CIGB-300, a synthetic peptide-based drug that targets the CK2 phosphoaceptor domain. Translational and clinical research

CK2 represents an oncology target scientifically validated. However, clinical research with inhibitors of the CK2-mediated phosphorylation event is still insufficient to recognize it as a clinically validated target. CIGB-300, an investigational peptide-based drug that targets the phosphoaceptor site, binds to a CK2 substrate array in vitro but mainly to B23/nucleophosmin in vivo. The CIGB-300 proapoptotic effect is preceded by its nucleolar localization, inhibition of the CK2-mediated phosphorylation on B23/nucleophosmin and nucleolar disassembly. Importantly, CIGB-300 shifted a protein array linked to apoptosis, ribosome biogenesis, cell proliferation, glycolisis, and cell motility in proteomic studies which helped to understand its mechanism of action. In the clinical ground, CIGB-300 has proved to be safe and well tolerated in a First-in-Human trial in women with cervical malignancies who also experienced signs of clinical benefit. In a second Phase 1 clinical trial in women with cervical cancer stage IB2/II, the MTD and DLT have been also identified in the clinical setting. Interestingly, in cervical tumors the B23/nucleophosmin protein levels were significantly reduced after CIGB-300 treatment at the nucleus compartment. In addition, expanded use of CIGB-300 in case studies has evidenced antitumor activity when administered as compassional option. Collectively, our data outline important clues on translational and clinical research from this novel peptide-based drug reinforcing its perspectives to treat cancer and paving the way to validate CK2 as a promising target in oncology.     

Genotypic and Phenotypic Applications for the Differentiation and Species-Level Identification of Achromobacter for Clinical Diagnoses

The Achromobacter is a genus in the family Alcaligenaceae, comprising fifteen species isolated from different sources, including clinical samples. The ability to detect and correctly identify Achromobacter species, particularly A. xylosoxidans, and differentiate them from other phenotypically similar and genotypically related Gram-negative, aerobic, non-fermenting species is important for patients with cystic fibrosis (CF), as well as for nosocomial and other opportunistic infections. Traditional phenotypic profile-based analyses have been demonstrated to be inadequate for reliable identifications of isolates of Achromobacter species and genotypic-based assays, relying upon comparative 16S rRNA gene sequence analyses are not able to insure definitive identifications of Achromobacter species, due to the inherently conserved nature of the gene. The uses of alternative methodologies to enable high-resolution differentiation between the species in the genus are needed. A comparative multi-locus sequence analysis (MLSA) of four selected ‘house-keeping’ genes (atpD, gyrB, recA, and rpoB) assessed the individual gene sequences for their potential in developing a reliable, rapid and cost-effective diagnostic protocol for Achromobacter species identifications. The analysis of the type strains of the species of the genus and 46 strains of Achromobacter species showed congruence between the cluster analyses derived from the individual genes. The MLSA gene sequences exhibited different levels of resolution in delineating the validly published Achromobacter species and elucidated strains that represent new genotypes and probable new species of the genus. Our results also suggested that the recently described A. spritinus is a later heterotypic synonym of A. marplatensis. Strains were analyzed, using whole-cell Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS), as an alternative phenotypic profile-based method with the potential to support the identifications determined by the genotypic DNA sequence-based MLSA. The MALDI-TOF MS data showed good accordance in strain groupings and identifications by the MLSA data.     

Improved artificial origins for phage Φ29 terminal protein-primed replication. Insights into early replication events

The replication machinery of bacteriophage Φ29 is a paradigm for protein-primed replication and it holds great potential for applied purposes. To better understand the early replication events and to find improved origins for DNA amplification based on the Φ29 system, we have studied the end-structure of a double-stranded DNA replication origin. We have observed that the strength of the origin is determined by a combination of factors. The strongest origin (30-fold respect to wt) has the sequence CCC at the 3′ end of the template strand, AAA at the 5′ end of the non-template strand and 6 nucleotides as optimal unpairing at the end of the origin. We also show that the presence of a correctly positioned displaced strand is important because origins with 5′ or 3′ ssDNA regions have very low activity. Most of the effect of the improved origins takes place at the passage between the terminal protein-primed and the DNA-primed modes of replication by the DNA polymerase suggesting the existence of a thermodynamic barrier at that point. We suggest that the template and non-template strands of the origin and the TP/DNA polymerase complex form series of interactions that control the critical start of terminal protein-primed replication.     

A positively charged residue of phi29 DNA polymerase, highly conserved in DNA polymerases from families A and B, is involved in binding the incoming nucleotide

Alignment of the protein sequence of DNA-dependent DNA polymerases has allowed the definition of a new motif, lying adjacent to motif B in the direction of the N-terminus and therefore named pre-motif B. Both motifs are located in the fingers subdomain, shown to rotate towards the active site to form a dNTP-binding pocket in several DNA polymerases in which a closed ternary complex pol:DNA:dNTP has been solved. The functional significance of pre-motif B has been studied by site-directed mutagenesis of 29 DNA polymerase. The affinity for nucleotides of 29 DNA polymerase mutant residues Ile364 and Lys371 was strongly affected in DNA- and terminal protein-primed reactions. Additionally, mutations in Ile364 affected the DNA-binding capacity of 29 DNA polymerase. The results suggest that Lys371 of 29 DNA polymerase, highly conserved among families A and B, interacts with the phosphate groups of the incoming nucleotide. On the other hand, the role of residue Ile364 seems to be structural, being important for both DNA and dNTP binding. Pre-motif B must therefore play an important role in binding the incoming nucleotide. Interestingly, the roles of Lys371 and Ile364 were also shown to be important in reactions without template, suggesting that 29 DNA polymerase can achieve the closed conformation in the absence of a DNA template.     

