willebrand factor (vWF) is a multimeric protein involved in the adhesion of platelets to an injured vessel wall. vWF is synthesized by the endothelial cell and the megakaryocyte as a precursor protein (pro‐vWF) that consists of four repeated domains, denoted D1‐D2‐D′‐D3‐A1‐A2‐A3‐D4‐B1‐B2‐B3‐C1‐C2. Previously, we have defined the domains on the pro‐vWF molecule involved in dimerization as well as the domains involved in multimer assembly of vWF dimers. In the endothelial cell, part of the vWF multimers is stored in specialized organelles, the Weibel‐Palade bodies. By using immunoelectron microscopy, we demonstrate that upon expression of full‐length vWF cDNA, vWF‐containing organelles are encountered in monkey kidney CV‐1 cells that are morphologically similar to the endothelial‐specific Weibel‐Palade bodies. Expression in CV‐1 cells of a series of vWF cDNA deletion mutants, lacking one or more domains, revealed that only those vWF mutant proteins that are able to assemble into multimers are encountered in dense‐cored vesicles. Our data show that this process is independent of a particular domain on vWF and indicate that a ‘condensed’, multimeric vWF is required for targeting to the Weibel‐Palade body.

Spatial segregation of transport and signalling functions between human endothelial caveolae and lipid raft proteomes

Lipid rafts and caveolae are biochemically similar, specialized domains of the PM (plasma membrane) that cluster specific proteins. However, they are morphologically distinct, implying different, possibly complementary functions. Two-dimensional gel electrophoresis preceding identification of proteins by MS was used to compare the relative abundance of proteins in DRMs (detergent-resistant membranes) isolated from HUVEC (human umbilical-vein endothelial cells), and caveolae immunopurified from DRM fractions. Various signalling and transport proteins were identified and additional cell-surface biotinylation revealed the majority to be exposed, demonstrating their presence at the PM. In resting endothelial cells, the scaffold of immunoisolated caveolae consists of only few resident proteins, related to structure [CAV1 (caveolin-1), vimentin] and transport (V-ATPase), as well as the GPI (glycosylphosphatidylinositol)-linked, surface-exposed protein CD59. Further quantitative characterization by immunoblotting and confocal microscopy of well-known [eNOS (endothelial nitric oxide synthase) and CAV1], less known [SNAP-23 (23 kDa synaptosome-associated protein) and BASP1 (brain acid soluble protein 1)] and novel [C8ORF2 (chromosome 8 open reading frame 2)] proteins showed different subcellular distributions with none of these proteins being exclusive to either caveolae or DRM. However, the DRM-associated fraction of the novel protein C8ORF2 (approximately 5% of total protein) associated with immunoseparated caveolae, in contrast with the raft protein SNAP-23. The segregation of caveolae from lipid rafts was visually confirmed in proliferating cells, where CAV1 was spatially separated from eNOS, SNAP-23 and BASP1. These results provide direct evidence for the previously suggested segregation of transport and signalling functions between specialized domains of the endothelial plasma membrane.

EPR Spectrum at 4, 9 and 35 GHz of hydrogenase from Chromatium vinosum. Direct evidence for spin-spin interaction between Ni(III) and the ironsulphur cluster

Abstract EPR spectra at 4, 9 and 35 GHz of hydrogenase isolated from Chromatium vinosum have been compared. The spectra at 4 and 35 GHz confirmed our earlier conclusions, made from observations at 9 GHz (Albracht, S.P.J., Kalkman, M.L. and Slater, E.C. (1983) Biochim. Biophys. Acta 724, 309–316), that the irreversibly inactivated enzyme molecules in the preparation give rise to two EPR signals due to the independent non-interacting S = 1 2 systems of Ni(III) and a |3Fe-xS| cluster. It was observed that intact enzyme molecules show a complex EPR spectrum caused by a spin-coupled pair of Ni(III) and a |4Fe-4S|3+ cluster. The interaction energy is so weak (approx. 0.01 cm−1) that the 35 GHz spectra of both the Ni(III) and the |4Fe-4S|3+ cluster have the appearance of rather normal S = 1 2 spectra with additional splittings as a result of the spin-spin interaction. At lower microwave frequencies, the spectra become increasingly complex but phenomenologically they behave as expected for an exchange-coupled pair of dissimilar ions. The distance between the two spin systems is estimated to be at the most 1.2 nm. The spin-relaxation rate of the Ni(III) ion is dramatically enhanced as a result of the coupling to the rapidly relaxing Fe-S cluster. The g values and so presumably also the ligand fields of Ni in intact and irreversibly inactivated enzyme molecules are identical. This suggests that the specific coordination of the nickel in the enzyme is not the only requirement for activity with artificial electron donors or acceptors, and that the presence of a nearby, intact |4Fe-4S|3+(3+,2+) cluster might be another essential factor. From the g values and the probable function of Ni in the enzyme we propose, as a working hypothesis, that the nickel ion has five ligands provided by the protein in a square-pyramidal coordination.

