Modulation of the chemokines KC and MCP-1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice

Activation of the aryl hydrocarbon receptor (AhR) by TCDD may lead to the induction of proinflammatory cytokines in various cell types and organs such as liver leading to active chronic inflammation. Here we studied the expression of the chemokines keratinocyte chemoattractant (KC) and monocyte chemoattractant protein 1 (MCP-1) in different organs of mice after exposure to TCDD. TCDD exposure led to an early and clear induction of KC in liver and spleen on day 1 which was sustained over a period of 10 days. The level of MCP-1 mRNA was induced by TCDD on day 1 in spleen, lung, kidney, and liver, which was further increased at day 7. Increase of KC and MCP-1 at day 7 in liver, thymus, kidney, adipose, and heart was associated with elevated levels of the macrophage marker F4/80, indicating the infiltration of macrophages in these organs. Induction of KC requires a functional AhR since mice with a mutation in the AhR nuclear localization domain (AhR(nls)) were found to be resistant to TCDD-induced expression of KC. These results are the first showing the induction of the chemokines KC and MCP-1 in multiple organs of mice associated with an increase of the macrophage marker F4/80 indicating the involvement in TCDD's inflammatory response like infiltration of macrophages.     

Beta-galactosidase (Escherichia coli) has a second catalytically important Mg2+ site

It is shown here that Escherichia coli beta-galactosidase has a second Mg2+ binding site that is important for activity. Binding of Mg2+ to the second site caused the k(cat) (with oNPG as the substrate) to increase about 100 s(-1); the Km was not affected. The Kd for binding the second Mg2+ is about 10(-4)M. Since the concentration of free Mg2+ in E. coli is about 1-2 mM, the second site is physiologically significant. Non-polar substitutions (Ala or Leu) for Glu-797, a residue in an active site loop, eliminated the k(cat) increase. This indicates that the second Mg2+ site is near to Glu-797. The Ki values of transition state analogs were decreased by small but statistically significant amounts when the second Mg2+ site was occupied and Arrhenius plots showed that less entropic activation energy is required when the second site is occupied. These inhibitor and temperature results suggest that binding of the second Mg2+ helps to order the active site for stabilization of the transition state.     

The role of peroxiredoxin III on late stage of proerythrocyte differentiation

Peroxiredoxin III (Prdx III), the mitochondrial peroxidase, was preferentially expressed in murine erythroleukemia (MEL) cells. However, the mechanisms by which Prdx III regulates erythroid differentiation are unknown. In this study, K562 cells were differentiated by Ara-C treatment, and Prdx III was dramatically increased until day 5. We also investigated Prdx III expression pattern on in vitro erythropoiesis of human CD34(+) cells. When human CD34(+) cells became proerythrocyte on day 7, Prdx III was diminished, and then augmented on day 12. We established the stable sublines of Prdx III overexpression (O/E), and dominant-negative (D/N). The intracellular ROS level of Prdx III O/E cell line was lower than D/N stable cell lines. Moreover, Prdx III O/E cell line was placed in G1-arrest, but not D/N cell lines. Finally, the expression level of beta-globin and GATA-1 was dramatically increased in Prdx III O/E cell line.     

Exploring a series of monoethynylfluorenes as alkynylating reagents for mercuric ion: Synthesis, spectroscopy, photophysics and potential use in mercury speciation

New luminescent mononuclear mercury(II) mono- and dialkynylated complexes containing substituted fluorene and fluorenone units [R-CC-HgCH₃] and [R-CC-Hg-CC-R] (R = 9,9-dialkylfluorene-2-yl and fluoren-9-one-2-yl; alkyl = H, ethyl, hexyl, octyl, hexadecyl) were prepared in good yields by mercuration of terminal acetylene R-CCH with CH₃HgCl and HgCl₂ at room temperature via the dehydrohalogenation reaction. The structures of these organomercurial compounds were characterized by IR and NMR spectroscopies, elemental analysis and FAB mass spectrometry. Their optical and photoluminescence spectra were also studied. The structural features of one complex was elucidated by X-ray crystallography in which there is an indication of weak mercuriophicity among the molecules in the solid state. A new protocol is developed for derivatization of inorganic mercury(II) ion into dialkynyl mercury(II) compounds followed by the ready extraction into dichloromethane, which can be analyzed by HPLC technique using UV detection. These results have important implications in the development of analytical procedures for the determination of mercuric ion in aqueous solutions.     

