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The two chiral isomers of ethyl 5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl carbamate, NSC 613863 (R-isomer)-(1) and NSC 613862 (S-isomer)-(2) (CI980) and the three achiral analogs NSC 330770 (2-demethylated analog A), NSC 337238 and C179 are potent microtubule inhibitors. These ligands interact with tubulin overlapping the colchicine binding site. This study addresses the effects of recognition by tubulin on the conformational properties of the ligands. The near-UV CD (circular dichroism) band of the R-isomer was suppressed, while that of the S-isomer displayed a more intense negative band when these compounds were bound to tubulin. Interestingly, the three other initially achiral compounds became optically active upon binding to tubulin; particularly, analog A exhibited a negative CD band on the order of magnitude of chiral compounds. The CD changes are reversible, highly specific and actually permit measuring the binding of the ligands by tubulin. These CD changes are compatible with the deformation of the bound ligands. Fluorescence emission is strongly enhanced and blue shifted upon binding to tubulin. Water among a solvent series had a specific solvent effect, except on the 1,2-dehydro analogs NSC 337238 and C179, suggesting hydrogen bonding to N1. The emission of tubulin-bound R-isomer, S-isomer and analog A could be mimicked by solvent viscosity, supporting the notion that the intramolecular rotation between the pyridopyrazine and phenyl rings is frozen upon binding.

International audience; The mobile French Guiana coast is a shoreface region downdrift of the Amazon River, where enormous quantities of inorganic and organic materials are exchanged with the Atlantic Ocean. The rapid accumulation of these materials forms highly unstable shore-attached mud banks, which can be temporally emerged and then rapidly colonized and stabilized by microphytobenthos and opportunistic mangroves (i.e. Avicennia germinans). Mud banks are preferential sites for the accumulation and significant remineralization of organic matter (OM) due to intense erosion/deposition cycles and potential biological colonization. The distribution and sources of bulk sedimentary OM were characterized by elemental and isotopic analyses of four sediment cores, together with samples from three potential OM sources (mangrove plants, suspended particulate matter (SPM) and microphytobenthos), all collected from the landward face of the Macouria mud bank (French Guiana). Total organic carbon (TOC) and nitrogen (TN) concentrations in the sediment cores showed that OM sources were characterized by spatio-temporal variations in this mud bank. The relative contributions of mangrove plants, SPM and microphytobenthos were estimated using a three end-member mixing model based on [TN/TOC]atomic ratios and δ13C values. Sedimentary OM is mostly controlled by SPM associated with variable amounts of OM derived from mangrove plants and microphytobenthos. These variations could be explained by topography and bed elevation, which decrease submersion time and increase desiccation. Higher contributions of microphytobenthos are associated with black OM-rich laminae, identified in sedimentary cores and linked to temporal emersion phases of the mud bank, which favor the growth of benthic microalgae. This result is confirmed by the calculation of the average sediment accumulation rate (around 36.7 ± 14.8 cm yr− 1), taking into account the emersion of the study site every spring tide (a fortnightly cycle). This value is within the range of the previous results from other mud banks in French Guiana but is more than ten times greater than values generally obtained in other coastal contexts.

NSC 613863 (R)-(+) and NSC 613862 (S)-(-) (CI980) are two chiral isomers of ethyl 5-amino 2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl carbamate which have potent antitubulin activity. The S-isomer is a more potent antimitotic compound than the R-isomer, and the two isomers differ markedly in binding to tubulin [Leynadier, D., Peyrot, V., Sarrazin, M., Briand, C., Andreu, J. M., Rener, G. A., & Temple, C., Jr. (1993) Biochemistry 32, 10675-10682]. To understand the origin of such differences, we studied the interactions of three R- and S-isomer structural analogs which differ in C2 (the chiral carbon), i.e., C179, NSC 337238, and NSC 330770. C179 is a methylated dehydrogenated achiral compound. It bound to tubulin with an apparent affinity Ka of (2.29 +/- 0.17) x 10(4) M-1, inhibited tubulin polymerization in vitro at a half-inhibitory concentration (IC50) of 100 microM, and presented no GTPase activity. The substitution of -CH3 by -H leads to the NSC 337238 compound. It bound to tubulin with a higher affinity [Ka = (2.62 +/- 0.35) x 10(5) M-1] and inhibited tubulin polymerization at a lower concentration (IC50 = 14 microM). It presented no GTPase activity and induced the formation of abnormal polymers at a protein critical concentration (Cr) of 2 mg mL-1. NSC 330770, a demethylated hydrogenated molecule, interacted strongly with tubulin [Ka = (3.30 +/- 0.56) x 10(6) M-1].(ABSTRACT TRUNCATED AT 250 WORDS)

