Monosaccharide precursors for boosting chondroitin-like capsular polysaccharide production

Chondroitin sulfate is a well-known bioactive molecule, widely used as an anti-osteoarthritis drug, that is nowadays mainly produced by animal tissue sources with unsafe extraction procedures. Recent studies have explored an integrated biotechnological–chemical strategy to obtain a chondroitin sulfate precursor from Escherichia coli K4 capsular polysaccharide, demonstrating the influence of environmental and growth conditions on capsule synthesis. In this research work, the flexibility of the strain biosynthetic machinery was investigated to enhance the K4 capsular polysaccharide production by supplementing the growth medium with the monosaccharides (glucuronic acid, galactosamine and fructose) that constitute the chain. Shake flask experiments were performed by adding the sugars singularly or together, by testing monosaccharide different concentrations and times of addition and by observing the bacterial sugar consumption. A K4 capsular polysaccharide production enhancement, compared to the control, was observed in all cases of supplementation and, in particular, significant 68 and 57 % increases were observed when adding 0.385 mM glucuronic acid plus galactosamine or 0.385 mM fructose, respectively. Increased expression levels of the gene kfoC, coding for a K4 polymerase, evaluated in different growth conditions, confirmed the results at the molecular level. Furthermore, batch fermentations, performed in lab-scale reactors (2 L), allowed to double the K4 capsular polysaccharide production values obtained in shake flask conditions, by means of a strict control of the growth parameters.     

High cell density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered heparin production

A bioengineered heparin, as a replacement for animal-derived heparin, is under development that relies on the fermentative production of heparosan by Escherichia coli K5 and its subsequent chemoenzymatic modification using biosynthetic enzymes. A critical enzyme in this pathway is the mammalian 6-O-sulfotransferase (6-OST-1) which specifically sulfonates the glucosamine residue in a heparin precursor. This mammalian enzyme, previously cloned and expressed in E. coli, is required in kilogram amounts if an industrial process for bioengineered heparin is to be established. In this study, high cell density cultivation techniques were exploited to obtain recombinant 6-OST-1. Physiological studies were performed in shake flasks to establish optimized growth and production conditions. Induction strategies were tested in fed-batch experiments to improve yield and productivity. High cell density cultivation in 7-l culture, together with a coupled inducer strategy using isopropyl β-d-1-thiogalactopyranoside and galactose, afforded 482 mg?l^?1 of enzyme with a biomass yield of 16.2 mg?g_cdw ^?1 and a productivity of 10.5 mg?l^?1?h^?1.     

A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii

Haloarchaeal alcohol dehydrogenases are exciting biocatalysts with potential industrial applications. In this study, two alcohol dehydrogenase enzymes from the extremely halophilic archaeon Haloferax volcanii (HvADH1 and HvADH2) were homologously expressed and subsequently purified by immobilized metal-affinity chromatography. The proteins appeared to copurify with endogenous alcohol dehydrogenases, and a double Δadh2 Δadh1 gene deletion strain was constructed to prevent this occurrence. Purified HvADH1 and HvADH2 were compared in terms of stability and enzymatic activity over a range of pH values, salt concentrations, and temperatures. Both enzymes were haloalkaliphilic and thermoactive for the oxidative reaction and catalyzed the reductive reaction at a slightly acidic pH. While the NAD⁺-dependent HvADH1 showed a preference for short-chain alcohols and was inherently unstable, HvADH2 exhibited dual cofactor specificity, accepted a broad range of substrates, and, with respect to HvADH1, was remarkably stable. Furthermore, HvADH2 exhibited tolerance to organic solvents. HvADH2 therefore displays much greater potential as an industrially useful biocatalyst than HvADH1.     

