Human recombinant domain antibodies against multiple sclerosis antigenic peptide CSF114(Glc)

Multiple sclerosis (MS) is a chronic auto‐immune disease characterized by a damage to the myelin component of the central nervous system. Self‐antigens created by aberrant glycosylation have been described to be a key component in the formation of auto‐antibodies. CSF114(Glc) is a synthetic glucopeptide detecting in vitro MS‐specific auto‐antibodies, and it is actively used in diagnostics and research to monitor and quantify MS‐associated Ig levels. We reasoned that antibodies raised against this probe could have been relevant for MS. We therefore screened a human Domain Antibody library against CSF114(Glc) using magnetic separation as a panning method. We obtained and described several clones, and the one with the highest signals was produced as a 6×His‐tagged protein to properly study the binding properties as a soluble antibody. By surface plasmon resonance measurements, we evidenced that our clone recognized CSF114(Glc) with high affinity and specific for the glucosylated peptide. Kinetic parameters of peptide–clone interaction were calculated obtaining a value of K_D in the nanomolar range. Harboring a human framework, this antibody should be very well tolerated by human immune system and may represent a valuable tool for MS diagnosis and therapy, paving the way to new research strategies. Copyright © 2014 John Wiley & Sons, Ltd.     

Magnetic Nanoparticles from Magnetospirillum gryphiswaldense Increase the Efficacy of Thermotherapy in a Model of Colon Carcinoma

Magnetic nanoparticles (MNPs) are capable of generate heating power under the influence of alternating magnetic fields (AMF); this behaviour recently opened new scenarios for advanced biomedical applications, mainly as new promising tumor therapies. In this paper we have tested magnetic nanoparticles called magnetosomes (MNs): a class of MNPs naturally produced by magnetotactic bacteria. We extracted MNs from Magnetospirillum gryphiswaldense strain MSR-1 and tested the interaction with cellular elements and anti-neoplastic activity both in vitro and in vivo, with the aim of developing new therapeutic approaches for neoplastic diseases. In vitro experiments performed on Human Colon Carcinoma HT-29 cell cultures demonstrated a strong uptake of MNs with no evident signs of cytotoxicity and revealed three phases in the interaction: adherence, transport and accumulation in Golgi vesicles. In vivo studies were performed on subcutaneous tumors in mice; in this model MNs are administered by direct injection in the tumor volume, then a protocol consisting of three exposures to an AMF rated at 187 kHz and 23kA/m is carried out on alternate days, over a week. Tumors were monitored by Magnetic Resonance Imaging (MRI) to obtain information about MNs distribution and possible tissue modifications induced by hyperthermia. Histological analysis showed fibrous and necrotic areas close to MNs injection sites in mice subjected to a complete thermotherapy protocol. These results, although concerning a specific tumor model, could be useful to further investigate the feasibility and efficacy of protocols based on MFH. Magnetic nanoparticles naturally produced and extracted from bacteria seem to be promising candidates for theranostic applications in cancer therapy.     

Perturbation of cytochrome P450, generation of oxidative stress and induction of DNA damage in Cyprinus carpio exposed in situ to potable surface water

Epidemiological evidence suggests a link between consumption of chlorinated drinking water and various cancers. Chlorination of water rich in organic chemicals produces carcinogenic organochlorine by-products (OBPs) such as trihalomethanes and haloacetic acids. Since the discovery of the first OBP in the 1970s, there have been several investigations designed to determine the biological effects of single chemicals or small artificial OBP combinations. However, there is still insufficient information regarding the general biological response to these compounds, and further studies are still needed to evaluate their potential genotoxic effects. In the current study, we evaluated the effect of three drinking water disinfectants on the activity of cytochrome P450 (CYP)-linked metabolizing enzymes and on the generation of oxidative stress in the livers of male and female Cyprinus carpio fish (carp). The fish were exposed in situ for up 20 days to surface water obtained from the Trasmene lake in Italy. The water was treated with 1-2 mg/L of either sodium hypochlorite (NaClO) or chlorine dioxide (ClO2) as traditional disinfectants or with a relatively new disinfectant product, peracetic acid (PAA). Micronucleus (MN) frequencies in circulating erythrocytes from the fish were also analysed as a biomarker of genotoxic effect. In the CYP-linked enzyme assays, a significant induction (up to a 57-fold increase in the deethylation of ethoxyresorufin with PAA treatment) and a notable inactivation (up to almost a 90% loss in hydroxylation of p-nitrophenol with all disinfectants, and of testosterone 2beta-hydroxylation with NaClO) was observed in subcellular liver preparations from exposed fish. Using the electron paramagnetic resonance (EPR) spectroscopy radical-probe technique, we also observed that CYP-modulation was associated with the production of reactive oxygen species (ROS). In addition, we found a significant increase in MN frequency in circulating erythrocytes after 10 days of exposure of fish to water treated with ClO2, while a non-significant six-fold increase in MN frequency was observed with NaClO, but not with PAA. Our data suggest that the use of ClO2 and NaClO to disinfect drinking water could generate harmful OBP mixtures that are able to perturb CYP-mediated reactions, generate oxidative stress and induce genetic damage. These data may provide a mechanistic explanation for epidemiological studies linking consumption of chlorinated drinking water to increased risk of urinary, gastrointestinal and bladder cancers.     