Several fluorescence properties of two enantiomers, NSC 613862 (S)-(-) and NSC 613863 (R)-(+), have been compared. Even though the two isomers showed the same fluorescence behavior in solution in different solvents, drastic differences were observed after binding to purified calf brain tubulin. Binding measurements for the two compounds were performed both by fluorescence spectroscopy and by column gel permeation, a direct method of measurement. For both isomers, the binding was characterized by the presence of one high-affinity binding site with an apparent association constant of (3.2 +/- 0.5) x 10(6) M-1 and (4.1 +/- 0.9) x 10(6) M-1 for the R- and S-isomer, respectively, and by several low-affinity sites. Both isomers were also shown to induce GTPase activity in tubulin. The high-affinity binding site seems to be the same for the two isomers. Moreover, fluorescence competition experiments suggest at least a partial overlap of the colchicine and podophyllotoxin site. To explain the differences in fluorescence behavior after binding to tubulin, we hypothesize that the R-isomer is positioned differently in its binding locus as compared with the S-isomer.

MDL 27048 [trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2- methyl-2-propen-1-one] fluoresces when bound to tubulin but not in solution. This effect has been investigated and found to be mimicked by viscous solvents. Therefore, MDL 27048 appears to be a fluorescent compound whose intramolecular rotational relaxation varies as a function of microenvironment viscosity. The binding parameters of MDL 27048 to tubulin have been firmly established by fluorescence of the ligand, quenching of the protein fluorescence, and gel equilibrium chromatography. The apparent binding equilibrium constant was (2.75 +/- 0.45) x 10(6)M-1, and the binding site number was 0.81 +/- 0.12 (10 mM sodium phosphate-0.1 mM GTP, pH 7.0, at 25 degrees C). The binding is exothermic. The binding of MDL 27048 overlaps the colchicine and podophyllotoxin binding sites. Binding of MDL 27048 to the colchicine site was also measured by competition with MTC [2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one] , a well-characterized reversibly binding probe of the colchicine site [Andreu et al. (1984) Biochemistry 23, 1742-1752; Bane et al., (1984) J. Biol. Chem. 259, 7391-7398]. In contrast with close analogues of colchicine, MDL 27048 and podophyllotoxin neither affected the far-ultraviolet circular dichroism spectrum of tubulin, within experimental error, nor induced tubulin GTPase activity. Like podophyllotoxin, an excess of MDL 27048 over tubulin induced no abnormal cooperative polymerization of tubulin, which is characteristic of colchicine binding.(ABSTRACT TRUNCATED AT 250 WORDS)

When NADPH was added in excess to a bovine liver DHFR solution, a fluorescence peak due to an energy transfer mechanism was apparent at 450 nm. It did not vary over time. The intrinsic fluorescence peak of DHFR at 320 nm was quenched and this phenomenon increased over the time-course after NADPH addition. This result was ascribed to a slow DHFR conformational change induced by NADPH binding, which has never been previously described in such a long time scale (more than 30 min). A kinetic scheme accounting for this mechanism has been proposed. Furthermore, this interconversion between two protein conformers led to an increase in the initial apparent rate of the enzymatic reaction catalyzed by DHFR.

