New functions of XPC in the protection of human skin cells from oxidative damage

Xeroderma pigmentosum (XP) C is involved in the recognition of a variety of bulky DNA-distorting lesions in nucleotide excision repair. Here, we show that XPC plays an unexpected and multifaceted role in cell protection from oxidative DNA damage. XP-C primary keratinocytes and fibroblasts are hypersensitive to the killing effects of DNA-oxidizing agents and this effect is reverted by expression of wild-type XPC. Upon oxidant exposure, XP-C primary keratinocytes and fibroblasts accumulate 8,5'-cyclopurine 2'-deoxynucleosides in their DNA, indicating that XPC is involved in their removal. In the absence of XPC, a decrease in the repair rate of 8-hydroxyguanine (8-OH-Gua) is also observed. We demonstrate that XPC-HR23B complex acts as cofactor in base excision repair of 8-OH-Gua, by stimulating the activity of its specific DNA glycosylase OGG1. In vitro experiments suggest that the mechanism involved is a combination of increased loading and turnover of OGG1 by XPC-HR23B complex. The accumulation of endogenous oxidative DNA damage might contribute to increased skin cancer risk and account for internal cancers reported for XP-C patients.     

The structure of the Lingo-1 ectodomain, a module implicated in central nervous system repair inhibition

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 A2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.     

Recombinant expression and biochemical characterization of the unique elongating beta-ketoacyl-acyl carrier protein synthase involved in fatty acid biosynthesis of Plasmodium falciparum using natural and artificial substrates

The human malaria parasite Plasmodium falciparum synthesizes fatty acids by using a type II synthase that is structurally different from the type I system found in eukaryotes. Because of this difference and the vital role of fatty acids, the enzymes involved in fatty acid biosynthesis of P. falciparum represent interesting targets for the development of new antimalarial drugs. beta-Ketoacyl-acyl carrier protein (ACP) synthase (PfFabBF), being the only elongating beta-ketoacyl-ACP synthase in P. falciparum, is a potential candidate for inhibition. In this study we present the cloning, expression, purification, and characterization of PfFabBF. Soluble protein was obtained when PfFabBF was expressed as a NusA fusion protein in Escherichia coli BL21(DE3)-CodonPlus-RIL cells under conditions of osmotic stress. The fusion protein was purified by affinity and ion exchange chromatography. Various acyl-P. falciparum acyl carrier protein (PfACP) substrates were tested for their specific activities, and their kinetic parameters were determined. Activity of PfFabBF was highest with C(4:0)- to C(10:0)-acyl-PfACPs and decreased with use of longer chain acyl-PfACPs. Consistent with the fatty acid synthesis profile found in the parasite cell, no activity could be detected with C(16:0)-PfACP, indicating that the enzyme is lacking the capability of elongating acyl chains that are longer than 14 carbon atoms. PfFabBF was found to be specific for acyl-PfACPs, and it displayed much lower activities with the corresponding acyl-CoAs. Furthermore, PfFabBF was shown to be sensitive to cerulenin and thiolactomycin, known inhibitors of beta-ketoacyl-ACP synthases. These results represent an important step toward the evaluation of P. falciparum beta-ketoacyl-ACP synthase as a novel antimalaria target.     

Structural characterization of the lipid A region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide

The lipid A components of Aeromonas salmonicida subsp. salmonicida from strains A449, 80204-1 and an in vivo rough isolate were isolated by mild acid hydrolysis of the lipopolysaccharide. Structural studies carried out by a combination of fatty acid, electrospray ionization-mass spectrometry and nuclear magnetic resonance analyses confirmed that the structure of lipid A was conserved among different isolates of A. salmonicida subsp. salmonicida. All analyzed strains contained three major lipid A molecules differing in acylation patterns corresponding to tetra-, penta- and hexaacylated lipid A species and comprising 4'-monophosphorylated beta-2-amino-2-deoxy-d-glucopyranose-(1-->6)-2-amino-2-deoxy-d-glucopyranose disaccharide, where the reducing end 2-amino-2-deoxy-d-glucose was present primarily in the alpha-pyranose form. Electrospray ionization-tandem mass spectrometry fragment pattern analysis, including investigation of the inner-ring fragmentation, allowed the localization of fatty acyl residues on the disaccharide backbone of lipid A. The tetraacylated lipid A structure containing 3-(dodecanoyloxy)tetradecanoic acid at N-2',3-hydroxytetradecanoic acid at N-2 and 3-hydroxytetradecanoic acid at O-3, respectively, was found. The pentaacyl lipid A molecule had a similar fatty acid distribution pattern and, additionally, carried 3-hydroxytetradecanoic acid at O-3'. In the hexaacylated lipid A structure, 3-hydroxytetradecanoic acid at O-3' was esterified with a secondary 9-hexadecenoic acid. Interestingly, lipid A of the in vivo rough isolate contained predominantly tetra- and pentaacylated lipid A species suggesting that the presence of the hexaacyl lipid A was associated with the smooth-form lipopolysaccharide.     

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status

Fuel ethanol production from plant biomass hydrolysates by Saccharomyces cerevisiae is of great economic and environmental significance. This paper reviews the current status with respect to alcoholic fermentation of the main plant biomass-derived monosaccharides by this yeast. Wild-type S. cerevisiae strains readily ferment glucose, mannose and fructose via the Embden–Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass hydrolysates into ethanol is a major challenge in metabolic engineering. The most abundant of these compounds is xylose. Recent metabolic and evolutionary engineering studies on S. cerevisiae strains that express a fungal xylose isomerase have enabled the rapid and efficient␣anaerobic fermentation of this pentose. l-Arabinose fermentation, based on the expression of a prokaryotic pathway in S. cerevisiae, has also been established, but needs further optimization before it can be considered for industrial implementation. In addition to these already investigated strategies, possible approaches for metabolic engineering of galacturonic acid and rhamnose fermentation by S. cerevisiae are discussed. An emerging and major challenge is to achieve the rapid transition from proof-of-principle experiments under ‘academic’ conditions (synthetic media, single substrates or simple substrate mixtures, absence of toxic inhibitors) towards efficient conversion of complex industrial substrate mixtures that contain synergistically acting inhibitors.     

