New thioderivatives of gossypol and gossypolone,as prodrugs of cytotoxic agents

New dithiane or dithiolane derivatives of gossypol and gossypolone were synthesized with dithiolethane or dithiolpropane in the presence of BF(3)/Et(2)O. These thioderivatives exhibited low toxicity on KB cells (human epidermoid carcinoma cells of the mouth). They react easily with electrophiles in aprotic solvents to regenerate gossypolone or to form dehydrogossypoldithianes and dehydrogossypoldithiolanes, which display higher toxicity on KB cells. In addition, the low toxicity of gossypol thioderivatives was reversed by nitric oxide donors in physiological media. These experiments suggest that gossypol and gossypolone dithianes and dithiolanes can be used as prodrugs that target tumor cells surrounded by high concentrations of nitric oxide.     

Amyloid-like fibril formation by polyQ proteins: a critical balance between the polyQ length and the constraints imposed by the host protein

Nine neurodegenerative disorders, called polyglutamine (polyQ) diseases, are characterized by the formation of intranuclear amyloid-like aggregates by nine proteins containing a polyQ tract above a threshold length. These insoluble aggregates and/or some of their soluble precursors are thought to play a role in the pathogenesis. The mechanism by which polyQ expansions trigger the aggregation of the relevant proteins remains, however, unclear. In this work, polyQ tracts of different lengths were inserted into a solvent-exposed loop of the β-lactamase BlaP and the effects of these insertions on the properties of BlaP were investigated by a range of biophysical techniques. The insertion of up to 79 glutamines does not modify the structure of BlaP; it does, however, significantly destabilize the enzyme. The extent of destabilization is largely independent of the polyQ length, allowing us to study independently the effects intrinsic to the polyQ length and those related to the structural integrity of BlaP on the aggregating properties of the chimeras. Only chimeras with 55Q and 79Q readily form amyloid-like fibrils; therefore, similarly to the proteins associated with diseases, there is a threshold number of glutamines above which the chimeras aggregate into amyloid-like fibrils. Most importantly, the chimera containing 79Q forms amyloid-like fibrils at the same rate whether BlaP is folded or not, whereas the 55Q chimera aggregates into amyloid-like fibrils only if BlaP is unfolded. The threshold value for amyloid-like fibril formation depends, therefore, on the structural integrity of the β-lactamase moiety and thus on the steric and/or conformational constraints applied to the polyQ tract. These constraints have, however, no significant effect on the propensity of the 79Q tract to trigger fibril formation. These results suggest that the influence of the protein context on the aggregating properties of polyQ disease-associated proteins could be negligible when the latter contain particularly long polyQ tracts.     

Solution structure of reduced horse heart cytochrome c

 In the frame of a broad study on the structural differences between the two redox forms of cytochromes to be related to the electron transfer process, the NMR solution structure of horse heart cytochrome c in the reduced form has been determined. The structural data obtained in the present work are compared to those already available in the literature on the same protein and the presence of conformational differences is discussed in the light of the experimental method employed for the structure determination. Redox-state dependent changes are analyzed and in particular they are related to the role of propionate-7 of the heme. Also some hydrogen bonds are changed upon reduction of the heme iron. A substantial similarity is observed for the backbone fold, independently of the oxidation state. At variance, some meaningful differences are observed in the orientation of a few side chains. These changes are related to those found in the case of the highly homologous cytochrome c from Saccharomyces cerevisiae. The exchangeability of the NH protons has been investigated and found to be smaller than in the case of the oxidized protein. We think that this is a characteristic of reduced cytochromes and that mobility is a medium for molecular recognition in vivo. Received: 8 June 1998 / Accepted: 13 October 1998     

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.     

Homo erectus from the Yunxian and Nankin Chinese sites: Anthropological insights using 3D virtual imaging techniques

Recent applications of 3D virtual imaging techniques in human palaeontology have increased the possibilities and the accuracy of anthropological analysis. Two examples are given for the reconsideration of fossils discovered more than 20 years ago, thanks to this new technology. The Lower and Middle Pleistocene skulls from Yunxian and Nankin in China, which were damaged in the process of fossilization, have been virtually reconstructed. A detailed reinvestigation has been conducted by considering those reconstructed skulls and their unpublished characters (i.e., inner anatomical features inaccessible until now). The results of this analysis provide new information about the early hominids of China and contribute to the discussion of variability in Homo erectus.     

A concise guide to cDNA microarray analysis

Microarray expression analysis has become one of the most widely used functional genomics tools. Efficient application of this technique requires the development of robust and reproducible protocols. We have optimized all aspects of the process, including PCR amplification of target cDNA clones, microarray printing, probe labeling and hybridization, and have developed strategies for data normalization and analysis.     

Comparative functional analysis of the human macrophage chitotriosidase

This work analyses the chitin-binding and catalytic domains of the human macrophage chitotriosidase and investigates the physiological role of this glycoside hydrolase in a complex mechanism such as the innate immune system, especially its antifungal activity. Accordingly, we first analyzed the ability of its chitin-binding domain to interact with chitin embedded in fungal cell walls using the β-lactamase activity reporter system described in our previous work. The data showed that the chitin-binding activity was related to the cell wall composition of the fungi strains and that their peptide-N-glycosidase/zymolyase treatments increased binding to fungal by increasing protein permeability. We also investigated the antifungal activity of the enzyme against Candida albicans. The antifungal properties of the complete chitotriosidase were analyzed and compared with those of the isolated chitin-binding and catalytic domains. The isolated catalytic domain but not the chitin-binding domain was sufficient to provide antifungal activity. Furthermore, to explain the lack of obvious pathologic phenotypes in humans homozygous for a widespread mutation that renders chitotriosidase inactive, we postulated that the absence of an active chitotriosidase might be compensated by the expression of another human hydrolytic enzyme such as lysozyme. The comparison of the antifungal properties of chitotriosidase and lysozyme indicated that surprisingly, both enzymes have similar in vitro antifungal properties. Furthermore, despite its more efficient hydrolytic activity on chitin, the observed antifungal activity of chitotriosidase was lower than that of lysozyme. Finally, this antifungal duality between chitotriosidase and lysozyme is discussed in the context of innate immunity.     

Mechanisms of drug-induced allergic contact dermatitis

Allergic contact dermatitis is induced by a wide variety of drugs that trigger specific immune responses following topical exposure. Identified chemical stuctures involved in such reactions include the mercuric and thiosalicylic acid groups of thimerosal, the diphenylketone group of the anti-inflammatory drug ketoprofen, the amide or ester structure of local anesthetics, and the side-chain and thiazolidine ring of -lactams. The T cell responses to such compounds involve CD4+ and CD8+ + T lymphocytes and also CD4–/CD8– + T cells. Although "T helper 2" cytokine production by drug-specific human T cells from patients with allergic contact dermatitis has been described, T helper 1-like and T cytotoxic 1-like responses clearly play key roles in this cutaneous reaction.