Structural elements of an NRPS cyclization domain and its intermodule docking domain

Epothilones are thiazole-containing natural products with anticancer activity that are biosynthesized by polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) enzymes EpoA–F. A cyclization domain of EpoB (Cy) assembles the thiazole functionality from an acetyl group and L-cysteine via condensation, cyclization, and dehydration. The PKS carrier protein of EpoA contributes the acetyl moiety, guided by a docking domain, whereas an NRPS EpoB carrier protein contributes L-cysteine. To visualize the structure of a cyclization domain with an accompanying docking domain, we solved a 2.03-Å resolution structure of this bidomain EpoB unit, comprising residues M1-Q497 (62 kDa) of the 160-kDa EpoB protein. We find that the N-terminal docking domain is connected to the V-shaped Cy domain by a 20-residue linker but otherwise makes no contacts to Cy. Molecular dynamic simulations and additional crystal structures reveal a high degree of flexibility for this docking domain, emphasizing the modular nature of the components of PKS-NRPS hybrid systems. These structures further reveal two 20-Å-long channels that run from distant sites on the Cy domain to the active site at the core of the enzyme, allowing two carrier proteins to dock with Cy and deliver their substrates simultaneously. Through mutagenesis and activity assays, catalytic residues N335 and D449 have been identified. Surprisingly, these residues do not map to the location of the conserved HHxxxDG motif in the structurally homologous NRPS condensation (C) domain. Thus, although both C and Cy domains have the same basic fold, their active sites appear distinct

Structural elements of an NRPS cyclization domain and its intermodule docking domain

Epothilones are thiazole-containing natural products with anticancer activity that are biosynthesized by polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) enzymes EpoA-F. A cyclization domain of EpoB (Cy) assembles the thiazole functionality from an acetyl group and l-cysteine via condensation, cyclization, and dehydration. The PKS carrier protein of EpoA contributes the acetyl moiety, guided by a docking domain, whereas an NRPS EpoB carrier protein contributes l-cysteine. To visualize the structure of a cyclization domain with an accompanying docking domain, we solved a 2.03-A resolution structure of this bidomain EpoB unit, comprising residues M1-Q497 (62 kDa) of the 160-kDa EpoB protein. We find that the N-terminal docking domain is connected to the V-shaped Cy domain by a 20-residue linker but otherwise makes no contacts to Cy. Molecular dynamic simulations and additional crystal structures reveal a high degree of flexibility for this docking domain, emphasizing the modular nature of the components of PKS-NRPS hybrid systems. These structures further reveal two 20-A-long channels that run from distant sites on the Cy domain to the active site at the core of the enzyme, allowing two carrier proteins to dock with Cy and deliver their substrates simultaneously. Through mutagenesis and activity assays, catalytic residues N335 and D449 have been identified. Surprisingly, these residues do not map to the location of the conserved HHxxxDG motif in the structurally homologous NRPS condensation (C) domain. Thus, although both C and Cy domains have the same basic fold, their active sites appear distinct

Measurement of Oxygenated Polycyclic Aromatic Hydrocarbons Associated with a Size-Segregated Urban Aerosol

Size-segregated atmospheric particles were collected in Boston, MA, using a micro-orifice impactor. The samples were analyzed for oxygenated polycyclic aromatic hydrocarbons (OPAH) using gas chromatography/mass spectrometry. Seven PAH ketones (1-acenaphthenone, 9-fluorenone, 11H-benzo[a]fluoren-11-one, 7H-benzo[c]fluoren-7-one, 11H-benzo[b]fluoren-11-one, benzanthrone, and 6H-benzo[cd]pyrene-6-one), four PAH diones (1,4-naphthoquinone, phenanthrenequinone, 5,12-naphthacenequinone, and benzo[a]pyrene-6,12-dione), and one PAH dicarboxylic acid anhydride (naphthalic anhydride) were identified. Seven additional compounds with mass spectra typical of OPAH were tentatively identified. OPAH were generally distributed among aerosol size fractions based on molecular weight. Compounds with molecular weights between 168 and 208 were ap proximately evenly distributed between the fine (aerodynamic diameter, D_p, 2 μm) particles. OPAH with molecular weights of 248 and greater were associated primarily with the fine aerosol fraction. Most OPAH were distributed with particle size in a broad, unimodal hump similar to the the distributions observed for PAH in the same samples. These results suggest that OPAH are initially associated with fine particles after formation by either combustion or gas phase photooxidation and then partition to larger particles by vaporization and sorption. Two OPAH were distributed in bimodal distributions with peaks at D_p ≈ 2 μm and D_p ≈ 2 μm. These bimodal distributions may be indicative of sorption behavior different from PAH and other OPAH