Melatonin supplementation decreases prolactin synthesis and release in rat adenohypophysis: correlation with anterior pituitary redox state and circadian clock mechanisms

In the laboratory rat, a number of physiological parameters display seasonal changes even under constant conditions of temperature, lighting, and food availability. Since there is evidence that prolactin (PRL) is, among the endocrine signals, a major mediator of seasonal adaptations, the authors aimed to examine whether melatonin administration in drinking water resembling in length the exposure to a winter photoperiod could affect accordingly the 24-h pattern of PRL synthesis and release and some of their anterior pituitary redox state and circadian clock modulatory mechanisms. Melatonin (3?μg/mL drinking water) or vehicle was given for 1 mo, and rats were euthanized at six time intervals during a 24-h cycle. High concentrations of melatonin (>2000 pg/mL) were detected in melatonin-treated rats from beginning of scotophase (at 21:00?h) to early photophase (at 09:00?h) as compared with a considerably narrower high-melatonin phase observed in controls. By cosinor analysis, melatonin-treated rats had significantly decreased MESOR (24-h time-series average) values of anterior pituitary PRL gene expression and circulating PRL, with acrophases (peak time) located in the middle of the scotophase, as in the control group. Melatonin treatment disrupted the 24-h pattern of anterior pituitary gene expression of nitric oxide synthase (NOS)-1 and -2, heme oxygenase-1 and -2, glutathione peroxidase, glutathione reductase, Cu/Zn- and Mn-superoxide dismutase, and catalase by shifting their acrophases to early/middle scotophase or amplifying the maxima. Only the inhibitory effect of melatonin on pituitary NOS-2 gene expression correlated temporally with inhibition of PRL production. Gene expression of metallothionein-1 and -3 showed maxima at early/middle photophase after melatonin treatment. The 24-h pattern of anterior pituitary lipid peroxidation did not vary after treatment. In vehicle-treated rats, Clock and Bmal1 expression peaked in the anterior pituitary at middle scotophase, whereas that of Per1 and Per2 and of Cry1 and Cry2 peaked at the middle and late photophase, respectively. Treatment with melatonin raised mean expression of anterior pituitary Per2, Cry1, and Cry2. In the case of Per1, decreased MESOR was observed, although the single significant difference found between the experimental groups when analyzed at individual time intervals was increase at early scotophase in the anterior pituitary of melatonin-treated rats. Melatonin significantly phase-delayed expression of Per1, Per2, and Cry1, also phase-delayed the plasma corticosterone circadian rhythm, and increased the amplitude of plasma corticosterone and thyrotropin rhythms. The results indicate that under prolonged duration of a daily melatonin signal, rat anterior pituitary PRL synthesis and release are depressed, together with significant changes in the redox and circadian mechanisms controlling them. (Author correspondence: danielcardinali@uca.edu.ar ; danielcardinali@fibertel.com.ar ).     

Antagonistic Activity of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> ATCC 4356 on the Growth and Adhesion/Invasion Characteristics of Human <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

The aim of this research was to determine the potential probiotic activity of Lactobacillus acidophilus ATCC 4356 against several human Campylobacter jejuni isolates. The ability to inhibit the pathogen’s growth was evaluated by co-culture experiments as well as by antimicrobial assays with cell-free culture supernatant (CFCS), while interference with adhesion/invasion to intestinal Caco-2 cells was studied by exclusion, competition, and displacement tests. In the co-culture experiments L. acidophilus ATCC 4356 strain reduced the growth of C. jejuni with variable percentages of inhibition related to the contact time. The CFCS showed inhibitory activity against C. jejuni strains, stability to low pH, and thermal treatment and sensitivity to proteinase K and trypsin. L. acidophilus ATCC 4356 was able to reduce the adhesion and invasion to Caco-2 cells by most of the human C. jejuni strains. Displacement and exclusion mechanisms seem to be the preferred modalities, which caused a significant reduction of adhesion/invasion of pathogens to intestinal cells. The observed inhibitory properties of L. acidophilus ATCC 4356 on growth ability and on cells adhesion/invasion of C. jejuni may offer potential use of this strain for the management of Campylobacter infections.     

miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration

During keratinocyte differentiation and stratification, cells undergo extensive remodeling of their actin cytoskeleton, which is important to control cell mobility and to coordinate and stabilize adhesive structures necessary for functional epithelia. Limited knowledge exists on how the actin cytoskeleton is remodeled in epithelial stratification and whether cell shape is a key determinant to trigger terminal differentiation. In this paper, using human keratinocytes and mouse epidermis as models, we implicate miR-24 in actin adhesion dynamics and demonstrate that miR-24 directly controls actin cable formation and cell mobility. miR-24 overexpression in proliferating cells was sufficient to trigger keratinocyte differentiation both in vitro and in vivo and directly repressed cytoskeletal modulators (PAK4, Tks5, and ArhGAP19). Silencing of these targets recapitulated the effects of miR-24 overexpression. Our results uncover a new regulatory pathway involving a differentiation-promoting microribonucleic acid that regulates actin adhesion dynamics in human and mouse epidermis.