Divergent enzyme kinetics and structural properties of the two human mitochondrial creatine kinase isoenzymes

The mitochondrial isoenzymes of creatine kinase (MtCK), ubiquitous uMtCK and sarcomeric sMtCK, are key enzymes of oxidative cellular energy metabolism and play an important role in human health and disease. Very little is known about uMtCK in general, or about sMtCK of human origin. Here we have heterologously expressed and purified both human MtCK isoenzymes to perform a biochemical, kinetic and structural characterization. Both isoenzymes occurred as octamers, which can dissociate into dimers. Distinct Stokes' radii of uMtCK and sMtCK in solution were indicative for conformational differences between these equally sized proteins. Both human MtCKs formed 2D-crystals on cardiolipin layers, which revealed further subtle differences in octamer structure and stability. Octameric human sMtCK displayed p4 symmetry with lattice parameters of 145 A, indicating a 'flattening' of the octamer on the phospholipid layer. pH optima and enzyme kinetic constants of the two human isoenzymes were significantly different. A pronounced substrate binding synergism (Kd > Km) was observed for all substrates, but was most pronounced in the forward reaction (PCr production) of uMtCK and led to a significantly lower Km for creatine (1.01 mM) and ATP (0.11 mM) as compared to sMtCK (creatine, 7.31 mM; ATP, 0.68 mM).     

The role of alcohols as solvents in the genotoxicity testing of alpha,beta-unsaturated ketones in the SOS chromotest

alpha,beta-Unsaturated ketones are bifunctional compounds which form promutagenic 1,N(2)-propanodeoxyguanosine adducts like carcinogenic alpha,beta-unsaturated aldehydes and are mutagenic and genotoxic like these aldehydes. They are important industrial chemicals, are found in our environment and are widespread in our food. We investigated the SOS repair inducing activities of five ketones in the SOS chromotest and compared these results with that of the Ames test. Alkyl substitution at the beta-position of the alpha, beta-unsaturated carbonyl moiety leads to a decrease or loss in genotoxicity. Genotoxicity is higher if using ethanol as solvent instead of dimethylsulfoxide (DMSO). An increasing effect is also observed with methanol and n-propanol. Addition of the alcohol dehydrogenase inhibitor 4-methylpyrazole does not significantly influence the genotoxicity indicating that it is unlikely that the solvent effect depends on competitive inhibition of alcohol dehydrogenase by the alcohols used as solvents. Since other possible explanations e.g. ketal formation or solubility effects are also unlikely, the mechanism of this solvent effect observed with three different E. coli PQ-strains remains unresolved. No significant difference in genotoxicity of ethyl vinyl ketone was found between the strains PQ 37 and PQ 243 indicating that base excision repair does not play a role in the repair of 1,N(2)-propanodeoxyguanosine adducts, the main adducts of the alpha,beta-unsaturated ketones.     

Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies

 A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid. Received: 9 August 1999 / Accepted: 28 September 1999     

Fatty acids and signalling in endothelial cells

The endothelium is critical for the maintenance of a proper vessel function. Disturbances of endothelial function, called endothelial dysfunction, have serious implications, and lead to the development of atherosclerosis. It is well established that the risk for atherosclerosis development is influenced by nutritional factors such as the intake of certain fatty acids. Due to the fundamental role of the endothelium for atherosclerosis development, it is, therefore, likely that fatty acids directly influence the function of endothelial cells. The present review aims to explain the divergent effects of different types of fatty acids on cardiovascular disease risk by summarizing in vitro-data on the effects of fatty acids on (1) important signalling pathways involved in the modulation of endothelial cell function, and (2) endothelial cell functional properties, namely vasoactive mediator release and mononuclear cell recruitment, both of which are typically dysregulated during endothelial dysfunction.     

