HEHEHE-tagged affibody molecule may be purified by IMAC, is conveniently labeled with [⁹⁹(m)Tc(CO)₃](+), and shows improved biodistribution with reduced hepatic radioactivity accumulation

Affibody molecules are a class of small (ca. 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging of therapeutic targets in vivo. A hexahistidine tag at the N-terminus streamlines development of new imaging probes by enabling facile purification using immobilized metal ion affinity chromatography (IMAC), as well as convenient [??(m)Tc(CO)?](+)-labeling. However, previous studies in mice have demonstrated that Affibody molecules labeled by this method yield higher liver accumulation of radioactivity, compared to the same tracer lacking the hexahistidine tag and labeled by an alternative method. Two variants of the HER2-binding Affibody molecule Z(HER)?(:)??? were made in an attempt to create a tagged tracer that could be purified by immobilized metal affinity chromatography, yet would not result in anomalous hepatic radioactivity accumulation following labeling with [??(m)Tc(CO)?](+). In one construct, the hexahistidine tag was moved to the C-terminus. In the other construct, every second histidine residue in the hexahistidine tag was replaced by the more hydrophilic glutamate, resulting in a HEHEHE-tag. Both variants, denoted Z(HER)?(:)???-H? and (HE)?-Z(HER)?(:)???, respectively, could be efficiently purified using IMAC and stably labeled with [??(m)Tc(CO)?](+) and were subsequently compared with the parental H?-Z(HER)?(:)??? having an N-terminal hexahistidine tag. All three variants were demonstrated to specifically bind to HER2-expressing cells in vitro. The hepatic accumulation of radioactivity in a murine model was 2-fold lower with [??(m)Tc(CO)?](+)-Z(HER2:342)-H? compared to [??(m)Tc(CO)?](+)-H?-Z(HER)?(:)???, and more than 10-fold lower with [??(m)Tc(CO)?](+)-(HE)?-Z(HER)?(:)???. These differences translated into appreciably superior tumor-to-liver ratio for [??(m)Tc(CO)?](+)-(HE)?-Z(HER)?(:)??? compared to the alternative conjugates. This information might be useful for development of other scaffold-based molecular imaging probes.     

Synthesis and biological evaluation of tetracyclic fluoroquinolones as antibacterial and anticancer agents

A simple and efficient synthesis of 6-fluoro-4-oxopyrido[2,3-a]carbazole-3-carboxylic acids (13a–e) and a structurally related 6-fluoro-4-oxothieno[2′,3′:4,5]pyrrolo[3,2-h]quinoline (13f) was achieved via Stille arylation of 7-chloro-6-fluoro-8-nitro-4-oxoquinoline-3-carboxylate and a subsequent microwave-assisted phosphite-mediated Cadogan reaction. The new compounds were tested for their in vitro antimicrobial and antiproliferative activity. The ability of 13a–f to inhibit the activity of DNA gyrase and topoisomerase IV was also investigated. The thieno isostere (13f) emerged as the most active antibacterial, while the 9-fluoro derivative (13e) was the most potent against multidrug-resistant staphylococci. Compounds 13a, 13c–f displayed growth inhibition against MCF-7 breast tumor and A549 non-small cell lung cancer cells coupled with an absence of cytotoxicity toward normal human-derm fibroblasts (HuDe). Compound 13e was the most active anticancer against MCF-7 cells, with greater potency than ellipticine (IC₅₀ 0.8 and 1.6 μM, respectively). The most active compounds in this series show promise as dual acting anticancer and antibacterial chemotherapeutics.     

Aerobic Biodegradation of N-Nitrosodimethylamine by the Propanotroph Rhodococcus ruber ENV425