Measurement of C_(24)H_(14) Polycyclic Aromatic Hydrocarbons Associated with a Size-Segregated Urban Aerosol

Six-ring C_(24)H_(14) (MW 302) polycyclic aromatic hydrocarbons (PAH), some of which are potent mutagens, are present in urban aerosols. Size-segregated atmospheric aerosol samples from Boston, MA, were analyzed for C_(24)H_(14) PAH by gas chromatography/mass spectrometry. Eleven peaks were found with mass to charge ratios of 302; of these, eight were identified using authentic standards. Five of the peaks were quantified. For each of these five, the distributions with respect to particle size were bimodal with the majority of the mass associated with accumulation mode particles (0.3−1.0 μm) and a smaller fraction of the mass associated with ultrafine mode particles (0.09−0.14 μm). These distribu tions are similar to those observed for PAH of molecular weight 252−278 in the same sample but different from those of benzo[ghi]perylene (MW 276) and coronene (MW 300), which were associated to a greater degree with ultrafine particles. The data suggest that C_(24)H_(14) PAH repartition to larger particles by vaporization and sorption more rapidly than do benzo[ghi]perylene and coronene. The total concentration of C_(24)H_(14) PAH (1.5 ng/m^3) was comparable to that of benzo[a]pyrene in the same sample. Because of their mutagenicities, C_(24)H_(14) PAH may make a contribution to the genotoxicity of urban aerosols comparable to that of benzo[a]pyrene

Quantitative Analysis of Histone Modifications: Formaldehyde Is a Source of Pathological N6-Formyllysine That Is Refractory to Histone Deacetylases

Aberrant protein modifications play an important role in the pathophysiology of many human diseases, in terms of both dysfunction of physiological modifications and the formation of pathological modifications by reaction of proteins with endogenous electrophiles. Recent studies have identified a chemical homolog of lysine acetylation, N[superscript 6]-formyllysine, as an abundant modification of histone and chromatin proteins, one possible source of which is the reaction of lysine with 3′-formylphosphate residues from DNA oxidation. Using a new liquid chromatography-coupled to tandem mass spectrometry method to quantify all N[superscript 6]-methyl-, -acetyl- and -formyl-lysine modifications, we now report that endogenous formaldehyde is a major source of N[superscript 6]-formyllysine and that this adduct is widespread among cellular proteins in all compartments. N[superscript 6]-formyllysine was evenly distributed among different classes of histone proteins from human TK6 cells at 1–4 modifications per 10[superscript 4] lysines, which contrasted strongly with lysine acetylation and mono-, di-, and tri-methylation levels of 1.5-380, 5-870, 0-1400, and 0-390 per 10[superscript 4] lysines, respectively. While isotope labeling studies revealed that lysine demethylation is not a source of N[superscript 6]-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N[superscript 6]-formyllysine, with use of [[superscript 13]C,[superscript 2]H[subscript 2]]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources. Inhibitors of class I and class II histone deacetylases did not affect the levels of N[superscript 6]-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10%) with a peptide substrate containing the formyl adduct. These data suggest that N[superscript 6]-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary modification

Isoniazid hair concentrations in children with tuberculosis: a proof of concept study

Assessing treatment adherence and quantifying tuberculosis drug exposure among children is challenging. We undertook a “proof of concept” study to assess the drug concentrations of isoniazid in hair as a therapeutic drug monitoring tool. Children <12 years of age initiated on thrice-weekly treatment including isoniazid (10 mg/kg) for newly diagnosed tuberculosis were enrolled. Isoniazid concentrations in hair were measured using liquid chromatography-tandem mass spectrometry at 1, 2, 4 and 6 months after tuberculosis treatment initiation. We found that isoniazid hair concentrations in all children on thrice weekly isoniazid were detectable and displayed variability across a dynamic range

Synthesis and characterization of Sn‑doped TiO2 flm for antibacterial applications

