Identification and quantification of blood–brain barrier transporters in isolated rat brain microvessels

The blood–brain barrier (BBB ) maintains brain homeostasis by tightly regulating the exchange of molecules with systemic circulation. It consists primarily of microvascular endothelial cells surrounded by astrocytic endfeet, pericytes, and microglia. Understanding the make‐up of transporters in rat BBB is essential to the translation of pharmacological and toxicological observations into humans. In this study, experimental workflows are presented in which the optimization of (a) isolation of rat brain microvessels (b) enrichment of endothelial cells, and (c) extraction and digestion of proteins were evaluated, followed by identification and quantification of BBB proteins. Optimization of microvessel isolation was indicated by 15‐fold enrichment of endothelial cell marker Glut1 mRNA , whereas markers for other cell types were not enriched. Filter‐aided sample preparation was shown to be superior to in‐solution sample preparation (10251 peptides vs. 7533 peptides). Label‐free proteomics was used to identify nearly 2000 proteins and quantify 1276 proteins in isolated microvessels. A combination of targeted and global proteomics was adopted to measure protein abundance of 6 ATP‐binding cassette and 27 solute carrier transporters. Data analysis using proprietary Progenesis and open access MaxQuant software showed overall agreement; however, Abcb9 and Slc22a8 were quantified only by MaxQuant, whereas Abcc9 and Abcd3 were quantified only by Progenesis. Agreement between targeted and untargeted quantification was demonstrated for Abcb1 (19.7 ± 1.4 vs. 17.8 ± 2.3) and Abcc4 (2.2 ± 0.7 vs. 2.1 ± 0.4), respectively. Rigorous quantification of BBB proteins, as reported in this study, should assist with translational modeling efforts involving brain disposition of xenobiotics.

     

G-quadruplexes with (4n?- 1) guanines in the G-tetrad core: formation of a G-triad·water complex and implication for small-molecule binding

G-quadruplexes are non-canonical structures of nucleic acids, in which guanine bases form planar G-tetrads (G·G·G·G) that stack on each other in the core of the structure. G-quadruplexes generally contain multiple times of four (4n) guanines in the core. Here, we study the structure of G-quadruplexes with only (4n - 1) guanines in the core. The solution structure of a DNA sequence containing 11 guanines showed the formation of a parallel G-quadruplex involving two G-tetrads and one G-triad with a vacant site. Molecular dynamics simulation established the formation of a stable G-triad·water complex, where water molecules mimic the position of the missing guanine in the vacant site. The concept of forming G-quadruplexes with missing guanines in the core broadens the current definition of G-quadruplex-forming sequences. The potential ability of such structures to bind different metabolites, including guanine, guanosine and GTP, in the vacant site, could have biological implications in regulatory functions. Formation of this unique binding pocket in the G-triad could be used as a specific target in drug design.     

Genetic determinism of reproductive fitness traits under drought stress in the model legume <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Fitness traits that determine the reproductive ability of individuals and the persistence of populations are affected by drought stress. Medicago truncatula that commonly encounters drought stress in its natural area, and for which large natural diversity and genetic tools are available, is a suitable species to investigate genetic determinism of fitness traits under stress. In a common garden, three successive cycles of short drought stress were applied after flowering, during the reproductive stage that is the most susceptible to drought for that species. Ten genotypes derived from natural populations and a mapping population were used to investigate the genetic determinism of vegetative and reproductive traits as components of fitness. A large genetic variation was observed and transgressive genotypes (more resistant or more susceptible than the parental genotypes) were found in the mapping population. Fitness traits were reduced by 5–74% in drought condition compared to well-watered condition. The most affected characters were total pod number per plant and total pod weight per plant. A total of 49 QTL, explaining between 6 and 38% of phenotypic variation for vegetative and reproductive fitness traits, were detected on all chromosomes except chromosome 6. A major QTL for flowering date (R ² of 19 and 38%) that co-located with QTL for reproductive fitness traits were found on chromosome 7. In this study, no major QTL specific to drought-stressed or well-watered conditions were detected. We, thus, showed that QTL explaining fitness traits were numerous with small effects, in accordance with the genetic determinism of a complex trait.     