FAM111A Mutations Result in Hypoparathyroidism and Impaired Skeletal Development

Kenny-Caffey syndrome (KCS) and the similar but more severe osteocraniostenosis (OCS) are genetic conditions characterized by impaired skeletal development with small and dense bones, short stature, and primary hypoparathyroidism with hypocalcemia. We studied five individuals with KCS and five with OCS and found that all of them had heterozygous mutations in FAM111A. One mutation was identified in four unrelated individuals with KCS, and another one was identified in two unrelated individuals with OCS; all occurred de novo. Thus, OCS and KCS are allelic disorders of different severity. FAM111A codes for a 611 amino acid protein with homology to trypsin-like peptidases. Although FAM111A has been found to bind to the large T-antigen of SV40 and restrict viral replication, its native function is unknown. Molecular modeling of FAM111A shows that residues affected by KCS and OCS mutations do not map close to the active site but are clustered on a segment of the protein and are at, or close to, its outer surface, suggesting that the pathogenesis involves the interaction with as yet unidentified partner proteins rather than impaired catalysis. FAM111A appears to be crucial to a pathway that governs parathyroid hormone production, calcium homeostasis, and skeletal development and growth.     

Effete,a Drosophila Chromatin-Associated Ubiquitin-Conjugating Enzyme That Affects Telomeric and Heterochromatic Position Effect Variegation

Drosophila telomeres are elongated by the transposition of telomere-specific retrotransposons rather than telomerase activity. Proximal to the terminal transposon array, Drosophila chromosomes contain several kilobases of a complex satellite DNA termed telomere-associated sequences (TASs). Reporter genes inserted into or next to the TAS are silenced through a mechanism called telomere position effect (TPE). TPE is reminiscent of the position effect variegation (PEV) induced by Drosophila constitutive heterochromatin. However, most genes that modulate PEV have no effect on TPE, and systematic searches for TPE modifiers have so far identified only a few dominant suppressors. Surprisingly, only a few of the genes required to prevent telomere fusion have been tested for their effect on TPE. Here, we show that with the exception of the effete (eff; also called UbcD1) mutant alleles, none of the tested mutations at the other telomere fusion genes affects TPE. We also found that mutations in eff, which encodes a class I ubiquitin-conjugating enzyme, act as suppressors of PEV. Thus, eff is one of the rare genes that can modulate both TPE and PEV. Immunolocalization experiments showed that Eff is a major constituent of polytene chromosomes. Eff is enriched at several euchromatic bands and interbands, the TAS regions, and the chromocenter. Our results suggest that Eff associates with different types of chromatin affecting their abilities to regulate gene expression.     

A plant derived multifunctional tool for nanobiotechnology based on Tomato bushy stunt virus

Structure, size, physicochemical properties and production strategies make many plant viruses ideal protein based nanoscaffolds, nanocontainers and nano-building blocks expected to deliver a multitude of applications in different fields such as biomedicine, pharmaceutical chemistry, separation science, catalytic chemistry, crop pest control and biomaterials science. Functionalization of viral nanoparticles through modification by design of their external and internal surfaces is essential to fully exploit the potentiality of these objects. In the present paper we describe the development of a plant derived multifunctional tool for nanobiotechnology based on Tomato bushy stunt virus. We demonstrate the ability of this system to remarkably sustain genetic modifications and in vitro chemical derivatizations of its outer surface, which resulted in the successful display of large chimeric peptides fusions and small chemical molecules, respectively. Moreover, we have defined physicochemical conditions for viral swelling and reversible viral pore gating that we have successfully employed for foreign molecules loading and retention in the inner cavity of this plant virus nanoparticles system. Finally, a production and purification strategy from Nicotiana benthamiana plants has been addressed and optimized.     