Simple sol–gel method has been exploited to deposit Sn-doped TiO2 thin flms on glass substrates. The resultant coatings were characterized by X-ray difraction (XRD), UV–visible techniques (UV–Vis), Fourier transform infrared spectroscopy (FTIR), and photoluminescence analysis (PL). The XRD pattern reveals an increase in crystallite size of the prepared samples with the increasing doping concentration. A decrease in doping concentrating resulted in the decrease in bandgap values. The diferent chemical bonds on these flms were identifed from their FTIR spectra. The photoluminescence analysis shows an increase in the emission peak intensity with increasing dopant concentration, and this can be attributed to the efect created due to surface states. The prepared samples were tested as antibacterial agent toward both Gram-positive and Gram-negative bacteria like S.aureus (Staphylococcus aureus) and E.coli (Escherichia coli), respectively. The size of the inhibition zones indicates that the sample shows maximum inhibitory property toward E.coli when compared to S.aureus

Mechanisms and consequences of TGF-ß overexpression by podocytes in progressive podocyte disease

In patients with progressive podocyte disease, such as focal segmental glomerulosclerosis (FSGS) and membranous nephropathy, upregulation of transforming growth factor-ß (TGF-ß) is observed in podocytes. Mechanical pressure or biomechanical strain in podocytopathies may cause overexpression of TGF-ß and angiotensin II (Ang II). Oxidative stress induced by Ang II may activate the latent TGF-ß, which then activates Smads and Ras/extracellular signal-regulated kinase (ERK) signaling pathways in podocytes. Enhanced TGF-ß activity in podocytes may lead to thickening of the glomerular basement membrane (GBM) by overproduction of GBM proteins and impaired GBM degradation in podocyte disease. It may also lead to podocyte apoptosis and detachment from the GBM, and epithelial-mesenchymal transition (EMT) of podocytes, initiating the development of glomerulosclerosis. Furthermore, activated TGF-ß/Smad signaling by podocytes may induce connective tissue growth factor and vascular endothelial growth factor overexpression, which could act as a paracrine effector mechanism on mesangial cells to stimulate mesangial matrix synthesis. In proliferative podocytopathies, such as cellular or collapsing FSGS, TGF-ß-induced ERK activation may play a role in podocyte proliferation, possibly via TGF-ß-induced EMT of podocytes. Collectively, these data bring new mechanistic insights into our understanding of the TGF-ß overexpression by podocytes in progressive podocyte disease

Loss of expression of TGF-βs and their receptors in chronic skin lesions induced by sulfur mustard as compared with chronic contact dermatitis patients

<p>Abstract</p> <p>Background</p> <p>Sulfur mustard (SM) is a blister-forming agent that has been used as a chemical weapon. Sulfur mustard can cause damage in various organs, especially the skin, respiratory system, and eyes. Generally, the multiple complications of mustard gas result from its alkalizing potency; it reacts with cellular components like DNA, RNA, proteins, and lipid membranes.</p> <p>TGF-β is a multi-functional cytokine with multiple biological effects ranging from cell differentiation and growth inhibition to extracellular matrix stimulation, immunosuppression, and immunomodulation. TGF-β has 3 isoforms (TGF-β 1, 2, 3) and its signaling is mediated by its receptors: R1, R2 and intracellular Smads molecules.</p> <p>TGF-β has been shown to have anti-inflammatory effects. TGF-βs and their receptors also have an important role in modulation of skin inflammation, proliferation of epidermal cells, and wound healing, and they have been implicated in different types of skin inflammatory disorders.</p> <p>Methods</p> <p>Seventeen exposed SM individuals (48.47 ± 9.3 years), 17 chronic dermatitis patients (46.52 ± 14.6 years), and 5 normal controls (44.00 ± 14.6 years) were enrolled in this study.</p> <p>Evaluation of TGF-βs and their receptors expressions was performed by semiquantitative RT-PCR. Only TGF1was analyzed immunohistochemically.</p> <p>Results</p> <p>Our results showed significant decreases in the expression percentages of TGF-β 1, 2 and R1, R2 in chemical victims in comparison with chronic dermatitis and normal subjects and significant decreases in the intensity of R1 and R2 expressions in chemical victims in comparison with chronic dermatitis and normal controls. (P value < 0.05)</p> <p>Conclusions</p> <p>TGF-βs and their receptors appear to have a noticeable role in chronic inflammatory skin lesions caused by sulfur mustard.</p