The propanotroph Rhodococcus ruber ENV425 was observed to rapidly biodegrade N-nitrosodimethylamine (NDMA) after growth on propane, tryptic soy broth, or glucose. The key degradation intermediates were methylamine, nitric oxide, nitrite, nitrate, and formate. Small quantities of formaldehyde and dimethylamine were also detected. A denitrosation reaction, initiated by hydrogen atom abstraction from one of the two methyl groups, is hypothesized to result in the formation of n-methylformaldimine and nitric oxide, the former of which decomposes in water to methylamine and formaldehyde and the latter of which is then oxidized further to nitrite and then nitrate. Although the strain mineralized more than 60% of the carbon in [¹⁴C]NDMA to ¹⁴CO₂, growth of strain ENV425 on NDMA as a sole carbon and energy source could not be confirmed. The bacterium was capable of utilizing NDMA, as well as the degradation intermediates methylamine and nitrate, as sources of nitrogen during growth on propane. In addition, ENV425 reduced environmentally relevant microgram/liter concentrations of NDMA to <2 ng/liter in batch cultures, suggesting that the bacterium may have applications for groundwater remediation.N-Nitrosodimethylamine (NDMA) is a potent carcinogen that has recently been detected in groundwater, wastewater, and drinking water (1, 2, 17, 18). It forms as a disinfection byproduct in wastewater and drinking water treated with chloramine and other disinfectants (17, 18, 43). NDMA has also been found to be present in aquifers at several military sites that have used 1,1-dimethylhydrazine, a component of liquid rocket propellant that contained NDMA as an impurity (6, 9). Although there is presently no federal maximum contaminant level for NDMA in drinking water, a risk assessment conducted by the U.S. Environmental Protection Agency suggested that concentrations as low as 0.7 ng/liter can increase lifetime cancer risk by 1 × 10⁻⁶ (34). In addition, California currently has a 10 ng/liter notification level for NDMA concentrations in drinking water and has recently recommended an even lower public health goal of 3 ng/liter (3, 20). Thus, the presence of even trace concentrations of this chemical in drinking water represents a potential public health concern.The rates and extents of NDMA biodegradation in natural environments, including surface water, sludges, and soils, are highly variable. In some studies, the compound has been reported to be recalcitrant or only partially biodegraded (16, 30, 31); in others, fairly rapid and extensive biodegradation has been previously observed (2, 13, 22, 40). Few studies have been conducted to examine NDMA biodegradation in groundwater. However, the persistence of NDMA derived originally from 1,1-dimethylhydrazine-based rocket fuel over decades in some groundwater aquifers (e.g., Rocky Mountain Arsenal, CO; former Air Force Plant PJKS, CO; and Aerojet Superfund Site, CA) suggests that this molecule can be very recalcitrant (8, 9, 35). At sites where biodegradation has been observed, the organisms responsible and the microbial degradation pathways are largely unknown.The metabolism of NDMA and other nitrosamines by mammals has received extensive study. NDMA requires metabolic activation to the methyldiazonium ion (a strong alkylating agent) to exert its genotoxic effects (1, 19, 34). This activation reaction is catalyzed by specific isozymes of the cytochrome P-450-dependent mixed-function oxidase system and proceeds through an initial α-hydroxylation reaction. Alternately, NDMA can be oxidized by the P-450 system via a denitrosation route, which does not result in the formation of a highly carcinogenic intermediate (11, 28, 37).The bacterial transformation of NDMA has not been studied in significant detail. Several bacteria expressing broad-specificity monooxygenase enzymes have been reported to degrade NDMA via cometabolism. These include the propanotrophs Rhodococcus sp. strain RHA1 (25, 26) and Rhodococcus ruber ENV425 (29) as well as Mycobacterium vaccae JOB5 (25), the methanotroph Methylosinus trichosporium OB3b (42), and the toluene oxidizer Pseudomonas mendocina KR1 (7). We recently characterized the pathway of NDMA transformation used by P. mendocina KR1, a bacterium that utilizes the enzyme toluene-4-monooxygenase (T4MO) to cometabolically degrade NDMA and other anthropogenic pollutants (7, 38). The pathway of NDMA transformation by KR1 differs from the two pathways described for mammals. A majority of the NDMA metabolized by T4MO in this strain is oxidized to N-nitrodimethylamine (NTDMA) and then further to N-nitromethylamine (NTMA), which accumulates as a terminal product (7).In this report, we describe the pathway used by the propanotroph R. ruber ENV425 to catabolize NDMA. This strain was originally isolated from turf soil, where propane was used as the sole carbon source, and was previously reported to oxidize methyl tertiary-butyl ether and other gasoline oxygenates (27). Our data show that the pathway of NDMA degradation mediated by strain ENV425 differs from that mediated by P. mendocina KR1. Rather, the pathway used for transformation of NDMA by ENV425 appears to be similar to the denitrosation pathway catalyzed by various P-450 isozymes in mammals, resulting in the production of nitric oxide (NO), nitrite, nitrate, formaldehyde, formate, and methylamine (MA) (11, 12, 28, 39). A significant fraction of the carbon in the NDMA molecule was released as CO₂ by strain ENV425, although growth on NDMA could not be confirmed. However, the bacterium was observed to utilize NDMA as well as the NDMA-degradation intermediates MA and nitrate as sources of nitrogen during growth on propane as a sole carbon and energy source.     

