The Flowering Unit in the Synflorescences of Amaranthaceae

The structure of the synflorescence and the flowering units in Amaranthaceae are characterized. The synflorescence is polytelic. In the flowering unit we recognize the main florescence and the enrichment zone. The florescences may consist of: (1) Fully developed partial florescences bearing three or more flowers; (2) Partial florescences reduced to one or a few fertile flowers having prophylls with more or less modified axillary productions; or (3) No partial florescences but solitary flowers having prophylls with no axillary productions. We described the flowering unit in species with florescences bearing a solitary flower and the flowering unit in species with florescences bearing partial florescences. Hypothesized developmental processes are described, with a view to finding relationships among different models characterized in the family as well as defining characters for cladistic studies, which may be useful to depict all the variations observed.     

Antitumor activity of some natural flavonoids and synthetic derivatives on various human and murine cancer cell lines

The effect of various natural flavonoids, cinnamic acid derivatives, and a series of synthetic flavones on cell proliferation was evaluated in vitro in a panel of established human and murine tumor cell lines. The most potent antiproliferative agents were caffeic acid n-butyl ester (12) > 2'-nitroflavone (26) > caffeic acid ethyl ester (11) approximately = 2',6-dinitroflavone (27) > apigenin (3) > 3'-bromoflavone (20) approximately = 2'-fluoro-6-bromoflavone (31). Some compounds showed a moderate effect, the order of cytotoxic activities being chrysin (2) > 2'-fluoro-6-chloroflavone (30) approximately = 2'-chlorochrysin (32) > alpha-naphthoflavone (7) > beta-naphthoflavone (8) approximately = 6-chloroflavone (14) approximately = 6-bromoflavone (15) approximately = 4'-nitroflavone (23). A structure-activity relationship analysis of each group of compounds was performed. None of the natural or synthetic compounds tested affected the proliferation of epithelial cells derived from normal mammary gland of mice or fibroblastic cells from mouse embryo, suggesting a selective action against tumor cells.     

N-domain of human adhesion/growth-regulatory galectin-9: Preference for distinct conformers and non-sialylated N-glycans and detection of ligand-induced structural changes in crystal and solution

Human tandem-repeat-type galectin-9 is a potent adhesion/growth-regulatory effector via lectin capacity of its N- and C-terminal domains. This bioactivity prompted further crystallographic study of the N-domain, combined with analysis in solution. Binding of lactose markedly increased the N-domain's resistance to thermal denaturation. Crystallography revealed its intimate contact profile, besides detecting an extension of the β-sandwich fold by an antiparallel β-strand F0 aligned to the C-terminal F1 strand. Ligand accommodation in its low-energy conformation leads to a movement of Arg87's side chain. As consequence, the ligand's glucose moiety and Arg87 become hydrogen bonded. The resulting predictions for spatial parameters in solution were verified by determining (a) the pattern of magnetization transfer from the protein to protons of lactose and Forssman disaccharide by NMR spectroscopy and (b) the ellipticity changes at wavelengths characteristic for Trp/Tyr residues in near-UV CD spectroscopy. Whereas solid-phase assays confirmed a previously noted tendency for homo- and heterotypic aggregation, gel filtration and ultracentrifugation disclosed monomeric status in solution, in line with crystallographic data. Using cell mutants with defects in glycosylation, this lectin domain was shown to preferentially bind N-glycans without α2,3-sialylation. Since proximal promoter sequences were delineated to diverge markedly among galectin genes and resulting differences in expression profiles were exemplarily documented immunohistochemically, the intrafamily diversification appears to have assigned this protein to a characteristic expression and activity profile among galectins. Our data thus take the crystallographic information to the level of the lectin in solution and in tissues by a strategic combination of spectroscopic and cell/histochemical assays.     