The human colon adenocarcinoma HT29-D4 cell line is an interesting model for studies on epithelial cell differentiation. Undifferentiated cells are malignant proliferating cells, whereas differentiated cells act like epithelial polarized cells. In the present study, we first characterized the action of taxoids on the microtubular network of HT29-D4 cells according to the state of differentiation. Microtubular bundles were found in undifferentiated cells but not in differentiated cells, even with 500-fold higher taxoid concentrations for 96 h. This finding led us to study changes in microtubules according to the polarity status of the cell. E-MAP-115 was expressed only in differentiated cells; expression of β-tubulin isotypes was altered in them relative to undifferentiated cells. Classes I, II, III, IVa and IVb isotypes were expressed in both phenotypes; however, differentiated epithelial cells displayed a specific increase in class III β-tubulin. Thus, the increase in expression of this β-tubulin isotype in differentiated cells is not restricted to neuronal cells. Moreover, these expression changes may reflect a higher stability of microtubular network in differentiated cells, which may explain the lower activity of anti-microtubule agents, independently of the mitotic process. These results indicate that the composition of microtubules should be considered as one of the criteria involved in the response of tumour cells to chemotherapy with anti-microtubule agents. © 1999 Cancer Research Campaign

It has been shown that ethyl 5-amino-l,2-dihydro-2-methyl-3-phenyl pyrido (3,4-b)pyrazin-7-yl carbamate (NSC 370147) inhibits microtubule formation, that this racemic compound binds to tubulin at or near the colchicine site, and that it enhances the binding of Vincristine to tubulin1. It appeared of interest to evaluate the activity of the R-enantiomer (NSC 613863) and the S-enantiomer (NSC 613862, CI 980). Hence, their interactions were examined at molecular level. Furthermore, we compared the binding properties of these new compounds to those of colchicine and colchicine analogues, which are firmly established. The influence of chirality on the polymerization of microtubule proteins and tubulin is also discussed.

Phillipson, J., Sarrazin-Comans M. & Stomatopoulos C., 1983: Food consumption by Microtus agrestis and the unsuitability of faecal analysis for the determination of food preference. Acta theriol., 28 26: 397—416 [With 6 Tables, 4 Figs. & Plate XIII] In the laboratory adult Microtus agrestis (Linnaeus, 1761) consumed an average of 21.4 g wet wt. (=6.3 g dry wt.) of fresh grass indiv"1 d" 1 with an annual mean digestibility of 52.8%. When four common species of food grass were on offer consumption in Autumn and Winter was directly proportional to availability. A preference for the more "succulent" species was exhibited in Spring and Summer. Over the year digestibility coefficients ranged between 33.6 and 67.8%, the highest values occurring in Spring and Summer. Faecal analysis suggested an order of food preference different to that determined by direct observation, the differences being attributable to the differential "desirability" and "digestibility" of the food grasses during the course of the year.

The interaction of cis-dichlorodiammineplatinum (II) (cisplatin) with human serum albumin (HSA), dissolved in phosphate buffer with or without sodium chloride (0ṁ1 M) has been examined at pH 7ṁ4 and μ = 0ṁ154. Equal volumes of cisplatin and HSA solutions were incubated at 37 °C for various times and filterable platinum concentrations versus time measured by flameless atomic absorption spectrophotometry. Binding kinetics differed depending on the buffer solutions used and on the time elapsing between cisplatin dissolution and outset of incubation with HSA. Experimental data were fitted to a theoretical equation used to calculate the number of nucleophilic sites per HSA molecule. Titrations of the HSA sulphydryl group content before and after incubation with a cisplatin solution were made, from which it was shown that the lone SH-group of the HSA macromolecule is involved in cisplatin binding. We also studied HSA's sensitivity towards denaturing agents when it was complexed with cisplatin. This sensitivity was decreased upon cisplatin binding. Also, the binding capacities of HSA and the HSA-Pt(II) complex to both tryptophan and warfarin were compared to determine the possible influence of cisplatin upon the binding to HSA of other drugs; this influence was negligible.