F.t.-i.r. and laser-Raman spectra of thymine and thymidine

F.t.-i.r. and laser-Raman spectra of thymine and thymidine in the solid state were recorded. Assignments were proposed for the frequencies observed. The influence of the deoxy sugar on the vibrations of the nucleoside are discussed as a function of its particular puckering. The aim of this work is to elucidate the differences between the molecules constituting the nucleic acids, in order the better to comprehend their biological functions.     

The oxidation of adrenaline and noradrenaline by the two forms of monoamine oxidase from human and rat brain

The selective monoamine oxidase inhibitors clorgyline and (−)-deprenyl were used to study the distribution of monoamine oxidase-A and -B (MAO-A, MAO-B) activities towards (−)-noradrenaline and (+),(−)-adrenaline in homogenates from seven different regions of human brain. The activities towards 5-hydroxytryptamine and 2-phenethylamine, which are essentially specific substrates for the A- and B-forms, respectively, under the conditions used in this work, were also determined. Noradreanline and adrenaline were substrates for both forms of the enzyme in all regions studied. The total MAO activity was found to be highest in the hypothalamus and lowest in the cerebellar cortex. Use of the selective MAO inhibitors clorgyline and (−)-deprenyl also showed adrenaline and noradrenaline to be substrates for both forms of the enzyme in rat brain. In human cerebral cortex and rat brain the two forms were found to have similar K_m-values and maximum velocities towards adrenaline. These values for the two forms were also found to be similar in human cerebral cortex when noradrenaline was used as the substrate. In contrast MAO-A showed a significantly lower K_m and a higher maximum velocity towards noradrenaline in rat brain. These results suggest that the rat may not provide a close model of the human for studies on the effects of MAO inhibitors on brain noradrenaline metabolism.     

A kinetic and spectral study of the alkaline transitions of chloroperoxidase

The optical spectrum of chloroperoxidase in the near ultraviolet and visible region was studied from pH 6 to 12. Chloroperoxidase undergoes a first transition which is irreversible at pH 7 and a second transition near pH 11. The second transition is reversible provided the incubation period above pH 11 is kept as short as possible. The spectral properties of the intermediates were studied in the Soret region by means of a rapid scan apparatus. The rates of the transitions were measured in a stopped-flow apparatus. The pH dependence of both the spectra and the rate constants indicate that at least three ionizations are involved in the first alkaline transition.     

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and favin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.Abbreviations ABZ albendazole - B[a]P benzo[a]pyrene - HPLC high-performance liquid chromatography - MC 3-methylcholanthrene - MFO mixed-function oxidase - UDPGT UDP-glucuronyltransferase     

Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 2. Site-directed mutagenesis of the xylose binding site.

Site-directed mutagenesis in the active site of xylose isomerase derived from Actinoplanes missouriensis is used to investigate the structural and functional role of specific residues. The mutagenesis work together with the crystallographic studies presented in detail in two accompanying papers adds significantly to the understanding of the catalytic mechanism of this enzyme. Changes caused by introduced mutations emphasize the correlation between substrate specificity and cation preference. Mutations in both His 220 and His 54 mainly affect the catalytic rate constant, with catalysis being severely reduced but not abolished, suggesting that both histidines are important, but not essential, for catalysis. Our results thus challenge the hypothesis that His 54 acts as an obligatory catalytic base for ring opening; this residue appears instead to be implicated in governing the anomeric specificity. With none of the active site histidines acting as a catalytic base, the role of the cations in catalyzing proton transfer is confirmed. In addition, Lys 183 appears to play a crucial part in the isomerization step, by assisting the proton shuttle. Other residues also are important but to a lesser extent. The conserved Lys 294 is indirectly involved in binding the activating cations. Among the active site aromatic residues, the tryptophans (16 and 137) play a role in maintaining the general architecture of the substrate binding site while the role of Phe 26 seems to be purely structural.     

Localization of calcium and microfilament changes in mechanically stressed cells

We combined fluorescence labeling, digital image processing, and micromanipulation to investigate the intracellular events induced by inflicting a mechanical stress on rat basophilic leukemia cells. Our findings were as follows:

1. Most cells displayed a localized calcium rise in response to micropipet aspiration. This represented an average threefold increase as compared to resting level, and it was observed during the first 10 s following aspiration. A slow return to initial level occurred within about 3 min. Further, this calcium rise involved a mobilization of intracellular stores, since it was not prevented by adding a calcium chelator into the extracellular medium.
2. All micropipet-aspirated cells displayed a local accumulation of microfilaments, with a preferential localization in the cell protrusions or near the pipet tips.
3. No absolute correlation was found between the localization of calcium rise and cytoskeletal accumulation.
4. Cell deformability was decreased when intracellular calcium was maintained at a constant (high or low) level with ionomycin and/or EGTA.

