The Metabolism of Aluminum Citrate and Biosynthesis of Oxalic Acid in <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

¹³CNMR and ¹HNMR studies revealed that aluminum citrate (Al-citrate) was metabolized intracellularly and that oxalic acid was an important product in the Al-stressed cells. This dicarboxylic acid was produced via the oxidation of glyoxylate, a precursor generated through the cleavage of isocitrate. In the control cells, citrate was biotransformed essentially with the aid of regular tricarboxylic cycle (TCA) enzymes. However, these control cells were able neither to uptake nor to metabolize Al-citrate. Al-stressed cells obtained at 38–40 h of growth showed maximal Al-citrate uptake and biotransforming activities. At least a fourfold increase in the activity of the enzyme isocitrate lyase (ICL, E. C. 4.1.3.1) has been observed in the Al-stressed cells compared with the control cells. The transport of Al-citrate was sensitive to p-dinitrophenol and sodium azide, but not to dicyclohexylcarbodiimide. Experiments with the dye 9-aminoacridine revealed that the translocation of Al-citrate led to an increase in intracellular pH. Thus, it appears that after the uptake of Al-citrate, this complex is metabolized intracellularly. Received: 13 August 2002 / Accepted: 4 September 2002     

Characterization and quality control of recombinant adenovirus vectors for gene therapy

Highly purified recombinant adenovirus undergoes routine quality controls for identity, potency and purity prior to its use as a gene therapy vector. Quantitative characterization of infectivity is measurable by the expression of the DNA binding protein, an early adenoviral protein, in an immunofluorescence bioassay on permissive cells as a potency determinant. The specific particle count, a key quality indicator, is the total number of intact particles present compared to the number of infectious units. Electron microscopic analysis using negative staining gives a qualitative biophysical analysis of the particles eluted from anion-exchange HPLC. One purity assessment is accomplished via the documented presence and relative ratios of component adenoviral proteins as well as potential contaminants by reversed-phase HPLC of the intact virus followed by protein peak identification using MALDI-TOF mass spectrometry and subsequent data mining. Verification of the viral genome is performed and expression of the transgene is evaluated in in vitro systems for identity. Production lots are also evaluated for replication-competent adenovirus prior to human use. For adenovirus carrying the human IL-2 transgene, quantitative IL-2 expression is demonstrated by ELISA and cytokine potency by cytotoxic T lymphocyte assay following infection of permissive cells. Both quantitative and qualitative analyses show good batch to batch reproducibility under routine test conditions using validated methods.     

The killer shrimp,Dikerogammarus villosus,invading European Alpine Lakes: A single main source but independent founder events with an overall loss of genetic diversity

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Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.     

Morphological vs. molecular delineation of taxa across montane regions in Europe: the case study of Gammarus balcanicus Schäferna, (Crustacea: Amphipoda)

Mountainous areas are characterized by substantial biodiversity and endemicity due to their complex geological history and habitat fragmentation. Hence, it can be assumed that particularly high species richness can be found in organisms with limited dispersal capabilities that inhabit mountain streams. A number of scientific papers focus on molecular phylogeography or traditional taxonomy of species or species groups inhabiting such habitats. However, there is a lack of studies that integrate morphological and molecular data to identify and delineate cryptic species. For practical reasons, uncovering cryptic diversity is crucial in taxa used in biomonitoring. Distinct species, hard to separate based on morphology only, may have different tolerance ranges towards a variety of factors. Thus, our goal is to combine the two approaches to reveal potential patterns of diversification within a species widely distributed across European mountains: the amphipod crustacean Gammarus balcanicus. The data were obtained from 13 populations spread across the range of the species. Individuals were initially ascribed to G. balcanicus based on conventional fauna key morpho‐anatomical diagnostic features and were further analysed for 23 additional features to explore any putative diversification. Morphometric data were analysed with use of the multiple correspondence analysis and anova . Molecular distances were calculated for 551‐bp‐long COI sequences. Test for isolation by distance was performed for both morphological and molecular data. The morphometric studies showed that some of the analysed features differed significantly between populations, although there was only a weak correlation between the morphological divergence and the between‐population geographical distances. Moreover, high morphological diversity was present within sites. A set of 42 COI haplotypes was identified among the 135 individuals sequenced. No haplotype was shared among populations. The molecular p‐distances within the nine localities presenting more than one haplotype were either almost null (ca. <0.003 for 7 localities) or relatively low (ca. 0.01–0.02 for 2 localities). In opposite, the molecular p‐distances between localities were mostly at a high level (94% of pairwise comparisons being >0.14), similar as between other well‐defined species of the genus Gammarus. Surprisingly, G. balcanicus appears to be polyphyletic based on topology of the neighbour‐joining tree. The level of genetic distance between localities was not correlated with their geographical proximity. Globally, combining spatial patterns of morphological versus molecular divergence indicates a high level of cryptic diversity within a species conventionally defined based upon fauna key morphological features. In this context, the name G. balcanicus should be applied only to the population from locus typicus, while the other populations represent a number of putative distinct species. We may expect that such phenomenon would apply also to other animal taxa with conserved morphology, which are widespread over different mountain ranges in Europe.     