EPR evidence for direct interaction of carbon monoxide with nickel in hydrogenase from Chromatium vinosum

Abstract Exposure of reduced hydrogenase from Chromatium vinosum to carbon monoxide resulted in two new EPR-detectable nickel species, one of which was sensitive to light. Illumination of H 2 -reduced enzyme and of CO-treated enzyme caused reversible photodissociation, resulting in the same nickel species. Although the rate of this photodissociation reaction H 2 -reduced enzyme in 2 H 2 O was nearly 6-times slower than in H 2 O (Van der Zwaan, J.W., Albracht, S.P.J., Fontijn, R.D. and Slater, E.C. (1985) FEBS Lett. 179, 271–277), no difference could be observed in the case of the CO-treated enzyme. It was concluded that hydrogen, possibly as a hybride ion, and CO can bind to nickel at the same ligand position. This is in agreement with the fact that CO is a competitive inhibitor of the enzyme.

A new EPR signal of nickel in Methanobacterium thermoautotrophicum

Abstract The two main EPR signals detectable in suspensions of whole cells of Methanobacterium thermoautotrophicum strain Marburg have both been assigned to nickel by growth of the bacterium on a medium enriched in 61 Ni ( I = 3/2). One of the signals could be identified as the soluble hydrogenase, that has earlier been characterized (Albracht, S.P.J.; Graf, E.-G. and Thauer, R.K. (1982) FEBS Lett. 140, 311–313). The other signal is new. Its g values and its hyperfine structure indicate that it represents nickel in a tetragonally distorted octahedral ligand field with 4 equivalent N atoms in the equatorial plane. From the available EPR data, it could not be established whether the nickel ion is in the monovalent or trivalent state. The nickel is attached to a soluble protein. Purified methyl-coenzyme M reductase displays the same signal. The spin concentration of the species in various preparations of the enzyme accounted for at most one-fifth of the concentration of the protein-bound factor F 430 . It is proposed that the signal represents F 430 in intact, active methyl-coenzyme M reductase molecules.

Direct evidence for sulphur as a ligand to nickel in hydrogenase: an EPR study of the enzyme from Wolinella succinogenes enriched in 33S

A preparation of hydrogenase from Wolinella (formerly Vibrio) succinogenes enriched in 33S to at least 70% has been studied by EPR spectroscopy. The sulphur isotope, which has a nuclear spin of 32, clearly broadened the spectrum of the Fe-S cluster. Resolved hyperfine splitting was observed in one of the lines of the EPR spectrum of Ni(III). It was concluded that this was due to interaction with one 33S nucleus. Also the appearance of the high-field line of the EPR signal of Ni(I), either before or after photodissociation of the nickel-hydrogen bond, indicated hyperfine interaction with one 33S nucleus. No indication has been observed for nitrogen hyperfine interaction in any of the EPR spectra in this study. The data provide independent proof of earlier conclusions from EXAFS studies by two other groups (Lindahl, P.A., Kojima, N. Hausinger, R.P., Fox J.A., Teo, B.K., Walsh C.T. and Orme-Johnson, W.H. (1984) J. Am. Chem. Soc., 106, 3062–3064, and Scott R.A., Wallin, S.A., Czechowsky, M., DerVartanian, D.V., LeGall, J., Peck, H.D.,Jr. and Moura, I. (1984) J. Am. Chem. Soc. 106, 6864–6865) that sulphur is present in the direct coordination of nickel in hydrogenase. The EPR data provide evidence for the presence of only one, or possibly two, S ligands.

Destruction and reconstitution of the activity of hydrogenase from Chromatium vinosum

The specific activity and the EPR spectra of purified hydrogenase from Chromatium vinosum vary from preparation to preparation. It has been found that both properties are dependent on the redox state of specific redox groups, probably thiols, in the enzyme. In defect enzyme molecules the proposed thiol groups are oxidized to an -S-S- bridge. As a result, the activity of the enzyme in the assay with viologens is minimal and nickel and the iron-sulphur cluster in the oxidized enzyme are present as two non-interacting S = 12 systems, Ni(III) and a [3Fe-4S]1+ cluster. In intact enzyme the -S-S- bridge does not exist. Such enzyme molecules are 15- (H2-production reaction) or 65- (H2-uptake reaction) times as active as defect molecules. Intact oxidized enzyme can accomodate a spin-coupled Ni(III)/[4Fe-4S]3+ pair. It can be converted to defect molecules by partial reduction with phenazine methosulphate plus ascorbate in air at 40–50°C. The reverse transition is induced by anaerobic incubation with a disulphide-reducing agent at 50°C.