It is concluded that cells have a general ability to respond to mechanical stimulation by a coordinated set of events. More parameters must be studied before the mechanisms of cell shape regulation are fully understood.     

Conformational studies of an undecapeptide reproducing the consensus sequence around the cleavage site of the RXVRG endoprotease from Xenopus laevis skin

Two synthetic fragments, corresponding to the 4–9 and 4–14 sequences of a tetradecapeptide used as a model to test the RXVRG-endoprotease activity from Xenopus laevis skin, have been studied by two-dimensional nmr spectroscopies, correlated spectroscopy, and nuclear Overhauser effect (NOE) spectroscopy. Both peptides wore the 5–9 consensus sequence found in several hormonal precursors. The nmr data for the 4–9 hexapeptide did not indicate any particular organization, either in water or in dimethylsulfoxide (DMSO), whereas, the 4–14 undecapeptide, a substrate for the RXVRG endoprotease, showed, in DMSO solution, significant trends of structural organization involving the amino acids pertaining to the consensus domain. From variations of integrated NOE peaks with temperature, the appearent interproton correlation times τ_c were estimated and the maxima observed with Va17, the central residue in the consensus sequence. A defined tertiary structure in that domain was also supported by medium-and long-range NOEs between As6 and Arg8, Glu4 and Gly9, and by the likely involvement of Arg8 and Gly9 NHs in intramolecular hydrogen bonds. Most of these observations could be rationalized by an equilibrium between a 5–3 β-turn and a 9 → 4 H-bonded loop. The predominance of one rotamer for the Cα-Cβ bond was established in four residues. Finally, the average ? and ψ angles were derived from two models taking, or not, into account variations in the correlation times along the sequence. This allowed us to discuss the artifacts generated by using an average correlation time through the whole molecule. © 1994 John Wiley & Sons, Inc.     

Cell cycle dependent distribution of a centrosomal antigen at the perinuclear MTOC or at the kinetochores of higher plant cells

Compelling evidence has been obtained in favour of the idea that the nuclear surface of higher plant cells is a microtubule-nucleating and/or organizing site (MTOC), in the absence of defined centrosomes. How these plant MTOC proteins are redistributed and function during the progression of the cell cycle remains entirely unknown. Using a monoclonal antibody (mAb 6C6) raised against isolated calf thymus centrosomes and showing apparent reaction with the plant nuclear surface, we followed the targeted antigen distribution during mitosis and meiosis of higher plants. Immunoblot analysis of protein fractions from Allium root meristematic cell extracts probed with mAb 6C6 reveals a polypeptide of an apparent Mr of 78000. In calf centrosome extracts, a polypeptide of comparable molecular mass is found in addition to a major antigen of Mr 180000 after mAb 6C6 immunoblotting. During mitotic initiation, the plant antigen is prominent on the periphery of the prophase nucleus. When the nuclear envelope breaks down, the antigen suddenly becomes associated with the centromere-kinetochores until late anaphase. In telophase, when the nuclear envelope is being reconstructed, it is no longer detected at the kinetochores but is solely associated again with the nuclear surface. This antigen displays a unique spatial and temporal distribution, which may reflect the pathway of plant protein(s) between the nuclear surface and the kinetochores under cell cycle control. So far, such processes have not been described in higher plant cells. These observations shed light on the putative activity of the plant kinetochore as a protein transporter. They also suggest that a plant centrosome-like antigen may have different cytoskeletal related functions depending on cell cycle regulated changes in its subcellular distribution.Abbreviations mAb monoclonal antibody - MSB microtubule stabilizing buffer - TBS Tris buffered saline - MTOC microtubule organizing centre     

Dedifferentiation and microtubule reorganization in the apical cell protoplast ofSphacelaria (Phaeophyceae)

Summary The apical cell ofSphacelaria, a tip-growing filamentous brown alga, and its protoplast constitute a model for the investigation of the consequences of cell wall removal on microtubular cytoskeletal organization and cell polarity. In the apical cell, the microtubular cytoskeleton is strongly polarized and, in most cases, extends from two centrosomes to the cortex where it constitutes a fine meshwork. Observations of microtubule dynamics throughout the cell cycle emphasize the coincidence between orientation of the mitotic axis and cell polarity. Just after protoplast isolation, dramatic alterations of initial polarity are observed, whatever the mitotic stage. In particular, the coincidence between cytoplasmic polarity and polarity of the system nucleus-centrosomes is lost in most cases. 12–24 h after protoplast isolation, the cell shows a more symmetrical organization while a dense cortical microtubular network spreads out concomitantly with wall reformation. Our discussion emphasizes the possible relationship between cell polarity and cell totipotency, and the relevance of such a model for higher plant studies.