Small conformationally restricted piperidine N-arylsulfonamides as orally active gamma-secretase inhibitors

The design and development of a new class of small 2,6-disubstituted piperidine N-arylsulfonamide gamma-secretase inhibitors is reported. Lowering molecular weight including the use of conformational constraint led to compounds with less CYP 3A4 liability compared to early leads. Compounds active orally in lowering Abeta levels in Tg CRND8 mice were identified as potential treatments for Alzheimer's disease.     

Amino-caprolactam derivatives as gamma-secretase inhibitors

A series of amino-caprolactam sulfonamides were developed from a screening hit. Compounds with good in vitro and in vivo gamma-secretase activity are reported.     

Are clownfish groups composed of close relatives? An analysis of microsatellite DNA variation in Amphiprion percula

A central question of evolutionary ecology is: why do animals live in groups? Answering this question requires that the costs and benefits of group living are measured from the perspective of each individual in the group. This, in turn, requires that the group's genetic structure is elucidated, because genetic relatedness can modulate the individuals’ costs and benefits. The clown anemonefish, Amphiprion percula, lives in groups composed of a breeding pair and zero to four nonbreeders. Both breeders and nonbreeders stand to gain by associating with relatives: breeders might prefer to tolerate nonbreeders that are relatives because there is little chance that relatives will survive to breed elsewhere; nonbreeders might prefer to associate with breeders that are relatives because of the potential to accrue indirect genetic benefits by enhancing anemone and, consequently, breeder fitness. Given the potential benefits of associating with relatives, we use microsatellite loci to investigate whether or not individuals within groups of A. percula are related. We develop seven polymorphic microsatellite loci, with a number of alleles (range 2–24) and an observed level of heterozygosity (mean = 0.5936) sufficient to assess fine‐scale genetic structure. The mean coefficient of relatedness among group members is 0.00 ± 0.10 (n = 9 groups), and there are no surprising patterns in the distribution of pairwise relatedness. We conclude that A. percula live in groups of unrelated individuals. This study lays the foundation for further investigations of behavioural, population and community ecology of anemonefishes which are emerging as model systems for evolutionary ecology in the marine environment.     

In vivo selection for the directed evolution of L-rhamnulose aldolase from L-rhamnulose-1-phosphate aldolase (RhaD)

Dihydroxyacetone phosphate (DHAP)-dependent aldolases have been widely used for organic synthesis. The major drawback of DHAP-dependent aldolases is their strict donor substrate specificity toward DHAP, which is expensive and unstable. Here we report the development of an in vivo selection system for the directed evolution of the DHAP-dependent aldolase, L-rhamnulose-1-phosphate aldolase (RhaD), to alter its donor substrate specificity from DHAP to dihydroxyacetone (DHA). We also report preliminary results on mutants that were discovered with this screen. A strain deficient in the L-rhamnose metabolic pathway in Escherichia coli (DeltarhaDAB, DE3) was constructed and used as a selection host strain. Co-expression of L-rhamnose isomerase (rhaA) and rhaD in the selection host did not restore its growth on minimal plate supplemented with L-rhamnose as a sole carbon source, because of the lack of L-rhamnulose kinase (RhaB) activity and the inability of WT RhaD aldolase to use unphosphorylated L-rhamnulose as a substrate. Use of this selection host and co-expression vector system gives us an in vivo selection for the desired mutant RhaD which can cleave unphosphorylated L-rhamnulose and allow the mutant to grow in the minimal media. An error-prone PCR (ep-PCR) library of rhaD gene on the co-expression vector was constructed and introduced into the rha-mutant, and survivors were selected in minimal media with l-rhamnose (MMRha media). An initial round of screening gave mutants allowing the selection strain to grow on MMRha plates. This in vivo selection system allows rapid screening of mutated aldolases that can utilize dihydroxyacetone as a donor substrate.     

