G. Gercken

Development of lipids during a spring plankton bloom in the northern North Sea: I. Particulate fatty acids

Abstract The plankton spring bloom in the northern North Sea was extensively investigated during a period of three months in 1976 at a fixed station occupied by the R.V. “Meteor”. Samples of different depth-profiles, representative of the phytoplankton development, were collected eleven times to analyze the concentration of fatty acids of the particulate matter. The water column was divided into an upper and lower layer according to the thermocline depths, because different processes take place in these layers. During the exponential growth phase the fatty acid concentration rose only slightly due to increases in polyunsaturated fatty acids (18:4, 20:5, 22:6), which are typical for marine plankton. With the exhaustion of nutrients the biochemical composition changed and the fatty acid concentration increased sharply from about 3 to 20 μmol C dm − finally to about 30% of the particulate carbon. The main proportion consisted of oleic acid (28.3%) and palmitic acid (24.2%). The first phytoplankton bloom, dominated by diatoms (Chaetoceros species), was characterized by the increase in fatty acids with 16 carbon atoms, whereas during the second smaller bloom, with dinoflagellates as the main species, more fatty acids with 18 carbon atoms occurred. After the stationary growth phase the phytoplankton biomass strongly decreased, resulting in an increase of particulate matter below the thermocline. The fatty acid pattern there was similar to that during the stationary phase of the phytoplankton bloom in the upper layer.

Novel Aspects of Intrinsic and Extrinsic Aging of Human Skin: Beneficial Effects of Soy Extract¶

Abstract Biochemical and structural changes of the dermal connective tissue substantially contribute to the phenotype of aging skin. To study connective tissue metabolism with respect to ultraviolet (UV) exposure, we performed an in vitro (human dermal fibroblasts) and an in vivo complementary DNA array study in combination with protein analysis in young and old volunteers. Several genes of the collagen metabolism such as Collagen I, III and VI as well as heat shock protein 47 and matrix metalloproteinase-1 are expressed differentially, indicating UV-mediated effects on collagen expression, processing and degradation. In particular, Collagen I is time and age dependently reduced after a single UV exposure in human skin in vivo. Moreover, older subjects display a lower baseline level and a shorter UV-mediated increase in hyaluronan (HA) levels. To counteract these age-dependent changes, cultured fibroblasts were treated with a specific soy extract. This treatment resulted in increased collagen and HA synthesis. In a placebo-controlled in vivo study, topical application of an isoflavone-containing emulsion significantly enhanced the number of dermal papillae per area after 2 weeks. Because the flattening of the dermal–epidermal junction is the most reproducible structural change in aged skin, this soy extract appears to rejuvenate the structure of mature skin.

Hydrolysis of cellooligosaccharides by Trichoderma reesei cellobiohydrolases: Experimental data and kinetic modeling

Abstract The hydrolysis of cellooligosaccharides with a degree of polymerization (DP) up to 8 by cellobiohydrolases (CBH) I and II from Trichoderma reesei MCG 77 was investigated. Both enzymes degraded the oligomeric substrates according to a “multiple attack” mechanism without release of significant amounts of intermediate hydrolysis products with a DP higher than 3. CBH I was determined not to be a real 1,4-β- d -glucan-cellobiohydrolase because glucose was produced from all oligomeric substrates. The maximum initial velocity of substrate degradation by CBH I was highest for DP 6. Km values decreased from DP 4 to 6 and remained almost constant for higher DPs, indicating that the active site of CBH I spans at least six glucosyl-units. Based on the known three-dimensional structure of the core protein of CBH II, the product ratio of cellobiose to cellotriose released by the action of CBH II on different cellooligosaccharides could be explained and theoretically predicted. A β-glycosidic bond at the nonreducing end of the substrate was found to be necessary for recognition by CBH I as well as CBH II because α- d -glucosyl-(1,4)-cellopentaose was not degraded by both enzymes. A mathematical model based on a reaction-rate-dependent, reversible loss of active enzyme, interpreted in terms of nonproductive substrate binding, was derived. This non-steady-state model was valid to predict concentration-time course data for the hydrolysis of all oligomeric substrates by CBH I and CBH II very well. The time-dependent substrate degradation and product formation clearly deviated from integrated Michaelis-Menten kinetics.

