Novel B-ring modified allocolchicinoids of the NCME series: design,synthesis, antimicrotubule activity and cytotoxicity

Two new series of allocolchicinoids mimicking the structure of (-)-N-acetylcolchinol O-methyl ether (2, NCME) were synthesized and evaluated for their abilities to inhibit tubulin assembly. Possible antitumor properties resulting thereof were evaluated in vitro on the human MCF-7 breast cancer cell line. The first series of NCME-derivatives was brought about by extending the seven membered B-ring to novel semisynthetic variations with a nitrogen containing eight-membered B-ring similar, for example, to the artificial, potent steganacin aza-analogue 3. In the second series the seven-membered B-ring of NCME (2) was modified by annulation with a heterocyclic ring system. The racemic ketone 7a serving as key precursor involved in the syntheses of all the target NCME variants 9-13 and 15, 16 was easily transformed into the eight-membered B-ring lactams 9 and 10 via a Beckmann rearrangement of the corresponding E-oxime 8. The tetrazole annulated congener 11 was prepared via azidotrimethylsilane-mediated Schmidt rearrangement. Treatment of educt 7a with Bredereck's reagent led to the enamino ketone 14, which was easily converted into the pyrazole- or pyrimidine-annulated allocolchicinoids 15 and 16. Remarkably, all the allocolchicinoids 9-13 with an azocin-B-ring affected the tubulin/microtubule equilibrium only moderately. In contrast, the novel heterocycle annulated seven membered B-ring variants 15 and 16 proved to be highly potent tubulin-inhibitory, antimitotic agents. Interaction with tubulin occured at concentrations similar to those observed for colchicine (1) or the lead NCME (2). In all cases the antiproliferative effects correlated roughly with the inhibition of tubulin assembly.     

Interaction of a Partially Disordered Antisigma Factor with Its Partner,the Signaling Domain of the TonB-Dependent Transporter HasR

Bacteria use diverse signaling pathways to control gene expression in response to external stimuli. In Gram-negative bacteria, the binding of a nutrient is sensed by an outer membrane transporter. This signal is then transmitted to an antisigma factor and subsequently to the cytoplasm where an ECF sigma factor induces expression of genes related to the acquisition of this nutrient. The molecular interactions involved in this transmembrane signaling are poorly understood and structural data on this family of antisigma factor are rare. Here, we present the first structural study of the periplasmic domain of an antisigma factor and its interaction with the transporter. The study concerns the signaling in the heme acquisition system (Has) of Serratia marcescens. Our data support unprecedented partially disordered periplasmic domain of an anti-sigma factor HasS in contact with a membrane-mimicking environment. We solved the 3D structure of the signaling domain of HasR transporter and identified the residues at the HasS−HasR interface. Their conservation in several bacteria suggests wider significance of the proposed model for the understanding of bacterial transmembrane signaling.     

A new species of the Boophis rappiodes group (Anura,Mantellidae) from the Sahamalaza Peninsula,northwest Madagascar,with acoustic monitoring of its nocturnal calling activity

A new species of treefrog of the Boophis rappiodes group (Anura, Mantellidae) is described from the Sahamalaza – Iles Radama National Park in northwest Madagascar. This new species is green in colour with bright red speckling across its head and dorsum; similar in morphology to other species of this group including: B. bottae, B. rappiodes, B. erythrodactylus and B. tasymena. The new species can be distinguished by its advertisement call and by a genetic divergence of more than 4.9% in the analysed mitochondrial 16S rRNA gene fragment. Its call consists of two note types: a trill and a click; although similar sounding to B. bottae, the trill note of the new species has a faster pulse rate while the click note is predominantly two-pulsed rather than three. All individuals were detected from the banks of two streams in Ankarafa Forest. The new species represents the only member of the B. rappiodes group endemic to Madagascar’s western coast, with the majority of other members known from the eastern rainforest belt. Despite its conspicuous call, it has not been detected from other surveys of northwest Madagascar and it is likely to be a local endemic to the peninsula. The ranges of two other amphibian species also appear restricted to Sahamalaza, and so the area seems to support a high level of endemicity. Although occurring inside a National Park, this species is highly threatened by the continuing decline in the quality and extent of its habitat. Due to these threats it is proposed that this species should be classified as Critically Endangered according to the IUCN Red List criteria.     

SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.     

Factors Affecting Exocellular Polysaccharide Production by Lactobacillus delbrueckii subsp. bulgaricus Grown in a Chemically Defined Medium

We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains of Lactobacillus delbrueckii subsp. bulgaricus. The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source. EPS production was greatest during the stationary phase. Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same. The EPS from strain L. delbrueckii subsp. bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain L. delbrueckii subsp. bulgaricus CNRZ 416 contained galactose, glucose, and rhamnose. However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions, L. delbrueckii subsp. bulgaricus strains produced as much EPS in the CDM as in milk. Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar. The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.     