Targeted Deletion of Kynurenine 3-Monooxygenase in Mice: A NEW TOOL FOR STUDYING KYNURENINE PATHWAY METABOLISM IN PERIPHERY AND BRAIN*

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway (KP) of tryptophan degradation, has been suggested to play a major role in physiological and pathological events involving bioactive KP metabolites. To explore this role in greater detail, we generated mice with a targeted genetic disruption of Kmo and present here the first biochemical and neurochemical characterization of these mutant animals. Kmo^−/− mice lacked KMO activity but showed no obvious abnormalities in the activity of four additional KP enzymes tested. As expected, Kmo^−/− mice showed substantial reductions in the levels of its enzymatic product, 3-hydroxykynurenine, in liver, brain, and plasma. Compared with wild-type animals, the levels of the downstream metabolite quinolinic acid were also greatly decreased in liver and plasma of the mutant mice but surprisingly were only slightly reduced (by ∼20%) in the brain. The levels of three other KP metabolites: kynurenine, kynurenic acid, and anthranilic acid, were substantially, but differentially, elevated in the liver, brain, and plasma of Kmo^−/− mice, whereas the liver and brain content of the major end product of the enzymatic cascade, NAD⁺, did not differ between Kmo^−/− and wild-type animals. When assessed by in vivo microdialysis, extracellular kynurenic acid levels were found to be significantly elevated in the brains of Kmo^−/− mice. Taken together, these results provide further evidence that KMO plays a key regulatory role in the KP and indicate that Kmo^−/− mice will be useful for studying tissue-specific functions of individual KP metabolites in health and disease.     

The role of a groundwater bacterial community in the degradation of the herbicide terbuthylazine

A bacterial community in an aquifer contaminated by s- triazines was studied. Groundwater microcosms were treated with terbuthylazine at a concentration of 100 μg L⁻¹ and degradation of the herbicide was assessed. The bacterial community structure (abundance and phylogenetic composition) and function (carbon production and cell viability) were analysed. The bacterial community was able to degrade the terbuthylazine; in particular, Betaproteobacteria were involved in the herbicide biotransformation. Identification of some bacterial isolates by PCR amplification of the 16S rRNA gene revealed the presence of two Betaproteobacteria species able to degrade the herbicide: Advenella incenata and Janthinobacterium lividum . PCR detection of the genes encoding s -triazine-degrading enzymes indicated the presence of the atz A and atz B genes in A. incenata and the atz B and atz C genes in J. lividum . The nucleotide sequences of the PCR fragments of the atz genes from these strains were 100% identical to the homologous genes of the Pseudomonas sp. strain ADP. In conclusion, the results show the potential for the use of a natural attenuation strategy in the treatment of aquifers polluted with the terbuthylazine. The two bacteria isolated could facilitate the implementation of effective bioremediation protocols, especially in the case of the significant amounts of herbicide that can be found in groundwater as a result of accidental spills.     

Mechanical Properties of β-Catenin Revealed by Single-Molecule Experiments

β-catenin is a central component of the adaptor complex that links cadherins to the actin cytoskeleton in adherens junctions and thus, it is a good candidate to sense and transmit mechanical forces to trigger specific changes inside the cell. To fully understand its molecular physiology, we must first investigate its mechanical role in mechanotransduction within the cadherin system. We have studied the mechanical response of β-catenin to stretching using single-molecule force spectroscopy and molecular dynamics. Unlike most proteins analyzed to date, which have a fixed mechanical unfolding pathway, the β-catenin armadillo repeat region (ARM) displays low mechanostability and multiple alternative unfolding pathways that seem to be modulated by its unstructured termini. These results are supported by steered molecular dynamics simulations, which also predict its mechanical stabilization and unfolding pathway restrictions when the contiguous α-helix of the C-terminal unstructured region is included. Furthermore, simulations of the ARM/E-cadherin cytosolic tail complex emulating the most probable stress geometry occurring in vivo show a mechanical stabilization of the interaction whose magnitude correlates with the length of the stretch of the cadherin cytosolic tail that is in contact with the ARM region.     

Architecture of Ulex europaeus: Changes in the vertical distribution of organs in relation to plant height and season

Abstract. This paper is concerned with the vertical distribution of photosynthetic, woody, reproductive and dead organs of Ulex europaeus, the dominant pyrophytic species in most of the shrublands of Galicia (NW Spain). Frequency and weight data for the above‐mentioned organs were collected each season for individual plants up to 150 cm tall, in order to quantitatively characterize the vertical organ distribution in this species. The study also deals with regression equations that would allow the prediction of phytomass from easier measurements. Frequency values were recorded using a grid of quadrats situated on the plant's vertical axis, and clipping each individual into vertical layers of 6 cm to obtain phytomass values. The abundance and location of photosynthetic, woody and dead organs varies in the three height classes studied, both regarding frequency and phytomass. A marked decrease in the ratio between photosynthetic and total phytomass is also observed as plant height increases. Significant seasonal differences were recorded only for reproductive organs. A good fit was obtained for several regression equations; total phytomass can be predicted from the largest diameter of the shrub, or organ phytomass from total weight.