Cytotoxic farnesyl glycosides from Pittosporum pancheri

Bioassay guided purification of the ethanolic extract of the bark of New Caledonian Pittosporum pancheri Brongn. and Gris (Pittosporaceae) led to the isolation and characterization of two new farnesyl monoglycosides, pancherins A and B. The structure of these compounds were determined on the basis of spectroscopic studies. The new compounds displayed a significant activity in the in vitro cytotoxic assay against KB cancer cell line, and pancherin A inhibits weakly farnesyl protein transferase.     

Biochemical alterations in human cells irradiated with alpha particles delivered by macro- or microbeams

Irradiation of individual cell nuclei with charged-particle microbeams requires accurate identification and localization of cells using Hoechst staining and UV illumination before computer-monitored localization of each cell. Using Fourier-transform infrared microspectroscopy (FT-IRM), we investigated whether the experimental conditions used for cell recognition induce cellular changes prior to irradiation and compared biochemical changes and DNA damage after targeted and nontargeted irradiation with alpha particles delivered by macro- or microbeams, using gamma radiation as a reference. Molecular damage in single HaCaT cells was studied by means of FT-IRM and comet assay (Gault et al., Int. J. Radiat. Biol. 81, 767-779, 2005). Hoechst 33342-stained HaCaT cells were exposed to single doses of 2 Gy (239)Pu alpha particles from a broad-beam irradiator, five impacted alpha particles from a microbeam irradiator, or 6 Gy gamma rays from (137)Cs, each of which resulted in about 5% clonogenic survival. FT-IRM of control cells indicated that Hoechst binding to nuclear DNA induced subtle changes in DNA conformation, and its excitation under UV illumination induced a dramatic shift of the DNA conformation from A to B as well as major DNA damage as measured by the comet assay. Comparison of the FT-IRM spectra of cells exposed to gamma rays or alpha particles specifically targeted to the nucleus, alpha particles from a broad-beam irradiator revealed spectral changes corresponding to all changes in constitutive bases in nucleic acids, suggesting oxidative damage in these bases, as well as structural damage in the deoxyribose-phosphate backbone of DNA and the osidic structure of nucleic acids. Concomitantly, spectral changes specific to protein suggested structural modifications. Striking differences in IR spectra between targeted microbeam- and nontargeted macrobeam-irradiated cells indicated greater residual unrepaired or misrepaired damage after microbeam irradiation. This was confirmed by the comet assay data. These results show that FT-IRM, together with the comet assay, is useful for assessing direct radiation-induced damage to nucleic acids and proteins in single cells and for investigating the effects of radiation quality. Significantly, FT-IRM revealed that Hoechst 33342 binding to DNA and exposure to UV light induce a dramatic change in DNA conformation as well as DNA damage. These findings suggest that fluorochrome staining should be avoided in studies of ionizing radiation-induced bystander effects based on charged-particle microbeam irradiation. An alternative cell nucleus recognition system that avoids nuclear matrix damage and its possible contribution to propagation of biological effects from irradiated cells to neighboring nontargeted cells needs to be developed.     

Protein kinase CK2 phosphorylation of EB2 regulates its function in the production of Epstein-Barr virus infectious viral particles

The Epstein-Barr Virus (EBV) early protein EB2 (also called BMLF1, Mta, or SM) promotes the nuclear export of a subset of early and late viral mRNAs and is essential for the production of infectious virions. We show here that in vitro, protein kinase CK2alpha and -beta subunits bind both individually and, more efficiently, as a complex to the EB2 N terminus and that the CK2beta regulatory subunit also interacts with the EB2 C terminus. Immunoprecipitated EB2 has CK2 activity that phosphorylates several sites within the 80 N-terminal amino acids of EB2, including Ser-55, -56, and -57, which are localized next to the nuclear export signal. EB2S3E, the phosphorylation-mimicking mutant of EB2 at these three serines, but not the phosphorylation ablation mutant EB2S3A, efficiently rescued the production of infectious EBV particles by HEK293(BMLF1-KO) cells harboring an EB2-defective EBV genome. The defect of EB2S3A in transcomplementing 293(BMLF1-KO) cells was not due to impaired nucleocytoplasmic shuttling of the mutated protein but was associated with a decrease in the cytoplasmic accumulation of several late viral mRNAs. Thus, EB2-mediated production of infectious EBV virions is regulated by CK2 phosphorylation at one or more of the serine residues Ser-55, -56, and -57.     

Enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum explored by two-dimensional analysis: identification of seven genes encoding new dockerin-containing proteins

The enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum grown on crystalline cellulose as a sole carbon and energy source was explored by two-dimensional electrophoresis. The cellulolytic system of C. cellulolyticum is composed of at least 30 dockerin-containing proteins (designated cellulosomal proteins) and 30 noncellulosomal components. Most of the known cellulosomal proteins, including CipC, Cel48F, Cel8C, Cel9G, Cel9E, Man5K, Cel9M, and Cel5A, were identified by using two-dimensional Western blot analysis with specific antibodies, whereas Cel5N, Cel9J, and Cel44O were identified by using N-terminal sequencing. Unknown enzymes having carboxymethyl cellulase or xylanase activities were detected by zymogram analysis of two-dimensional gels. Some of these enzymes were identified by N-terminal sequencing as homologs of proteins listed in the NCBI database. Using Trap-Dock PCR and DNA walking, seven genes encoding new dockerin-containing proteins were cloned and sequenced. Some of these genes are clustered. Enzymes encoded by these genes belong to glycoside hydrolase families GH2, GH9, GH10, GH26, GH27, and GH59. Except for members of family GH9, which contains only cellulases, the new modular glycoside hydrolases discovered in this work could be involved in the degradation of different hemicellulosic substrates, such as xylan or galactomannan.     