Developmental expression of neuromodulators in the central complex of the grasshopper Schistocerca gregaria

The central complex is a major integrative region within the insect brain with demonstrated roles in spatial orientation, the regulation of locomotor behavior, and sound production. In the hemimetabolous grasshopper, the central complex comprises the protocerebral bridge, central body (CB), ellipsoid body, noduli, and accessory lobes, and this modular organization develops entirely during embryogenesis. From a biochemical perspective, a range of neuroactive substances has been demonstrated in these modules of the adult central complex, but little is known about their developmental expression. In this study, we use matrix‐assisted laser desorption/ionization‐imaging mass spectrometry on single brain slices to confirm the presence of several peptide families (tachykinin, allatostatin, periviscerokinin/pyrokinin, FLRFamide, and neuropeptide F) in the adult central complex and then use immunohistochemistry and histology to examine their developmental expression, together with that of the indolamin serotonin, and the endogenous messenger nitric oxide (NO; via its synthesizing enzyme). We find that each neuromodulator is expressed according to a unique, stereotypic, pattern within the various modules making up the central complex. Neuropeptides such as tachykinin (55%) and allatostatin (65%), and the NO‐synthesizing enzyme diaphorase (70%), are expressed earlier during embryonic development than the biogenic amine serotonin (80%), whereas periviscerokinin‐like peptides and FLRFamide‐like peptides begin to be expressed only postembryonically. Within the CB, these neuroactive substances are present in tangential projection neurons before they appear in columnar neurons. There is also no colocalization of serotonin‐positive and peptide‐positive projections up to the third larval instar during development, consistent with the clear dorsoventral layering of the neuropil we observe. Our results provide the first neurochemical fingerprint of the developing central complex in an hemimetabolous insect. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Timing of reproduction in a Darwin''s finch: temporal opportunism under spatial constraints

We investigated whether predation by the minor grison ( Galictis cuja , a small mustelid) played a key role in limiting a wild cavy population ( Cavia magna ), ultimately leading to its local extinction. Radio-telemetry and capture-mark-recapture techniques were used to estimate grison predation rates (kill rates), time-specific probabilities of apparent mortality (population loss rate), overall mortality and grison predation for the cavy population. Additionally, we present data on alternative prey species, grison diet and reproduction to show potential proximate mechanisms of grison predation on wild cavies. The predictions specified were mostly confirmed: (1) grison predation was responsible for almost 80% of the cavies killed by known predators; (2) grison predation probabilities paralleled those of overall mortality of cavies over time; and (3) also those of the apparent mortality of the population. Thus, the population dynamics and the local extinction of the cavy population were not due to emigration processes. (4) Grison predation rates were not density-dependent, but showed pronounced peaks during the austral summer. The grison mainly preyed on small mammals: two water-rat species and the wild cavies. When the availability of alternative prey decreased in summer, the grison appeared to specialise on cavies. The onset of grison reproduction was somewhat delayed in relation to the onset of cavy reproduction. The lack of alternative prey coincided with high grison food demands due to reproduction, leading to a very high predation pressure ultimately resulting in the local extinction of the cavy population. We conclude that grison predation was indeed the main factor driving changes of the cavy population studied and speculate why caviomorph rodents might be especially susceptible to local extinction processes.     

Bis‐ and Tetrakis(carboxylato)platinum(IV) Complexes with Mixed Axial Ligands – Synthesis,Characterization, and Cytotoxicity

A series of twelve novel diamminetetrakis(carboxylato)platinum(IV) and 18 novel bis(carboxylato)dichlorido(ethane‐1,2‐diamine)platinum(IV) complexes with mixed axial carboxylato ligands was synthesized and characterized by multinuclear ¹H‐, ¹³C‐, ¹⁵N‐, and ¹⁹⁵Pt‐NMR spectroscopy. Their cytotoxic potential was evaluated (by MTT assay) against three human cancer cell lines derived from ovarian teratocarcinoma (CH1/PA‐1), lung (A549), and colon carcinoma (SW480). In the cisplatin‐sensitive CH1/PA‐1 cancer cell line, diamminetetrakis(carboxylato)platinum(IV) complexes showed IC₅₀ values in the low micromolar range, whereas, for the most lipophilic compounds of the bis(carboxylato)dichlorido(ethane‐1,2‐diamine)platinum(IV) series, IC₅₀ values in the nanomolar range were found.     

Oxidative Damage of U937 Human Leukemic Cells Caused by Hydroxyl Radical Results in Singlet Oxygen Formation

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.