Characterization of hematopoietic potential of mesenchymal stem cells

Mesenchymal and hematopoietic tissues are important reservoirs of adult stem cells. The potential of tissue resident mesenchymal stem cells (MSCs) to differentiate into cells of mesodermal and ectodermal lineages has been reported previously. We examined the hypothesis that adherent adipose tissue resident mesenchymal stem cells (ASCs) are capable of generating cells with hematopoietic characteristics. When cultured in differentiation media, clonally isolated ASCs develop into cells with hematopoietic attributes. The hematopoietic differentiated cells (HD) express early hematopoietic (c‐kit, PROM1, CD4) as well as monocyte/macrophage markers (CCR5, CD68, MRC1, CD11b, CSF1R). Additionally, HD cells display functional characteristics of monocyte/macrophages such as phagocytosis and enzymatic activity of α‐Naphthyl Acetate Esterase. HD cells are also responsive to stimulation by IL‐4 and LPS as shown by increased CD14 and HLA‐DRB1 expressions and release of IL‐2, IL10, and TNF. Taken together, this study characterizes the potential of ASCs to generate functional macrophages in vitro, and therefore paves way for their possible use in cell therapy applications. J. Cell. Physiol. 225: 888–897, 2010. © 2010 Wiley‐Liss, Inc.     

Novel 3-fluoro-6-methoxyquinoline derivatives as inhibitors of bacterial DNA gyrase and topoisomerase IV

Novel (non-fluoroquinolone) inhibitors of bacterial type II topoisomerases (NBTIs) are an emerging class of antibacterial agents. We report an optimized series of cyclobutylaryl-substituted NBTIs. Compound 14 demonstrated excellent activity both in vitro (S. aureus MIC₉₀ = 0.125 μg/mL) and in vivo (systemic and tissue infections). Enhanced inhibition of Topoisomerase IV correlated with improved activity in S. aureus strains with mutations conferring resistance to NBTIs. Compound 14 also displayed an improved hERG IC₅₀ of 85.9 μM and a favorable profile in the anesthetized guinea pig model.     

Regulation of the Epithelial Na+ Channel by Membrane Tension

The sensitivity of αβγ rat epithelial Na⁺ channel (rENaC) to osmotically or mechanically induced changes of membrane tension was investigated in the Xenopus oocyte expression system, using both dual electrode voltage clamp and cell-attached patch clamp methodologies. ENaC whole-cell currents were insensitive to mechanical cell swelling caused by direct injection of 90 or 180 nl of 100-mM KCl. Similarly, ENaC whole-cell currents were insensitive to osmotic cell swelling caused by a 33% decrease of bathing solution osmolarity. The lack of an effect of cell swelling on ENaC was independent of the status of the actin cytoskeleton, as ENaC remained insensitive to osmotic and mechanical cell swelling in oocytes pretreated with cytochalasin B for 2–5 h. This apparent insensitivity of ENaC to increased cell volume and changes of membrane tension was also observed at the single channel level in membrane patches subjected to negative or positive pressures of 5 or 10 in. of water. However, and contrary to the lack of an effect of cell swelling, ENaC currents were inhibited by cell shrinking. A 45-min incubation in a 260-mosmol solution (a 25% increase of solution osmolarity) caused a decrease of ENaC currents (at −100 mV) from −3.42 ± 0.34 to −2.02 ± 0.23 μA (n = 6). This decrease of current with cell shrinking was completely blocked by pretreatment of oocytes with cytochalasin B, indicating that these changes of current are not likely related to a direct effect of cell shrinking. We conclude that αβγ rENaC is not directly mechanosensitive when expressed in a system that can produce a channel with identical properties to those found in native epithelia.     

Curtius Rearrangement Using Ultrasonication: Isolation of Isocyanates of Fmoc-Amino Acids and Their Utility for the Synthesis of Dipeptidyl Ureas

An efficient conversion of N^α-[(9–fluorenylmethyl)oxy]carbonyl (Fmoc) amino acid azides to the corresponding isocyanates using ultrasound is described. The Curtius rearrangement was accomplished using acid azides in toluene solution as well as solid powder at room temperature. All isocyanates synthesized have been obtained as crystalline solids and were characterized. Coupling of isocyanates with amino acid methyl ester hydrochloride salts in presence of N-methylmorpholine (NMM) resulted in Fmoc-protected dipeptidyl urea esters, which have been well characterized by ¹H NMR, ¹³C NMR and mass spectrometry.     

The slow S to M rise of chlorophyll a fluorescence reflects transition from state 2 to state 1 in the green alga Chlamydomonas reinhardtii

Photosynthesis Research - The green alga Chlamydomonas (C.) reinhardtii is a model organism for photosynthesis research. State transitions regulate redistribution of excitation energy between...     

