Desmosterol may replace cholesterol in lipid membranes

Recently, knockout mice entirely lacking cholesterol have been described as showing only a mild phenotype. For these animals, synthesis of cholesterol was interrupted at the level of its immediate precursor, desmosterol. Since cholesterol is a major and essential constituent of mammalian cellular membranes, we asked whether cholesterol with its specific impact on membrane properties might be replaced by desmosterol. By employing various approaches of NMR, fluorescence, and EPR spectroscopy, we found that the properties of phospholipid membranes like lipid packing in the presence of cholesterol or desmosterol are very similar. However, for lanosterol, a more distant precursor of cholesterol synthesis, we found significant differences in comparison with cholesterol and desmosterol. Our results show that, from the point of view of membrane biophysics, cholesterol and desmosterol behave identically and, therefore, replacement of cholesterol by desmosterol may not impact organism homeostasis.     

Energetics of primary and secondary electron transfer in Photosystem II membrane particles of spinach revisited on basis of recombination-fluorescence measurements

Photon absorption by one of the roughly 200 chlorophylls of the plant Photosystem II (PSII) results in formation of an equilibrated excited state (Chl200*) and is followed by chlorophyll oxidation (formation of P680+) coupled to reduction of a specific pheophytin (Phe), then electron transfer from Phe- to a firmly bound quinone (QA), and subsequently reduction of P680+ by a redox-active tyrosine residue denoted as Z. The involved free-energy differences (DeltaG) and redox potentials are of prime interest. Oxygen-evolving PSII membrane particles of spinach were studied at 5 degrees C. By analyzing the delayed and prompt Chl fluorescence, we determined the equilibrium constant and thus free-energy difference between Chl200* and the [Z+,QA-] radical pair to be -0.43+/-0.025 eV, at 10 mus after the photon absorption event for PSII in its S(3)-state. On basis of this value and previously published results, the free-energy difference between P680* and [P680+,QA-] is calculated to be -0.50+/-0.04 eV; the free-energy loss associated with electron transfer from Phe to QA is found to be 0.34+/-0.04 eV. The given uncertainty ranges do not represent a standard deviation or likely error, but an estimate of the maximal error. Assuming a QA-/QA redox potential of -0.08 V, the following redox-potential estimates are obtained: +1.25 V for P680/P680+; +1.21 V for Z/Z+ (at 10 mus); -0.42 V for Phe-/Phe; -0.58 V for P680*/P680+.     

Prediction of the Adaptability of Pseudomonas putida DOT-T1E to a Second Phase of a Solvent for Economically Sound Two-Phase Biotransformations

The strain Pseudomonas putida DOT-T1E was tested for its ability to tolerate second phases of different alkanols for their use as solvents in two-liquid-phase biotransformations. Although 1-decanol showed an about 10-fold higher toxicity to the cells than 1-octanol, the cells were able to adapt completely to 1-decanol only and could not be adapted in order to grow stably in the presence of a second phase of 1-octanol. The main explanation for this observation can be seen in the higher water and membrane solubility of 1-octanol. The hydrophobicity (log P) of a substance correlates with a certain partitioning of that compound into the membrane. Combining the log P value with the water solubility, the maximum membrane concentration of a compound can be calculated. With this simple calculation, it is possible to predict the property of an organic chemical for its potential applicability as a solvent for two-liquid-phase biotransformations with solvent-tolerant P. putida strains. Only compounds that show a maximum membrane concentration of less than 400mM, such as 1-decanol, seem to be tolerated by these bacterial strains when applied in supersaturating concentrations to the medium. Taking into consideration that a solvent for a two-liquid-phase system should possess partitioning properties for potential substrates and products of a fine chemical synthesis, it can be seen that 1-decanol is a suitable solvent for such biotransformation processes. This was also demonstrated in shake cultures, where increasing amounts of a second phase of 1-decanol led to bacteria tolerating higher concentrations of the model substrate 3-nitrotoluene. Transferring this example to a 5-liter-scale bioreactor with 10% (vol/vol) 1-decanol, the amount of 3-nitrotoluene tolerated by the cells is up to 200-fold higher than in pure aqueous medium. The system demonstrates the usefulness of two-phase biotransformations utilizing solvent-tolerant bacteria.     