Mechanisms of lipid peroxidation in human blood plasma: A kinetic approach

There is strong evidence that the oxidation of plasma lipoproteins plays an important role in atherogenesis. The exact mechanisms by which lipoprotein oxidation occurs in the presence of other plasma constituents, however, remains unclear. To investigate the role of different antioxidants for this process, we studied the oxidation of human plasma supplemented in vitro with physiological amounts of major plasma antioxidants alpha-tocopherol, ubiquinol-10 ascorbate, urate, bilirubin and albumin. The plasma was diluted 2-fold and oxidized by 3.75 mM Cu(II). The concentrations of the antioxidants, fatty acids, linoleic acid hydroperoxides and oxycholesterols in oxidizing plasma were measured. The oxidation was characterized by three consecutive phases similar to the known lag, propagation, and decomposition phases of low density lipoprotein oxidation. The rate of the initiation of oxidation as calculated from antioxidant consumption rates was raised by supplementation with alpha-tocopherol or ascorbate. The oxidation rate in the lag phase was lowered by supplementation with any of the antioxidants, whereas in the propagation phase the oxidation rate was slightly higher in supplemented than in unsupplemented plasma. The kinetic chain length in the lag phase was less than one in supplemented plasma and about one in unsupplemented plasma. The chain length in the propagation phase was between three and six for all plasma samples. A higher rate of urate consumption and a reduced rate of alpha-tocopherol consumption were found in plasma supplemented with ascorbate in comparison with unsupplemented plasma. These data suggest that: (i) the reduction of Cu(II) by alpha-tocopherol and ascorbate is a major initiating event in Cu(II)-catalyzed oxidation of human plasma; (ii) the following lag phase is caused by radical-scavenging effects of all antioxidants with alpha-tocopherol as a major lipophilic and urate as a major hydrophilic scavenger; (iii) interactions between antioxidants, such as regeneration of ascorbate by urate and of alpha-tocopherol by ascorbate, take place during the lag phase; (iv) in the absence of added antioxidants the oxidation in the lag phase can occur via a chain reaction; and (v) in the propagation phase the oxidation is not inhibited by antioxidants and occurs autocatalytically.

Quantitative preparation and gas chromatography of short and medium chain fatty acid benzyl esters (C1-C12)

Abstract The benzyl esters of short and medium chain fatty acids (C1—C12) were prepared with N,N′-dicyclohexyl-O-benzylisourea or with phenyldiazomethane. The extent of estertification was checked by using 14C-labeled fatty acids. The fatty acid benzyl esters were separated gas chromatographically on both SE-30 and EGSS-X columns. For quantification, the relative response for equal weights of fatty acids was determined. The mild esterification with phenyldiazomethane allows this method to be used for the assay of short and medium chain free fatty acids in biological materials.

Increase of bovine alveolar macrophage superoxide anion and hydrogen peroxide release by dusts of different origin.

The release of reactive oxygen intermediates (ROI) from bovine alveolar macrophages (BAM) after stimulation with heavy metal-containing dusts was investigated. BAM were obtained by postmortem lavages of bovine lungs. The dusts were collected from waste incineration, sewage sludge incineration, an electric power station, and from two different factories. Three quartz dusts were used as heavy metal-free controls. The dusts were fractionated by sieving and sedimentation and analyzed by electron microscopy, atomic absorption spectrometry (AAS), and atomic emission spectrometry with inductively coupled plasma (AES-ICP). Incubation of BAM with the dusts (12.5-1000 micrograms/ml medium) led to concentration-dependent increases in ROI release. The secretion of ROI was already seen after 15 min and lasted throughout the experiment up to 90 min, with the exception of a waste incinerator ash, which contained the highest contents of some heavy metals and where the release of ROI ceased after 60 min. We suggest that this dust exhibits simultaneously stimulating and inhibiting effects. The ratio of the secreted O2- and H2O2 varied, depending on the dust being investigated. The release of hydrogen peroxide correlated best, in descending order, with the content of iron, manganese, chromium, vanadium, and arsenic in the dusts.