Protection of rice against Nilaparvata lugens by direct toxicity of sodium selenate

Nilaparvata lugens is one of the most notorious pest insects of cultured rice, and outbreaks of N. lugens cause high economic losses each year. While pest control by chemical pesticides is still the standard procedure for treating N. lugens infections, excessive use of these insecticides has led to the emergence of resistant strains and high pesticide residues in plants for human consumption and the environment. Therefore, novel and environment-friendly pest control strategies are needed. In previous studies, selenium was shown to protect selenium-accumulating plants from biotic stress. However, studies on nonaccumulator (crop) plants are lacking. In this study, rice plants (Oryza sativa, Nipponbare) were treated with sodium selenate by seed priming and foliar spray and then infested with N. lugens. Brown planthoppers feeding on these plants showed increased mortality compared to those feeding on control plants. Treatment of the plants with sodium selenate did not affect the enzymes involved in the biosynthesis of the plant stress hormones jasmonic acid and salicylic acid, suggesting that the observed insect mortality cannot be attributed to the activation of these hormonal plant defenses. Feeding assays using an artificial diet supplemented with sodium selenate revealed direct toxicity toward N. lugens. With a low concentration of 6.5 ± 1.5 µM sodium selenate, half of the insects were killed after 3 days. In summary, sodium selenate treatment of plants can be used as a potential alternative pest management strategy to protect rice against N. lugens infestation through direct toxicity.     

The catalase activity of diiron adenine deaminase

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn²⁺ before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO₄. Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe^II/Fe^II]-ADE catalyzed the conversion of H₂O₂ to O₂ and H₂O. The values of k_cat and k_cat/K_m for the catalase activity are 200 s⁻¹ and 2.4 × 10⁴ M⁻¹ s⁻¹, respectively. [Fe^II/Fe^II]-ADE underwent more than 100 turnovers with H₂O₂ before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g_ave = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H₂O₂ by [Fe^II/Fe^II]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.     

Peroxynitrite has potent pulmonary vasodilator activity in the rat

Peroxynitrite (PN) worsens pathological conditions associated with oxidative stress. However, beneficial effects have also been reported. PN has been shown to demonstrate vasodilator as well as vasoconstrictor properties that are dependent upon the experimental conditions and the vascular bed studied. PN-induced vascular smooth muscle relaxation may involve the formation of nitric oxide (NO) donors. The present results show that PN has significant vasodilator activity in the pulmonary and systemic vascular beds, and that responses to PN were not attenuated by L-penicillamine (L-PEN), a PN scavenger, whereas responses to sodium nitroprusside (SNP) were decreased. PN had a small inhibitory effect on decreases in arterial pressure in response to the NO donors diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) and S-nitrosoglutathione (GSNO). PN partially reversed hypoxic pulmonary vasoconstriction. PN responses were attenuated by the soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and responses to PN and the PN precursor, 3-morpholinosydnonimine (SIN-1), were different. These data show that PN has potent pulmonary vasodilator activity in the rat, and provide evidence that a PN interaction with S-nitrosothiols is not the major mechanism mediating the response. These data suggest that responses to PN are mediated by the activation of sGC, and that PN has a small inhibitory effect on NO responses.     

Crystal structure of trehalose-6-phosphate phosphatase-related protein: biochemical and biological implications

We report here the crystal structure of a trehalose-6-phosphate phosphatase-related protein (T6PP) from Thermoplasma acidophilum, TA1209, determined by the dual-wavelength anomalous diffraction (DAD) method. T6PP is a member of the haloacid dehalogenase (HAD) superfamily with significant sequence homology with trehalose-6-phosphate phosphatase, phosphoserine phosphatase, P-type ATPases and other members of the family. T6PP possesses a core domain of known alpha/beta-hydrolase fold, characteristic of the HAD family, and a cap domain, with a tertiary fold consisting of a four-stranded beta-sheet with two alpha-helices on one side of the sheet. An active-site magnesium ion and a glycerol molecule bound at the interface between the two domains provide insight into the mode of substrate binding by T6PP. A trehalose-6-phosphate molecule modeled into a cage formed by the two domains makes favorable interactions with the protein molecule. We have confirmed that T6PP is a trehalose phosphatase from amino acid sequence, three-dimensional structure, and biochemical assays.     

Influence of zinc on cardiac and serum biochemical parameters in rabbits

The pattern of lipid profiles and organic constituents of cardiac and serum tissues of rabbits were studied on treatment with cholesterol, zinc and zinc + cholesterol. Total carbohydrate and total protein levels were decreased with elevated lipid levels in cholesterol fed rabbits. However, the zinc and cholesterol + zinc fed rabbits showed decreased lipid fractions in cardiac and serum tissues leading to reduced atherosclerotic process in rabbits. These results suggest that the zinc is acting as a hypolipidaemic and anti atherogenic agent in experimental rabbits.