Wide-field, motion-sensitive neurons and matched filters for optic flow fields

 The receptive field organization of a class of visual interneurons in the fly brain (vertical system, or VS neurons) shows a striking similarity to certain self-motion-induced optic flow fields. The present study compares the measured motion sensitivities of the VS neurons (Krapp et al. 1998) to a matched filter model for optic flow fields generated by rotation or translation. The model minimizes the variance of the filter output caused by noise and distance variability between different scenes. To that end, prior knowledge about distance and self-motion statistics is incorporated in the form of a “world model”. We show that a special case of the matched filter model is able to predict the local motion sensitivities observed in some VS neurons. This suggests that their receptive field organization enables the VS neurons to maintain a consistent output when the same type of self-motion occurs in different situations. Received: 14 June 1999 / Accepted in revised form: 20 March 2000     

PKC signaling in CF/T43 cell line: regulation of NKCC1 by PKC-delta isotype

Cystic fibrosis (CF) airway epithelial cells have a reduced mass of ether-linked diacylglycerols which might alter protein kinase C (PKC)-regulated Cl secretion. PKC regulation of basolateral Na-K-2Cl cotransport (NKCC1) was investigated in CF nasal polyp epithelial cells and a CF/T43 cell line to ascertain whether PKC signaling was altered in CF. NKCC1 was detected as bumetanide-sensitive (86)Rb influx. Methoxamine, a alpha(1)-adrenergic agonist, increased PKC activity in cytosol and a particulate fraction for a prolonged time period, as predicted from previous studies on the generation of diglycerides induced with methoxamine. Short-term stimulation of CF/T43 cells for 40 s promoted a shift in PKC-delta and -zeta to a particulate fraction, increased activity of immune complexes of cytosolic PKC-delta and of particulate PKC-zeta and increased activity of NKCC1. Pretreatment with antisense oligonucleotide to PKC-delta blocked methoxamine-stimulated PKC-delta activity, reduced PKC-delta mass by 61.4%, and prevented methoxamine-stimulated activity of NKCC1. Sense and missense oligonucleotide to PKC-delta and antisense oligonucleotide to PKC-zeta did not alter expression of PKC-delta or the effects of methoxamine. These results demonstrate that PKC-delta-dependent activation of NKCC1 is preserved in CF cells and suggest that regulation of NKCC1 is independent of low ether-linked diglyceride mass.     

Linkage Relationships and Haplotype Variation of the Major Histocompatibility Complex Class I <Emphasis Type="BoldItalic">A</Emphasis> Genes in the Cichlid Fish <Emphasis Type="BoldItalic">Oreochromis niloticus</Emphasis>

: The haplochromine cichlid species flocks of the East African Great Lakes are one of the best examples of adaptive radiation. Analysis of genetic variation among these species provides valuable information on species relationships and timing of speciation events. Although the haplochromine cichlids generally display little genetic variation, the major histocompatibility complex (Mhc) genes have been found to be highly variable. A study of the linkage relationships of the Mhc class I A genes in the cichlid fish Oreochromis niloticus was therefore undertaken. Class I loci were identified, and their segregation in seven mothers and their haploid embryos was determined. In total, 56 class I A sequences were found among the seven families. A strong concordance of segregation was observed in five haplotypes among the embryos, indicating a close linkage of all loci. The number of loci per haplotype varied from 11 to 17, while the total number of distinct loci found among all families was 22. These findings show that all class I A loci are linked in a single genetic cluster in O. niloticus.     

Sustained pre-TCR expression in Notch1IC-transgenic rats impairs T cell maturation and selection

Notch1 is involved in directing cell fate decisions in a variety of developmental scenarios. Extending previous experiments in mice, we generated transgenic rats expressing the intracellular domain of Notch1 in the thymus. Importantly, this leads to sustained expression of the pre-TCR throughout thymocyte development, accompanied by a reduction of alphabetaTCR complexes. In addition, re-expression of RAG-1 and RAG-2 in TCRbeta(+) cells is impaired, and the Valpha repertoire is altered. Consequently, thymocytes in transgenic rats do not undergo positive selection and largely fail to progress to the single positive stage. According to our model, the previously reported effects of Notch1 on the CD4/CD8 cell fate decision may be explained by a differential sensitivity of the two lineages toward altered TCR signaling.