Effects of high-fat diet exposure during fetal life on type 2 diabetes development in the progeny

Nutrition during fetal life is a critical factor contributing to diabetes development in adulthood. The aim of our study was to verify: 1) whether a high-fat (HF) diet in young adult mice induces alterations in beta-cell mass, proliferation, neogenesis, and apoptosis, as well as insulin sensitivity and secretion; 2) whether these alterations may be reversible after HF diet suspension; 3) the effects in a first (F1) and second generation (F2) of mice without direct exposure to a HF diet after birth. Type 2 diabetes developed in adult mice on a HF diet, in F1 mice that were HF diet-exposed during fetal or neonatal life, and in F2 mice whose mothers were HF diet-exposed during their fetal life. beta-cell mass, replication, and neogenesis were high in HF diet-exposed mice and decreased after diet suspension. beta-cell mass and replication remained high in F1 mice and decreased in F2 mice whose mothers were exposed to a HF diet. beta-cell neogenesis was present in adult mice on a HF diet and in F1 mice that were HF diet-exposed during fetal and/or neonatal life. We conclude that a HF diet during fetal life, particularly if combined with the same insult during the suckling period, can induce the type 2 diabetes phenotype, which can be directly transmitted to the progeny even in the absence of additional dietary insults.     

Low electromagnetic field (50 Hz) induces differentiation on primary human oral keratinocytes (HOK)

This work concerns the effect of low frequency electromagnetic fields (ELF) on biochemical properties of human oral keratinocytes (HOK). Cells exposed to a 2 mT, 50 Hz, magnetic field, showed by scanning electron microscopy (SEM) modification in shape and morphology; these modifications were also associated with different actin distribution, revealed by phalloidin fluorescence analysis. Moreover, exposed cells had a smaller clonogenic capacity, and decreased cellular growth. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression. The advance in differentiation was confirmed by a decrease of expression of epidermal growth factor (EGF) receptor in exposed cells, supporting the idea that exposure to electromagnetic field carries keratinocytes to higher differentiation level. These observations support the hypothesis that 50 Hz electromagnetic fields may modify cell morphology and interfere in differentiation and cellular adhesion of normal keratinocytes.     

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.     

Free radical release in C6 glial cells enriched in hexacosanoic acid: implication for X-linked adrenoleukodystrophy pathogenesis

Free radicals have been implicated in the etiopathology of some neurological and demyelinating diseases. To evaluate their involvement in the cerebral form of X-linked adrenoleukodystrophy (cerALD) disorder, characterised by very long chain fatty acid (VLCFA) accumulation, we utilised an in vitro model using rat C6 glial cells, enriched in hexacosenoic acid (C26:0, HA). Modified cells were incubated in presence of oxidative stressors, such as bacterial endotoxin lipopolisaccharides (LPS) and human oxidised low-density lipoprotein (ox-LDL), and the production of proinflammatory cytokines, nitrite, nitrate and superoxide was determined in the supernatants. The results show that modified cells produce higher amounts of nitric oxide (NO) products and superoxide compared to native C6 cells, supporting the role of free radicals as important pathophysiological modulator of the neuroinflammatory response in ALD. This hypothesis suggests that the cerebral damage in ALD could be due to intracellular signalling activated by interaction of exogenous factors with the particular membrane fatty acid composition.     

NMR solution structure of the acylphosphatase from Escherichia coli

The solution structure of Escherichia coli acylphosphatase (E. coli AcP), a small enzyme catalyzing the hydrolysis of acylphosphates, was determined by (1)H and (15)N NMR and restrained modelling calculation. In analogy with the other members of AcP family, E. coli AcP shows an alpha/beta sandwich domain composed of four antiparallel and one parallel beta-strand, assembled in a five-stranded beta-sheet facing two antiparallel alpha-helices. The pairwise RMSD values calculated for the backbone atoms of E. coli and Sulfolobus solfataricus AcP, Bovine common type AcP and Horse muscle AcP are 2.18, 5.31 and 5.12 A, respectively. No significant differences are present in the active site region and the catalytic residue side chains are consistently positioned in the structures.     

Structure of Pyridoxal Kinase from Sheep Brain and Role of the Tryptophanyl Residues

The primary structure of sheep brain pyridoxal kinase has been determined by direct chemical and physical methods. The enzyme contains 312 amino acid residues with an acetylated methionine at the N-terminus, yielding a molecular mass of 34,861 Da. The functional role played by the two tryptophanyl residues in positions 52 and 244 of the polypeptide chain has been investigated by fluorescence spectroscopy. The tryptophanyl residues are not completely exposed to the rapidly relaxing solvent and they are poorly accessible to collisional quenchers. Chemical modification with NBS abolishes the catalytic activity of the kinase. The amino acid sequence of the sheep brain enzyme shows high similarity (86.2% identity) with the human pyridoxal kinase recently reported [Hanna, Turner, and Kirkness, (1997), J. Biol. Chem. 272, 10756–10760]. Comparison of the mammalian proteins with bacterial and yeast putative pyridoxal kinases retrieved from the Swiss-Prot data bank shows a low degree of overall similarity. In particular, the putative ATP-binding domain is conserved, whereas the region that appears to be crucial in the binding of the pyridoxal substrate is not. Thus, the assignment of the bacterial and yeast cDNA-deduced proteins as pyridoxal kinases should be taken with caution.     

High-resolution magnetic resonance spectroscopy of the mouse vomeronasal organ

The chemical composition of the vomeronasal organ (VNO) was investigated by means of in vitro proton magnetic resonance spectroscopy (MRS) in prepubertal and adult mice of both sexes. Results demonstrate that MRS detects several chemical constituents in the VNO, showing their age- and sex-associated changes in concentration. Preliminary experiments also suggest the ability of MRS to show compositional changes in the VNO after pheromonal stimulation. MRS can serve as a useful technique to investigate vomeronasal chemoreception.