Development of Novel Rifampicin-Derived P-Glycoprotein Activators/Inducers. Synthesis,In Silico Analysis and Application in the RBE4 Cell Model,Using Paraquat as Substrate

P-glycoprotein (P-gp) is a 170 kDa transmembrane protein involved in the outward transport of many structurally unrelated substrates. P-gp activation/induction may function as an antidotal pathway to prevent the cytotoxicity of these substrates. In the present study we aimed at testing rifampicin (Rif) and three newly synthesized Rif derivatives (a mono-methoxylated derivative, MeORif, a peracetylated derivative, PerAcRif, and a reduced derivative, RedRif) to establish their ability to modulate P-gp expression and activity in a cellular model of the rat’s blood–brain barrier, the RBE4 cell line P-gp expression was assessed by western blot using C219 anti-P-gp antibody. P-gp function was evaluated by flow cytometry measuring the accumulation of rhodamine123. Whenever P-gp activation/induction ability was detected in a tested compound, its antidotal effect was further tested using paraquat as cytotoxicity model. Interactions between Rif or its derivatives and P-gp were also investigated by computational analysis. Rif led to a significant increase in P-gp expression at 72 h and RedRif significantly increased both P-gp expression and activity. No significant differences were observed for the other derivatives. Pre- or simultaneous treatment with RedRif protected cells against paraquat-induced cytotoxicity, an effect reverted by GF120918, a P-gp inhibitor, corroborating the observed P-gp activation ability. Interaction of RedRif with P-gp drug-binding pocket was consistent with an activation mechanism of action, which was confirmed with docking studies. Therefore, RedRif protection against paraquat-induced cytotoxicity in RBE4 cells, through P-gp activation/induction, suggests that it may be useful as an antidote for cytotoxic substrates of P-gp.     

COCCOLITH-ASSOCIATED POLYSACCHARIDES FROM CELLS OF EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

Coccoliths of Emiliania huxleyi (Lohmann) Hay and Mohler, a unicellular calcifying alga, consist of calcite closely associated with an acidic, Ca²⁺-binding polysaccharide. This polysaccharide is thought to play a regulatory role in coccolith synthesis by interfering with CaCO₃ crystallization. Here we show that the polysaccharides from three different strains, A 92, L and 92 D, all inhibit the precipitation of CaCO₃ in vitro to the same extent. The monosaccharide compositions of the A 92 and L polysaccharide are similar. The 92 D material, however, deviates from the other two: it contains significantly lower amounts of methylated sugars and ribose, and elevated levels of rhamnose and galactose. It also contains antigenic determinants not detected in the A 92 and L polysaccharides. In contrast to the latter two macromolecules the 92 D polysaccharide migrates as two bands upon polyacrylamide gel electrophoresis, possibly resulting from complexing with small amounts of protein. The coccolith polysaccharide from L cells, cultured at an elevated growth rate, also migrates as two bands. This phenomenon is due to an increase in molecular size distribution. The results suggest that certain properties of the molecule may be subject to variation without interfering with its function.