Metabolite status of the heart in acute insufficiency due to 1-fluoro-2,4-dinitrobenzene

Nach 1-Fluor-2,4-dinitrobenzol-Vergiftung von perfundierten Kaninchenherzen waren bei vollständiger Insuffizienz der ATP-Gehalt sehr signifikant und der Kreatinphosphatgehalt weniger stark vermindert. Glykogen-, Glucose- und Lactatgehalt unterschieden sich von den Kontrollwerten nicht.

Development of lipids during a spring plankton bloom in the northern North Sea: II. Dissolved lipids and fatty acids

Abstract During the spring plankton bloom (1976) in the northern North Sea the fatty acid and lipid distribution of the chloroform extractable fraction of seawater was analyzed by gas chromatography and thin-layer chromatography. The fatty acids in the seawater made up about 3% of the total dissolved organic matter. Maximum concentrations during the first phytoplankton bloom were in the range of 5 μmol C dm−3. Palmitic acid (30.4%) and oleic acid (21.4%) as well as myristic, stearic and palmitoleic acids were the main fatty acids. The concentrations of fatty acids were higher below the thermocline in the deeper layers of the water column. The oleic acid showed large fluctuations, especially below the thermocline and seemed to be bound in a polar fraction. Different lipid classes containing fatty acids were determined by thin-layer chromatography. An estimation showed that the free fatty acid fraction made up the main portion (47.9%), followed by the triacylglycerol fraction (22.8%) and a more polar fraction with 25.1%. The triacylglycerol fraction appears to be more stable than the other fraction in the water column.

Cytotoxicity of ester and ether lysophospholipids on Leishmania donovani promastigotes.

The cytotoxic activity of four ester lysophospholipids, three ether lysophospholipids, and two radylglycerols on Leishmania donovani promastigotes was determined by measuring the inhibition of cell growth. The 1-acyl lysophospholipids reduced cell growth to 50% of controls at concentrations of 6.4-10.9 microM. In contrast, 1-O-alkenyl-sn-glycero-3-phosphoethanolamine, 1-O-hexadecyl-sn-glycero-3-phosphocholine, and 1-O-hexadecyl-sn-glycerol already showed a 50% inhibition of growth at concentrations between 2.1 and 2.8 microM. Moreover, the unnatural alkyl lysophospholipid analogue 1-O-octadecyl-2-methoxy-sn-glycero-3-phosphocholine was even 10-fold more toxic. Incubations of L. donovani promastigotes with radioactively labelled ether lysophospholipids revealed a rapid uptake of these compounds and their incorporation into cellular lipids at a non-toxic concentration of 1.0 microM. An accumulation of the lysophospholipids in the cell due to insufficient metabolism may be the cause of its cytotoxic effect. The sensitivity of L. donovani cells towards ether lysophospholipids was found to be similar to that reported for tumor cells.

Tight Control of Matrix Metalloproteinase-1 Activity in Human Skin¶

Abstract Chronic ultraviolet irradiation leads to photoaging in human skin, which is associated with degradation of connective tissue. This is partly due to the fibroblast collagenase (matrix metalloproteinase 1 [MMP-1]). Using complementary DNA array technique we demonstrate that after UV irradiation, MMP-1, MMP-3 and the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) are time-dependently induced on the messenger RNA level in dermal fibroblasts in vitro and in vivo in human buttock skin. This increase in gene expression is paralleled by an increase of latent and active MMP-1 protein after low-dose UV-A exposure in vitro. In vivo the concentration of latent MMP-1 in suction blister fluids peaks 24 h after irradiation with 2 minimal erythema doses of solar simulated radiation. However, only a small proportion of MMP-1 in vitro (5.5 ± 1.5%) and in vivo is active, whereas the majority of MMP-1 remains in its inactive proform. Interestingly, in suction blister fluid the concentration and duration of TIMP-1 expression exceeds that of MMP-1. Taken together, these data indicate that MMP-1 activity is tightly regulated transcriptionally and posttranscriptionally. Furthermore, the pronounced individual differences in all targets investigated provide a possible explanation for the different susceptibility of individuals to UV exposure and, thus, to the clinical features of photodamage.