Flavonoids enantiomer distribution in different parts of goldenrod (Solidago virgaurea L.), lucerne (Medicago sativa L.) and phacelia (Phacelia tanacetifolia Benth.)

Plant material is a rich source of valuable compounds such as flavanones. Their different forms influence bioavailability and biological activity, causing problems with the selection of plant material for specific purposes. The purpose of this research was to determine selected flavanone (eriodictyol, naringenin, liquiritigenin, and hesperetin) enantiomer contents in free form and bonded to glycosides by an RP‐UHPLC‐ESI‐MS/MS method. Different parts (stems, leaves, and flowers) of goldenrod (Solidago virgaurea L.), lucerne (Medicago sativa L.), and phacelia (Phacelia tanacetifolia Benth.) were used. The highest content of eriodictyol was found in goldenrod flowers (13.1 μg/g), where it occurred mainly as the (S)‐enantiomer, and the greatest proportion of the total amount was bonded to glycosides. The richest source of naringenin was found to be lucerne leaves (4.7 μg/g), where it was mainly bonded to glycosides and with the (S)‐enantiomer as the dominant form. Liquiritigenin was determined only in lucerne, where the flowers contained the highest amount (1.2 μg/g), with the (R)‐enantiomer as dominant aglycone form and the (S)‐enantiomer as the dominant glycosylated form. The highest hesperetin content was determined in phacelia leaves (0.38 μg/g), where it was present in the form of a glycoside and only as the (S)‐enantiomer. A comparison of the different analyte forms occurring in different plant parts was performed for the first time.     

New steroidal saponins of Agave americana

Two new saponins, agavasaponin E and agavasaponin H have been isolated from the methanolic extract of Agave americana leaves and their structures elucidated. Agavasaponin E is 3-O-[β-d-xylopyranosyl-(1→2glc1)-α-l-rhamnopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→3glc 1)-β-d-glucopyranosyl-(1→4)-β-d-glucopyranosyl-(1→4)-α-d-galactopyranosyl]-(25R)-5α-spirostan-12-on-3β-ol, whereas agavasaponin H is 3-O-[β-d-xylopyranosyl-(1→2 glc 1)-α-l-rhamnopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→3 glc 1)-β-d-glucopyranosyl-(1→4)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranosyl]-26-O-[β-d-glucopyranosyl]-(25R)-5α-furostan-12-on-3β,22α,26-triol.     

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen,Legionella pneumophila

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila‐translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.     

Effect of tunicamycin on the biogenesis of hepatitis C virus glycoproteins

Hepatitis C virus (HCV) infects humans, with a prevalence around 3% of population, causing acute and chronic hepatitis and hepatocellular carcinoma. We studied the effect of inhibition of glycosylation on the assembly of the HCV particle. HCV possesses two envelope glycoproteins E1 and E2 that are highly modified by N-glycans. These glycan residues are crucial for viral entry and maturation of the progeny. Here, we examined the influence of inhibition of N-glycosylation on expression of E1 and E2. Since the propagation of HCV in cell culture is limited, we used a recombinant baculovirus producing viral-like particles in insect cells. Our data showed that blocking of N-glycan transfer to the nascent polypeptide chain with the antibiotic tunicamycin resulted in the loss of E1 and E2. We also found that a dose of tunicamycin that did not influence the cell viability significantly reduced the E2 level in infected cells. The results indicate that blocking of glycosylation at an early step efficiently reduces the assembly of HCV virions. Thus, we suggest that derivatives of tunicamycin that preferentially block glycosylation of viral proteins may become potential therapeutic agents against HCV.     

Application of F magnetic resonance to study the efficacy of fluorine labeled drugs in the three-dimensional cultured breast cancer cells

The cellular monitoring of tumor response to treatments is important for drug discovery and drug development in cancer therapy. We studied efficacy of Herceptin, a common breast cancer drug conjugated with a fluorine organic compound, perfluoro-15-crown-5-ether (PFCE) which easily forms biocompatible emulsions. Three new pharmaceutical forms of Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink were synthesized for the ex vivo study of their efficacy in breast cancer treatment. The emulsions were administered to 10⁹ cells mL⁻¹ of HER-2 positive human adenocarcinoma (MCF-7) cells and the same amount of human mammary epithelial cells (HMEC) cultured in three-dimensional (3D) geometry using hollow fiber bioreactor (HFB) device. Following drugs administration ex vivo, fluorine-19 magnetic resonance imaging (¹⁹F MRI) was applied for cells imaging to measure their viability and to study drug efficacy over 72 h. To ensure optimum drug tracking, HydraLink was used to provide stable binding affinity of emulsified Herceptin to receptor while cationic lipid (Lipofectamine) was used to enhance lipophilicity of the emulsions.After 72 h of treatment with Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink the viability of cells was 54 ± 2%, 49 ± 3%, 43 ± 5% and 42 ± 1%, respectively, as compared with control 93 ± 2%. The efficacy (EC₅₀) of Herceptin conjugated with emulsions was found to be 970 ± 13 μg mL⁻¹ for Herceptin/PFCE, 645 ± 11 μg mL⁻¹ for Herceptin/PFCE/Lipoplex, 678 ± 7 μg mL⁻¹ for Herceptin/PFCE/HydraLink and 1000 ± 3 μg mL⁻¹ for Herceptin. The results show that fluorine emulsions improved the efficacy of Herceptin and ¹⁹F signal intensity changes validated drug efficiency. The significant correlations between duration of treatments and MCF-7 cells viability were observed. While we studied breast cancer cells, the fluorine emulsions could be applied for treatment of other cancer cells overexpressing HER-2.     