Modifications of the chemical structure of terpenes in antiplasmodial and antifungal drug research

Pure natural monoterpenes were evaluated in vitro for their antiplasmodial activities against Plasmodium falciparum. Chemically modified terpenes were also tested to see whether the introduction of an alkyne, a cyclopropane, a diene, or a cyclopentenone moiety had an influence on the biological activity. The IC(50) obtained on a chloroquine-resistant strain of Plasmodium (FcM29-Cameroon) showed moderate activity, but with the alkyne and the cyclopentenone derivatives showing a promising enhancement of activity compared with the parent molecules. On the contrary, no antifungal activity was found in vitro using Candida albicans. Given the observed antiplasmodial activity of some of these modified monoterpenes, new monoterpene derivatives could be the basis for new antimalarial drugs to be researched.     

Rapid hit to lead evaluation of pyrazolo[3,4-d]pyrimidin-4-one as selective and orally bioavailable mGluR1 antagonists

Our HTS effort yielded a preferential mGluR1 pyrimidinone antagonist 1 with lead-like characteristics. Rapid hit to lead (HTL) study identified compounds with improved functional activity and selectivity such as 1b with little improvements in ADME properties. Addition of an aminosulfonyl group on the N-1 aromatic ring led to 2f, a compound with similar in vitro biochemical profiles as those of 1b but drastically improved in vitro ADME properties. These improvements were paralleled by rat PK study characterized by low clearance and quantitative bioavailability. Compound 2f represented a true lead-like molecule that is amenable for further lead optimization (LO) evaluation.     

V beta profiles in African children with acute cerebral or uncomplicated malaria: very focused changes among a remarkable global stability

T cells are thought to play a critical role in cerebral malaria pathogenesis. However, available evidences are restricted to rodent models in which V beta specific T cell expansion has been associated with neurological syndrome suggesting involvement of superantigens or dominant antigens. Using flow cytometry, we studied the peripheral V beta T cell repertoire of Ghanaian children with cerebral malaria, uncomplicated malaria and asymptomatic control children, to look for either expansion or deletion of specific V beta associated with cerebral malaria. At admission, the general pattern of the repertoire of the patients was very similar, with no major distortion compared to the control group a part a significant increase of the frequency of the V beta 21.3 subset correlating with disease severity and attributed to the CD4 subset. During convalescence very limited fluctuations were observed including a significant decrease of the V beta 21.3 subset and increase of the V beta 20 subset, a subset not detected at admission. The remarkable stability of the V beta repertoire observed in acute malaria either cerebral or uncomplicated argues against the idea that cerebral malaria would result from a T cell-mediated inflammatory shock syndrome driven by some dominant super-antigenic activity(ies). The significance of the reproducible increase of the CD4+V beta 21.3T cell subset deserves further investigations.     

Post-hatching parental care behaviour and hormonal status in a precocial bird

In birds, the link between parental care behaviour and prolactin release during incubation persists after hatching in altricial birds, but has never been precisely studied during the whole rearing period in precocial species, such as ducks. The present study aims to understand how changes in parental care after hatching are related to circulating prolactin levels in mallard hens rearing ducklings. Blood was sampled in hens over at least 13 post-hatching weeks and the behaviour of the hens and the ducklings was recorded daily until fledging. Contacts between hens and the ducklings, leadership of the ducklings and gathering of them steadily decreased over post-hatching time. Conversely, resting, preening and agonistic behaviour of hens towards ducklings increased. Plasma prolactin concentrations remained at high levels after hatching and then fell after week 6 when body mass and structural size of the young were close to those of the hen. Parental care behaviour declined linearly with brood age, showed a disruption of the hen-brood bond at week 6 post-hatching and was related to prolactin concentration according to a sigmoid function. Our results suggest that a definite threshold in circulating prolactin is necessary to promote and/or to maintain post-hatching parental care in ducks.     

Towards an integrated cellular and molecular: definition of breast cancers

Breast cancer is a major health problem as well as scientifically poorly understood. Our knowledge of breast cancer is however rapidly progressing in several directions. First, genomic studies are establishing a new molecular classification of breast cancers. Molecular subtypes have been identified and are being associated with the histoclinical forms of breast cancers. Second, genetic alterations are discovered and classified, generating new potential therapeutical targets. Third, mammary stem cells have been identified in the normal mammary epithelium. Their altered counterparts have been identified in tumors and are being characterized. These combined studies allow a new integrated cellular and molecular definition of breast cancers and a conceptual basis that will help the management of the disease.