An engineered folded PLP-bound monomer of Treponema denticola cystalysin reveals the effect of the dimeric structure on the catalytic properties of the enzyme

Cystalysin, a dimeric pyridoxal 5'-phosphate (PLP)-dependent lyase, is a virulence factor of the human oral pathogen Treponema denticola. Guided by bioinformatic analysis, two interfacial residues (Leu57 and Leu62) and an active site residue (Tyr64*), hydrogen-bonded with the PLP phosphate group of the neighboring subunit, have been mutated. The wild-type and the L57A, L62A, Y64*A, L57A/L62A, L57A/Y64*A, L57A/L62A/Y64*A mutants, all having a C-terminal histidine tag, have been constructed, expressed, and purified. The impact of these mutations on the dimeric state of cystalysin in the apo- and holo-form has been analyzed by size-exclusion chromatography. The results demonstrate that (i) Leu57 is more critical than Leu62 for apodimer formation, (ii) Tyr64*, more than Leu62, interferes with dimerization of holocystalysin without affecting that of apoenzyme, (iii) while each single mutation is inadequate in significantly altering the extent of monomerization of both apo- and holo-cystalysin, their combination leads to species which remain in a folded monomeric state at a reasonably high concentration in both the apo- and holo-forms. Although L57A/L62A or L57A/Y64*A, even to a different extent, are stimulated to dimer formation in the presence of either unproductive or productive ligands, L57A/L62A/Y64*A remains prevalently monomer at a concentration up to 50 microM. Kinetic analyses show that in this monomeric species the alpha,beta-eliminase, alanine racemase, and D-alanine half-transaminase activities are almost abolished, while the L-alanine half-transaminase activity is slightly enhanced when compared with that of wild-type. The structural basis of the stereospecific transaminase activity displayed by the engineered folded PLP-bound monomer has been analyzed and discussed.     

Structure–Activity Relationship of novel phenylacetic CXCR1 inhibitors

We reported recently the Structure–Activity Relationship (SAR) of a class of CXCL8 allosteric modulators. They invariably share a 2-arylpropionic moiety so far considered a key structural determinant of the biological activity. We show the results of recent SAR studies on a novel series of phenylacetic derivatives supported by a combined approach of mutagenesis experiments and conformational analysis. The results suggest novel insights on the fine role of the propionic/acetic chain in the modulation of CXCL8 receptors.     

Exploring bis-(indolyl)methane moiety as an alternative and innovative CAP group in the design of histone deacetylase (HDAC) inhibitors

In order to gather further knowledge about the structural requirements on histone deacetylase inhibitors (HDACi), starting from the schematic model of the common pharmacophore that characterizes this class of molecules (surface recognition CAP group—connection unit—linker region—Zinc Binding Group), we designed and synthesized a series of hydroxamic acids containing a bis-(indolyl)methane moiety. HDAC inhibition profile and antiproliferative activity were evaluated.     

A Targeted Constitutive Mutation in the Apc Tumor Suppressor Gene Underlies Mammary But Not Intestinal Tumorigenesis

Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/β-catenin signaling. Notably, genotype–phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/β-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/β-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc^+/1572T mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc^+/1572T mice suggests that specific dosages of Wnt/β-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.     

Greater risk of incident asthma cases in adults with Allergic Rhinitis and Effect of Allergen Immunotherapy: A Retrospective Cohort Study

Asthma and rhinitis are often co-morbid conditions. As rhinitis often precedes asthma it is possible that effective treatment of allergic rhinitis may reduce asthma progression.The aim of our study is to investigate history of allergic rhinitis as a risk factor for asthma and the potential effect of allergen immunotherapy in attenuating the incidence of asthma.Hospital-referred non-asthmatic adults, aged 18–40 years between 1990 and 1991, were retrospectively followed up until January and April 2000. At the end of follow up, available subjects were clinically examined for asthma diagnosis and history of allergen specific immunotherapy, second-hand smoking and the presence of pets in the household. A total of 436 non-asthmatic adults (332 subjects with allergic rhinitis and 104 with no allergic rhinitis nor history of atopy) were available for final analyses.The highest OR (odds ratio) associated with a diagnosis of asthma at the end of follow-up was for the diagnosis of allergic rhinitis at baseline (OR, 7.8; 95%CI, 3.1–20.0 in the model containing the covariates of rhinitis diagnosis, sex, second-hand smoke exposure, presence of pets at home, family history of allergic disorders, sensitization to Parietaria judaica; grass pollen; house dust mites; Olea europea: orchard; perennial rye; and cat allergens). Female sex, sensitization to Parietaria judaica and the presence of pets in the home were also significantly predictive of new onset asthma in the same model. Treatment with allergen immunotherapy was significantly and inversely related to the development of new onset asthma (OR, 0.53; 95%CI, 0.32–0.86).In the present study we found that allergic rhinitis is an important independent risk factor for asthma. Moreover, treatment with allergen immunotherapy lowers the risk of the development of new asthma cases in adults with allergic rhinitis.     

Physical energies to the rescue of damaged tissues

Rhythmic oscillatory patterns sustain cellular dynamics, driving the concerted action of regulatory molecules, microtubules, and molecular motors. We describe cellular microtubules as oscillators capable of synchronization and swarming,generating mechanical and electric patterns that impact biomolecular recognition.We consider the biological relevance of seeing the inside of cells populated by a network of molecules that behave as bioelectronic circuits and chromophores. We discuss the novel perspectives disclosed by mechanobiology,bioelectromagnetism, and photobiomodulation, both in term of fundamental basic science and in light of the biomedical implication of using physical energies to govern(stem) cell fate. We focus on the feasibility of exploiting atomic force microscopy and hyperspectral imaging to detect signatures of nanomotions and electromagnetic radiation(light), respectively, generated by the stem cells across the specification of their multilineage repertoire. The chance is reported of using these signatures and the diffusive features of physical waves to direct specifically the differentiation program of stem cells in situ, where they already are resident in all the tissues of the human body. We discuss how this strategy may pave the way to a regenerative and precision medicine without the needs for(stem) cell or tissue transplantation. We describe a novel paradigm based upon boosting our inherent ability for self-healing.