Characterization of loaded liposomes by size exclusion chromatography

This review focuses on the use of conventional (SEC) and high performance (HPSEC) size exclusion chromatography for the analysis of liposomes. The suitability of both techniques is examined regarding the field of liposome applications. The potentiality of conventional SEC is strongly improved by using a HPLC system associated to gel columns with a size selectivity range allowing liposome characterization in addition to particle fractionation. Practical aspects of size exclusion chromatography are described and a methodology based on HPSEC coupled to multidetection modes for on-line analysis of liposomes via label or substance encapsulation is presented. Examples of conventional SEC and HPSEC applications are described which concern polydispersity, size and encapsulation stability, bilayer permeabilization, liposome formation and reconstitution, incorporation of amphiphilic molecules. Size exclusion chromatography is a simple and powerful technique for investigation of encapsulation, insertion/interaction of substances from small solutes (ions, surfactants, drugs, etc.) up to large molecules (proteins, peptides and nucleic acids) in liposomes.     

Multimodal analysis of metals in copper–zinc superoxide dismutase isoforms separated on electrophoresis gels

It has been suggested that copper–zinc superoxide dismutase (CuZnSOD) isoforms of distinct isoelectric point (pI) could result from differences in their metallation state. Our aim was then to develop and validate analytical methods for the determination and understanding of metallation states in human CuZnSOD isoforms. To avoid metal losses during sample preparation steps, CuZnSOD isoforms were separated according to their pI using non-denaturing isoelectric focusing (IEF) gel electrophoresis. Metal quantification was directly performed in-gel. Cu/Zn ratios of CuZnSOD isoforms were quantified by Particle-Induced X-ray Emission (PIXE) and Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS). Cu/Zn ratios were measured close to the value of 1 as expected from the known stoichiometry of CuZnSOD with slight, but statistically significant, differences between acidic and basic isoforms. Overall, this study demonstrates that metal quantification can be performed directly on metalloproteins separated on electrophoresis gels.     

pH-induced conformational changes in chromatin subunits measured by circular dichroism and immunochemical reactivity

Changes in the conformational state of chromatin core particles from chicken erythrocytes were studied by both immunochemical and biophysical methods as a function of pH and ionic strength. When the pH of core particles in a solution of ionic strength 3, 60 or 220 mM was lowered from pH 7.5, a sharp transition in the circular dichroism spectrum of DNA monitored between 320 and 260 nm was observed at pH 6.65. This change in DNA ellipticity was totally reversible. Binding to core particles of antibodies specific for histones H2B, H2A, H3 and for the IRGERA (synthetic C-terminal) peptide of H3 was used to follow changes in histone antigenicity. Binding was studied in the pH range 7.5-5.35, and at ionic strength of 60 and 220 mM. A change in reactivity of some histone epitopes was observed around pH 6.2–6.5. However, the changes observed by circular dichroism and antibody binding pertain to different components of chromatin subunits and they probably reflect independent phenomena. The alteration in accessibility of these determinants at the surface of core particles was completely reversible and was dependent on ionic strength. The conformation changes in core particles occurring near physiological ionic strength and pH may reflect dynamic changes in chromatin structure that possess functional significance.     

Additive effects of metal excess and superoxide,a highly toxic mixture in bacteria

Heavy metal contamination is a serious environmental problem. Understanding the toxicity mechanisms may allow to lower concentration of metals in the metal-based antimicrobial treatments of crops, and reduce metal content in soil and groundwater. Here, we investigate the interplay between metal efflux systems and the superoxide dismutase (SOD) in the purple bacterium Rubrivivax gelatinosus and other bacteria through analysis of the impact of metal accumulation. Exposure of the Cd²⁺-efflux mutant ΔcadA to Cd²⁺ caused an increase in the amount and activity of the cytosolic Fe-Sod SodB, thereby suggesting a role of SodB in the protection against Cd²⁺. In support of this conclusion, inactivation of sodB gene in the ΔcadA cells alleviated detoxification of superoxide and enhanced Cd²⁺ toxicity. Similar findings were described in the Cu⁺-efflux mutant with Cu⁺. Induction of the Mn-Sod or Fe-Sod in response to metals in other bacteria, including Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Vibrio cholera and Bacillus subtilis, was also shown. Both excess Cd²⁺ or Cu⁺ and superoxide can damage [4Fe-4S] clusters. The additive effect of metal and superoxide on the [4Fe-4S] could therefore explain the hypersensitive phenotype in mutants lacking SOD and the efflux ATPase. These findings underscore that ROS defence system becomes decisive for bacterial survival under metal excess.