Improving simvastatin bioconversion in Escherichia coli by deletion of bioH

Simvastatin is an important cholesterol lowering compound and is currently synthesized from the natural product lovastatin via multistep chemical synthesis. We have previously reported the use of an Escherichia coli strain BL21(DE3)/pAW31 as the host for whole-cell biocatalytic conversion of monacolin J acid to simvastatin acid. During fermentation and bioconversion, unknown E. coli enzyme(s) hydrolyzed the membrane permeable thioester substrate dimethylbutyryl-S-methyl mercaptopropionate (DMB-S-MMP) to the free acid, significantly decreased the efficiencies of the whole-cell bioconversion and the downstream purification steps. Using the Keio K-12 Singe-Gene Knockout collection, we identified BioH as the sole enzyme responsible for the observed substrate hydrolysis. Purification and reconstitution of E. coli BioH activity in vitro confirmed its function. BioH catalyzed the rapid hydrolysis of DMB-S-MMP with kcat and Km values of 260+/-45 s(-1) and 229+/-26 microM, respectively. This is in agreement with previous reports that BioH can function as a carboxylesterase towards fatty acid esters. YT2, which is a delta bioH mutant of BL21(DE3), did not hydrolyze DMB-S-MMP during prolonged fermentation and was used as an alternative host for whole-cell biocatalysis. The rate of simvastatin acid synthesis in YT2 was significantly faster than in BL21(DE3) and 99% conversion of 15 mM simvastatin acid in less than 12 h was achieved. Furthermore, the engineered host required significantly less DMB-S-MMP to be added to accomplish complete conversion. Finally, simvastatin acid synthesized using YT2 can be readily purified from fermentation broth and no additional steps to remove the hydrolyzed dimethylbutyryl-S-mercaptopropionic acid is required. Together, the proteomic and metabolic engineering approaches render the whole-cell biocatalytic process more robust and economically attractive.     

Effect of luteinizing hormone (LH), pregnancy-specific protein B (PSPB), or arachidonic acid (AA) on secretion of progesterone and prostaglandins (PG) E (PGE; PGE(1) and PGE(2)) and F(2alpha) (PGF(2alpha)) by ovine corpora lutea of the estrous cycle or pregnancy in vitro

LH regulates luteal progesterone secretion during the estrous cycle in ewes and cows. However, PGE, not LH, stimulated ovine luteal progesterone secretion in vitro at day 90 of pregnancy and at day 200 in cows. The hypophysis is not obligatory after day 50 nor the ovaries after day 55 to maintain pregnancy in ewes. LH has been reported to regulate ovine placental PGE secretion up to day 50 of pregnancy and by pregnancy-specific protein B (PSPB) after day 50 of pregnancy. The objective of this experiment was to determine if and when a switch from LH to PGE occurred as the luteotropin regulating luteal progesterone secretion during pregnancy in ewes. Ovine luteal tissue slices of the estrous cycle (days 8, 11, 13, and 15) or pregnancy (days 8, 11, 13, 15, 20, 30, 40, 50, 60, and 90) were incubated in vitro with vehicle, LH, AA (precursor to PGE(2) and PGF(2alpha) synthesis), or PSPB in M199 for 4 h and 8 h. Concentrations of progesterone in jugular venous plasma of bred ewes increased (P< or =0.05) after day 50 and continued to increase through day 90. Secretion of progesterone by luteal tissue of non-bred ewes on days 8, 11, 13 and 15 and by bred ewes on days 8, 11, 13, 15, 20, 30, 40, and 50 was increased (P< or =0.05) by LH, but not by luteal tissue from pregnant ewes after day 50 (P> or =0.05). LH-stimulated progesterone secretion by luteal tissue from day 15 bred ewes was greater (P< or =0.05) than day 15 luteal tissue from non-bred ewes. Concentrations of progesterone in media were increased (P< or =0.05) when luteal tissue from pregnant ewes on day 50, 60, or 90 were incubated with AA or PSPB. Concentrations of PGE in media of non-bred ewes on days 8, 11, 13, or 15 and bred ewes on days 8 and 11 did not differ (P> or =0.05). Concentrations of PGE were increased (P< or =0.05) in media by luteal slices from bred ewes on days 13, 15, 20, 30, 40, 50, 60, and 90 of vehicle, LH, AA or PSPB-treated ewes. In addition, PSPB increased (P< or =0.05) PGE in media by luteal slices from pregnant ewes only on days 40, 50, 60, and 90. Concentrations of PGF(2alpha) were increased in media (P<0.05) of vehicle, AA, LH, or PSPB-treated luteal tissue from non-bred ewes and bred ewes on day 15 and by luteal tissue from bred ewes on days 20 and 30 after which concentrations of PGF(2alpha) in media declined (P< or =0.05) and did not differ (P> or =0.05) from non-bred or bred ewes on days 8, 11, or 13. It is concluded that LH regulates luteal progesterone secretion during the estrous cycle of non-bred ewes and up to day 50 of pregnancy, while only PGE regulates luteal progresterone secretion by ovine corpora lutea from days 50 to 90 of pregnancy. In addition, PSPB appears to regulate luteal secretion of progesterone from days 50 to 90 of pregnancy through stimulation of PGE secretion by ovine luteal tissue.