Role of carbohydrate moiety of bleomycin-A2 in caspase-3 activation and internucleosomal chromatin fragmentation in apoptosis of laryngeal carcinoma cells

Bleomycin (BLM), an antitumor antibiotic, is currently used during anticancer therapy. The therapeutic efficiency of BLM for the treatment of malignant tumors is related to its ability to cleave DNA. However, little is known about the biological activity of the glycannic moiety in BLM-induced cytotoxicity. In this study, cell death induced by BLM-A2 and deglycosylated BLM-A2 was studied in a laryngeal carcinoma cell line (HEp-2 cells). Our results indicate that HEp-2 cells showed morphological and biochemical changes associated with apoptosis in the presence of low concentrations of BLM-A2. In contrast, the same changes, except activation of caspase-3 and internucleosomal digestion of genomic DNA, were observed when cells were exposed to high concentrations of deglycosylated BLM-A2. These observations indicate that the glycannic moiety from the bleomycin molecule has important biological effects on the cytotoxicity of the drug.     

Chemical–Genetic Profiling of Imidazo[1,2-a]pyridines and -Pyrimidines Reveals Target Pathways Conserved between Yeast and Human Cells

Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines compose a class of compounds that target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo[1,2-a]pyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo[1,2-a]pyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo[1,2-a]pyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical–genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure–activity relationships.     

Anti-tubercular activity of ruthenium (II) complexes with polypyridines

A series of nine polypyridyl-ruthenium (II) complexes (N-ligands = 2,2'-bipyridines; 2,2'-6',2'-terpyridines, di-alkyloxy-2,2'-6,2-bipyridine-3,3'-di-carboxylates), were tested against Mycobacterium tuberculosis (MBT). The complex (11) showed remarkable activity against MBT as compared to other complexes, (1-10). The aquo ligand of complex (11), as opposed to other chloro and acetonitrile derivatives, appears to play a key role in the antitubercular potency of this new class of metal-based compounds.     

Mutually exclusive distribution of the sap and eag S-layer genes and the lytB/lytA cell wall hydrolase genes in Bacillus thuringiensis

Recently, two Bacillus thuringiensis strains were reported to synthesize parasporal inclusion bodies made not of the expected crystal (Cry) proteins but rather of the surface layer proteins (SLP) Sap (encoded by sap) and EA1 (encoded by eag), respectively. Whether the presence of the sap and eag genes is restricted to these two B. thuringiensis strains or ubiquitous in B. thuringiensis is unknown. We report here the distribution of the sap and eag genes in B. thuringiensis. Strains in the Bacillus cereus group were added for comparison purposes. We show that sap and eag are either present in tandem in 35% of the B. thuringiensis strains analysed and absent in 65% of the strains. When absent, a different tandem, the lytB/lytA cell wall hydrolase genes, is present. The distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes is not species-specific in B. thuringiensis, B. cereus and Bacillus weihenstephanensis. Bacillus anthracis and Bacillus mycoides harbor sap and eag but not lytB/lytA. The sap, eag and lytB/lytA genes were absent in Bacillus pseudomycoides. Clearly, the distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes in B. thuringiensis and in the Bacillus cereus group is mutually exclusive. We also showed that two genes involved in cell wall metabolism, csaA and csaB, are present not only upstream of the sap and eag S-layer genes, but also upstream of the lytB/lytA tandem in strains where sap and eag are absent. Bootstrapped neighbor-joining trees were inferred from the translated amino acid sequences of sap, eag and the tandem lytB/lytA, respectively.     

Cytotoxic activity of withanolides isolated from Tunisian Datura metel L

Withanolide-type steroids, withametelin Q (1) and 12α-hydroxydaturametelin B (2) along with three known withanolides, were isolated from leaves of Datura metel L. (Solanaceae). The respective structures, characterized mainly by NMR spectroscopy, were identified as (20R,22R,24R)-21,24-epoxy-1α,3β-dihydroxywitha-5,25(27)-dienolide-3-O-β-d-glucopyranoside (1) and (20R,22R,24R)-12α,21,27-trihydroxy-1-oxowitha-2,5,24-trienolide-27-O-β-d-glucopyranoside (2). The cytotoxicity of isolated compounds was evaluated against human lung carcinoma cells (A549) and human colorectal adenocarcinoma cells (DLD-1), respectively. Compound 2 exhibited cytotoxicity against A549 and DLD-1 cell lines, with IC₅₀ values of 7 and 2.0 μM, respectively. However, for compounds 6 and 7, cytotoxicities were higher against DLD-1 cells with IC₅₀ values of 0.6 and 0.7 μM. Both compounds blocked the cell cycle in the S-phase and induced apoptosis.