Identification of the N-linked glycosylation sites on the high density lipoprotein (HDL) receptor SR-BI and assessment of their effects on HDL binding and selective lipid uptake

The murine class B, type I scavenger receptor mSR-BI, a high density lipoprotein (HDL) receptor that mediates selective uptake of HDL lipids, contains 11 potential N-linked glycosylation sites and unknown numbers of both endoglycosidase H-sensitive and -resistant oligosaccharides. We have examined the consequences of mutating each of these sites (Asn --> Gln or Thr --> Ala) on post-translational processing of mSR-BI, cell surface expression, and HDL binding and lipid transport activities. All 11 sites were glycosylated; however, disruption of only two (Asn-108 and Asn-173) substantially altered expression and function. There was very little detectable post-translational processing of these two mutants to endoglycosidase H resistance and very low cell surface expression, suggesting that oligosaccharide modification at these sites apparently plays an important role in endoplasmic reticulum folding and/or intracellular transport. Strikingly, although the low levels of the 108 and 173 mutants that were expressed on the cell surface exhibited a marked reduction in their ability to transfer lipids from HDL to cells, they nevertheless bound nearly normal amounts of HDL. Indeed, the affinity of (125)I-HDL binding to the 173 mutant was similar to that of the wild-type receptor. Thus, N-linked glycosylation can influence both the intracellular transport and lipid-transporter activity of SR-BI. The ability to uncouple the HDL binding and lipid transport activities of mSR-BI by in vitro mutagenesis should provide a powerful tool for further analysis of the mechanism of SR-BI-mediated selective lipid uptake.     

Semi-automatic high-throughput determination of plasma protein binding using a 96-well plate filtrate assembly and fast liquid chromatography-tandem mass spectrometry

A semi-automatic, high-throughput method has been developed to rapidly assess plasma protein binding of new chemical entities in drug discovery phase. New chemical entities are mixed with plasma and the unbound fractions are separated from the bound fraction by ultrafiltration in a 96-well filtrate assembly. The unbound fractions are then analyzed by fast liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample handling is automated by a robotic system. Employing a cocktail approach where multiple new chemical entities are allowed to bind to plasma proteins in the same well has further increased the throughput. We have validated the method with 12 commercially available compounds. The plasma protein binding data obtained by this method are comparable with the literature values. This method enables the determination of protein binding for 32 compounds in one single experiment instead of 1-2 compounds using the conventional methods.     

GIP,a G-protein-coupled receptor interacting protein

A novel protein was cloned while screening for partners interacting with the second intracellular loop of the V2 vasopressin receptor (V2R). The protein was named GIP as in G-protein-coupled receptor Interacting Protein; the corresponding gene was located on the 17th chromosome where three exons encode for a 379-amino-acid protein.GIP subcellular localization was studied by immunocytochemistry and also using a biotinylating agent. The protein was found to be localized, at least in part, on the plasma membrane, probably in the form of a trimer.The results indicated that GIP is a transmembrane protein and the most part of the molecule is intracellular. Sequence homology inferred that GIP cytosolic domain is folded as a collagen-like helix followed by a globular domain.The interaction of the globular domain with the V2R was confirmed by pull-down experiments indicating that this structural motif can also interact with cytosolic proteins.     

Radiation-induced chromosome aberrations: insights gained from biophysical modeling

Enzymatic misrepair of ionizing-radiation-induced DNA damage can produce large-scale rearrangements of the genome, such as translocations and dicentrics. These and other chromosome exchange aberrations can cause major phenotypic alterations, including cell death, mutation and neoplasia. Exchange formation requires that two (or more) genomic loci come together spatially. Consequently, the surprisingly rich aberration spectra uncovered by recently developed techniques, when combined with biophysically based computer modeling, help characterize large-scale chromatin architecture in the interphase nucleus. Most results are consistent with a picture whereby chromosomes are mainly confined to territories, chromatin motion is limited, and interchromosomal interactions involve mainly territory surfaces. Aberration spectra and modeling also help characterize DNA repair/misrepair mechanisms. Quantitative results for mammalian cells are best described by a breakage-and-reunion model, suggesting that the dominant recombinational mechanism during the G(0)/G(1) phase of the cell cycle is non-homologous end-joining of radiogenic DNA double strand breaks. In turn, better mechanistic and quantitative understanding of aberration formation gives new insights into health-related applications.     

Chemotaxonomic features of lipophilic components inAdesmia species (Leguminosae)

Studies were performed on GC-MS to assess the lipophilic composition of sixAdesmia species representing two subgenera and three series. Normal fatty acids and hydrocarbons were mainly found, as well as acetylenic compounds, dibasic acids, cyclic hydrocarbons, high molecular weight alcohols and one sterol.