The kinetic deuterium isotope effect as applied to metabolic deactivation of imatinib to the des-methyl metabolite,CGP74588

There has recently been a burgeoning interest in impeding drug metabolism by replacing hydrogen atoms with deuterium to invoke a kinetic isotope effect. Imatinib, a front-line therapy for both chronic myeloid leukemia and of gastrointestinal stromal tumours, is often substantially metabolised via N-demethylation to the significantly less active CGP74588. Since deuterium–carbon bonds are stronger than hydrogen–carbon bonds, we hypothesised that the N-trideuteromethyl analogue of imatinib might be subject to a reduced metabolic turnover as compared to imatinib and lead to different pharmacokinetic properties, and hence improved efficacy, in vivo. Consequently, we investigated whether the N-trideuteromethyl analogue would maintain target inhibition and show a reduced propensity for N-demethylation in in vitro assays with liver microsomes and following oral administration to rats. The N-trideuteromethyl compound exhibited similar activity as a tyrosine kinase inhibitor as imatinib and similar efficacy as an antiproliferative in cellular assays. In comparison to imatinib, the trideuterated analogue also showed reduced N-demethylation upon incubation with both rat and human liver microsomes, consistent with a deuterium isotope effect. However, the reduced in vitro metabolism did not translate into increased exposure of the N-trideuteromethyl analogue following intravenous administration of the compound to rats and no significant difference was observed for the formation of the N-desmethyl metabolite from either parent drug.     

Divergent and convergent synthesis of polymannosylated dibranched antigenic peptide of the immunodominant epitope MBP(83–99)

Multiple antigenic peptide (MAP) systems are dendrimeric structures bearing multiple copies of identical or different peptide epitopes, and they have been demonstrated to show enhanced immunogenicity. Herein, we report the direct (divergent) and indirect (convergent) synthesis, using contemporary synthetic approaches, of a di-branched antigenic peptide (di-BAP) containing the immunodominant epitope MBP(83–99), which is implicated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The direct synthesis (di-BAP 1) was performed using microwave irradiation. The indirect synthesis (di-BAP 2) was carried out performing an efficient chemoselective coupling reaction through the formation of a thioether bond. Both di-BAPs were conjugated to polysaccharide mannan since mannosylation is a promising technique to achieve modulation in immune response. The conjugation was achieved through free amino groups of both di-BAPs via the formation of Schiff bases. The mannan-conjugated di-BAPs were further evaluated in vivo in a prophylactic vaccination protocol, prior to EAE induction in Lewis rats.     

New structure–activity relationship studies in a series of N,N-bis(cyclohexanol)amine aryl esters as potent reversers of P-glycoprotein-mediated multidrug resistance (MDR)

As a continuation of previous research on a new series of potent and efficacious P-gp-dependent multidrug resistant (MDR) reversers with a N,N-bis(cyclohexanol)amine scaffold, we have designed and synthesized several analogs by modulation of the two aromatic moieties linked through ester functions to the N,N-bis(cyclohexanol)amine, aiming to optimize activity and to extend structure–activity relationships (SAR) within the series. This scaffold, when esterified with two different aromatic carboxylic acids, gives origin to four geometric isomers (cis/trans, trans/trans, cis/cis and trans/cis).The new compounds were tested on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay. Most of them resulted in being potent modulators of the extrusion pump P-gp, showing potency values ([I]_0.5) in the submicromolar and nanomolar range. Of these, compounds 2b, 2c, 3d, 5a–d and 6d, showed excellent efficacy with a α_max close to 1. Selected compounds (2d, 3a, 3b, 5a–d) were further studied to evaluate their doxorubicin cytotoxicity potentiation (RF) on doxorubicin-resistant erythroleukemia K562 cells and were found able to enhance significantly doxorubicin cytotoxicity on K562/DOX cells.The results of both pirarubicin uptake and the cytotoxicity assay, indicate that the new compounds of the series are potent P-gp-mediated MDR reversers. They present a structure with a mix of flexible and rigid moieties, a property that seems critical to allow the molecules to choose the most productive of the several binding modes possible in the transporter recognition site.In particular, compounds 5c and 5d, similar to the already reported analogous isomers 1c and 1d,²⁹ are potent and efficacious modulators of P-gp-dependent MDR and may be promising leads for the development of MDR-reversal drugs.