Magnetic and other non-visual orientation mechanisms in some cave and surface urodeles

Animals adapted to light-deprived habitats might have improved non-visual sensory systems. Specimens of several cave-dwelling species of urodeles spontaneously and persistently align to natural or artificially-modified permanent magnetic fields. Video observations under dim infrared illumination revealed an obvious individual preference for one particular magnetic direction in every animal tested. Therefore, animals changed alignments predictably when the horizontal magnetic field vector (compass direction) was artificially reversed or deviated. When the vertical vector was compensated, individuals aligned axially. With the vertical vector reversed (inclination upward), either axial alignment was still typical, or the individuals behaved as with the horizontal vector reversed. However, reactions as to the natural field occurred as well. The findings suggest a receptor mechanism that needs both horizontal and vertical magnetic cues, but it is still an open question how and where the physical and physiological mechanisms of magnetic transduction and reception are realized. The visual system is likely not necessary because Proteus is ontogenetically deprived of eyesight, and the other species were blindfolded due to the faint infrared illumination. The results therefore tend to favor those putative receptor mechanisms, assumed to work by means of magnetite nano-elements. In sum, the ability to align within the geomagnetic field may be considered a prerequisite for magnetic orientation and is, among other sensory improvements, judged to be highly relevant as an important sensorial and ecological adaptation to light-deprived habitats.     

siRNAs containing 2′-fluorinated Northern-methanocarbacyclic (2′-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2′-F-NMC NTPs

We recently reported the synthesis of 2′-fluorinated Northern-methanocarbacyclic (2′-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2′-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2′-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5′ phosphate, suggesting that the 2′-F-NMC is a poor substrate for 5′ kinases. In mice, the 2′-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2′-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5′-phosphate mimic 5′-(E)-vinylphosphonate was attached to the 2′-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2′-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2′-F-NMC. Finally, the 5′-triphosphate of 2′-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic.     

Reversed-phase high-performance liquid chromatographic method for the determination of the 11-hydroxythromboxane B2 anomers equilibrium

An improved reversed-phase HPLC method for the separation and detection of both hemiacetalic 11-hydroxy anomers of thromboxane B₂ (TXB₂) is described. Water-acetonitrile mixtures have served as mobile phases. By diminishing stepwise the temperature of the chromatographic system from 40 to 0°C, the UV-absorbance profile of TXB₂ changed from one broad peak to two clearly separated narrow peaks corresponding to the two anomers existing in equilibrium. Modification of the mobile phase pH from 1.6 to 6.9 (0°C) resulted in different concentration ratios of the anomers. The equilibrium constant and the Gibbs free energy were calculated. The intermediate open aldehyde form of TXB₂ is unstable and, therefore, cannot be observed either by HPLC or by ¹H NMR measurements.     

Cell cycle re-entry of quiescent mammalian nuclei following heterokaryon formation

When 3T3 mouse fibroblasts are made quiescent by serum deprivation and are then fused with tsAF8 hamster fibroblasts synchronized by a combination of high temperature block and hydroxyurea, the nuclei of binucleated heterokaryons which are formed enter S phase asynchronously in media containing low levels of serum. The tsAF8 nuclei of these biphasic heterokaryons enter S phase shortly after fusion, as do the tsAFS nuclei of homokaryons in the same culture. In contrast, the nuclei of the biphasic heterokaryons which have been contributed by quiescent 3T3 enter S phase only after a lag following fusion. This suggests that the quiescent nucleus within the heterokaryon is stimulated by factor(s) from the more advanced cell to re-enter the cell cycle in the absence of serum. In contrast to factors which induce the immediate synthesis of DNA, these factors may be those responsible for the transition of a cell from a non-proliferating to a proliferating state.     

Excretion of metabolites by hydrogen bacteria

Summary D(–)-3-hydroxybutanoic acid was produced by a double mutant of Alcaligenes eutrophus, unable to synthesize poly-3-hydroxybutanoic acid and to utilize 3-hydroxybutanoate as a substrate. About 3.4 g of 3-hydroxybutanoate/l were produced under optimum conditions at pH 7.0 to 7.3, at a temperature of 30°C after exhaustion of ammonia and at restricted aeration rates allowing 10–12% of the maximum respiratory rate of the cells to occur. D(–)-3-hydroxybutanoic acid was identified by means of gas and ion exchange chromatography, IR-spectrometry and specific rotation.     

Comparative genomic in situ hybridization discloses recurrent gain of chromosome 4 in experimental gliomas of the rat.

The genetic characterization of experimental tumors is essential in order to evaluate their relevance as appropriate animal models for human neoplasms. We have used flow cytometry and a recently established Comparative Genomic in situ Hybridization (CGH) protocol for the rat (Kappler et al., 1998) to investigate chromosome copy number changes in five ethylnitrosourea induced gliomas of the rat. Flow cytometry showed aneuploid DNA indices in three of the tumors investigated. CGH analysis of primary tumors revealed whole chromosome and subchromosomal gains of rat chromosomes (RNO) 1, 2, 4, 6, 7, 10, 11, 12, and 13. Loss of RNO 5q23-->q35 was apparent in one tumor. High level copy number gains were not observed using CGH as well as semiquantitative PCR with Tgfa, Met and Hbb primers. Low copy number gain of RNO 4 represents the most common aberration, since it was detected in four of five tumors investigated. Three tumors showed gain of RNO 7, while two tumors showed gains of RNO 10q31-->qter and RNO 12q. Deletion of RNO 5q23-->q35 and gain of RNO 4 occurred mutually exclusively. Therefore, we conclude that these two alterations may represent different pathways in the pathogenesis of experimental gliomas in the rat. Findings are discussed in analogy to human gliomas.     

C-banding of Peromyscus constitutive heterochromatin persists following histone hyperacetylation

Approx. 35% of the DNA of cultured cells from the cactus mouse, Peromuscus eremicus, is contained in highly condensed constitutive heterochromatin which can be visualized in metaphase chromosomes stained by the C-band technique. Previous studies have shown this constitutive heterochromatin to contain a large proportion of underacetylated, arginine-rich histones, the majority of which can be hyperacetylated when cells are treated with butyrate. In order to determine whether this simulation of the acetylated state of euchromatin alters the cytological properties of constitutive heterochromatin as well, chromosomes from butyrate-treated cells have been examined. Because of the paucity of cells in butyrate-treated cultures, prematurely condensed chromosomes (PCCs) were produced from butyrate-treated cells by fusion with mitotic cells. In these PCCs, both the highly condensed nature and the ability to C-band were preserved in the hyperacetylated constitutive heterochromatin, suggesting that the subset of arginine-rich histones which is refractory to acetylation in the presence of butyrate may be responsible for the maintenance of the heterochromatic state. In addition, PCC analyses indicated that butyrate arrests Peromyscus cells in both the G1 and G2 phases of the cell cycle and confirmed the late-replicating pattern of constitutive heterochromatin.     

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Ohne Zusammenfassung

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Altered erythrocyte protein kinase C activity and membrane protein phosphorylation in chronic myelogenous leukemia

The membrane protein kinase C (PKC) content was found to be higher in erythrocytes form patients suffering from chronic myelogenous leukemia (CML) compared to normal erythrocytes. PKC activity was also higher in the cytosol and after translocation to the membrane, as assessed by histone phosphorylation. The increased PKC activity in CML erythrocytes was associated with abnormal phosphorylation of protein 4.1. Since phosphorylation-dephosphorylation mechanisms are likely candidates for controlling membrane protein associations, the altered PKC activity may be one of the factors responsible for altered thermal sensitivity and mechanical stability of CML erythrocytes.