Chinese hamster apurinic/apyrimidinic endonuclease (chAPE1) expressed in sf9 cells reveals that its endonuclease activity is regulated by phosphorylation

Apurinic/apyrimidinic endonuclease (APE), an essential DNA repair enzyme, initiates the base excision repair pathway by creating a nick 5' to an abasic site in double-stranded DNA. Although the Chinese hamster ovary cells remain an important model for DNA repair studies, the Chinese hamster APE (chAPE1) has not been studied in vitro in respect to its kinetic characteristics. Here we report the results of a kinetic study performed on cloned and overexpressed enzyme in sf9 cells. The kinetic parameters were fully compatible with the broad range of kinetic parameters reported for the human enzyme. However, the activity measures depended on the time point of the culture. We applied inductivity coupled plasma spectrometry to measure the phosphorylation level of chAPE1. Our data showed that a higher phosphorylation of chAPE1 in the expression host was correlated to a lower endonuclease activity. The phosphorylation of a higher activity batch of chAPE1 by casein kinase II decreased the endonuclease activity, and the dephosphorylation of chAPE1 by lambda phosphatase increased the endonuclease activity. The exonuclease activity of chAPE1 was not observed in our kinetic analysis. The results suggest that noticeable divergence in reported activity levels for the human APE1 endonuclease might be caused by unaccounted phosphorylation. Our data also demonstrate that only selected kinases and phosphatases exert regulatory effects on chAPE1 endonuclease activity, suggesting further that this regulatory mechanism may function in vivo to turn on and off the function of this important enzyme in different organisms.     

The structure of the R184A mutant of the inositol monophosphatase encoded by suhB and implications for its functional interactions in Escherichia coli

The Escherichia coli product of the suhB gene, SuhB, is an inositol monophosphatase (IMPase) that is best known as a suppressor of temperature-sensitive growth phenotypes in E. coli. To gain insights into these biological diverse effects, we determined the structure of the SuhB R184A mutant protein. The structure showed a dimer organization similar to other IMPases, but with an altered interface suggesting that the presence of Arg-184 in the wild-type protein could shift the monomer-dimer equilibrium toward monomer. In parallel, a gel shift assay showed that SuhB forms a tight complex with RNA polymerase (RNA pol) that inhibits the IMPase catalytic activity of SuhB. A variety of SuhB mutant proteins designed to stabilize the dimer interface did not show a clear correlation with the ability of a specific mutant protein to complement the DeltasuhB mutation when introduced extragenically despite being active IMPases. However, the loss of sensitivity to RNA pol binding, i.e. in G173V, R184I, and L96F/R184I, did correlate strongly with loss of complementation of DeltasuhB. Because residue 184 forms the core of the SuhB dimer, it is likely that the interaction with RNA polymerase requires monomeric SuhB. The exposure of specific residues facilitates the interaction of SuhB with RNA pol (or another target with a similar binding surface) and it is this heterodimer formation that is critical to the ability of SuhB to rescue temperature-sensitive phenotypes in E. coli.     

Vaccinia virus N1L protein resembles a B cell lymphoma-2 (Bcl-2) family protein

Poxviruses encode immuno-modulatory proteins capable of subverting host defenses. The poxvirus vaccinia expresses a small 14-kDa protein, N1L, that is critical for virulence. We report the crystal structure of N1L, which reveals an unexpected but striking resemblance to host apoptotic regulators of the B cell lymphoma-2 (Bcl-2) family. Although N1L lacks detectable Bcl-2 homology (BH) motifs at the sequence level, we show that N1L binds with high affinity to the BH3 peptides of pro-apoptotic Bcl-2 family proteins in vitro, consistent with a role for N1L in modulating host antiviral defenses.     

Determination of volatile organic compounds as biomarkers of lung cancer by SPME-GC-TOF/MS and chemometrics

A method for qualitative and quantitative the determination of concentrations volatile organic compounds (VOCs) in human breath samples using solid phase microextraction (SPME) and gas chromatography-time of flight-mass spectrometry (GC-TOF/MS) has been carried out. They are employed for the preconcentration, separation and analysis of biological samples. The technique to rapid determination compounds present in human air, at the level of parts per billion (ppb) is applied. This method was optimized and evaluated. It showed linear correlations ranging from 0.83 to 234.05 ppb, limit of detection in the range of 0.31 to 0.75 ppb and precision, expressed as the RSD, was less then 10.00%. The unique combination of statistical methods allowed reduce the number of compounds to significant ones only and indicate the potential way to find the biomarkers of the lung cancer. Presented an analytical and statistical methods for detection composition of exhaled air could be applied as a potential non-intrusive tool for screening of lung cancer.