Siderophore mediated iron(III) uptake in Gliocladium virens. 1. Properties of cis-fusarinine, trans-fusarinine, dimerum acid, and their ferric complexes

Gliocladium virens (ATCC 24290) produces two monohydroxamates (cis- and trans-fusarinine) and a dihydroxamate (dimerum acid) as the major siderophores in the culture filtrate. This fungus also produces minor quantities of three trihydroxamates (the deferri forms of ferricrocin, coprogen B, and coprogen). Structural features of the free ligands and the metal complexed forms of cis-fusarinine (cF), trans-fusarinine (tF), and dimerum acid (DA) have been investigated using electronic (visible), circular dichroism (CD), and NMR spectroscopy. In aqueous solution, in the pH range of 6.5-8.0, all of the ferric complexes of cF (and tF) exist as 3:1 chelates. Fe(cF)3 [or Fe(tF)3] forms both lambda and delta coordination isomers, but the former in a slight excess. DA forms a 3:2 ferric complex in the pH range of 5.0-8.0. Iron coordination in Fe2(DA)3 is predominantly delta. DA ligands in Ga2(DA)3 exist as two different conformers at a ratio of 2:1. In mixed solution cF, tF, and DA form a large number of homogeneous and heterogeneous Fe(III) chelates.     

Hydrogen peroxide generation and antioxidant enzyme activities in the leaves and roots of wheat cultivars subjected to long-term soil drought stress

Photosynthesis Research - The dynamics of the activity of catalase, ascorbate peroxidase, guaiacol peroxidase, and benzidine peroxidase, as well as the level of hydrogen peroxide in the vegetative...     

Serum IL-10 involved in morphine tolerance development during adjuvant-induced arthritis

Opioid receptors play an important role in modulation of hyperalgesia in inflamed tissues, but chronic morphine application induces such side effects as tolerance. There is near communications between cytokines and mu opioid receptor expression. This study was aimed to assess the role of serum IL-10 in morphine tolerance development during adjuvant-induced arthritis. Adjuvant arthritis (AA) was induced on day 0 by single injection of Complete Freund’s Adjuvant (CFA) into the rats’ hindpaw. Hyperalgesia, edema, and spinal mu opioid receptor (mOR) variations were assessed on 0, 7, 14, and 21 days of the study. For assessment of the morphine tolerance development, morphine effective dose (4 mg/kg) was administered from the 14th day after CFA injection and continued until the morphine post-dose paw withdrawal latency (PWL); it did not significantly differ from the baseline. For assessment of the effects of IL-10 on tolerance induction, a neutralizing dose (ND50) of anti-IL-10 was administered daily during different stages of the study. AA induction in the right hindpaw of rats resulted in unilateral inflammation and hyperalgesia within 21 days of the study. Anti-IL-10 antibody administration in the AA rats induced marked elevation of hyperalgesia compared to the AA control group. Our data also indicated that morphine effective anti-hyperalgesic dose significantly decreased in the AA rats compared to the control group, which this symptom was aligned with spinal mu opioid receptor (mOR) expression increase during AA. Moreover, there was a significant difference in morphine tolerance induction between the AA and control rats, and our results also demonstrated that IL-10 played an important role in tolerance-induction process. It can be concluded that morphine tolerance slowly progressed when administered morphine effective dose was reduced during AA chronic inflammation. On the other hand, it seems that increased level of serum IL-10 may affect morphine tolerance development during inflammation.     

Toxicity of Fe2O3 nanoparticles on human blood lymphocytes

Magnetic nanoparticles (NPs) are used to a large extent in the targeted delivery of therapeutic agents. In this study, we aimed to investigate the possible toxicity of Fe₂O ₃ NPs on human cells, including blood lymphocytes. We isolated blood lymphocytes from healthy humans using Ficoll polysaccharide and subsequently by gradient centrifugation. Then, the toxicity parameters, including cell viability, reactive oxygen species (ROS) formation, lipid peroxidation, cellular glutathione (GSH) level, mitochondrial and lysosomal damage, were measured in blood lymphocytes after exposure to Fe ₂O ₃ NPs. Our results indicated that Fe ₂O ₃ NPs significantly (dependent on concentration) reduced the cell viability, and the IC ₅₀ was determined to be 1 mM. With increasing concentrations, we found that Fe ₂O ₃ NPs–induced cell toxicity was associated with a significant increase in intracellular ROS and loss of mitochondrial membrane potential and lysosomal membrane leakiness. Consequently, these NPs at different concentrations affect GSH level and cause oxidative stress in human lymphocytes.