Adrenergic modulation of splenic macrophage cytokine release in polymicrobial sepsis

Enhanced adrenergic stimulation and catecholamine release are important components of the pathophysiology of sepsis. Under physiological conditions, adrenergic stimulation has been shown to be a negative regulator of proinflammatory cytokine production through increasing IL-10 production. Here we have investigated if adrenergic stimulation similarly inhibits TNF- and IL-6 production by splenic macrophages isolated from a polymicrobial sepsis model. Male B₆D₂F₁ mice were subjected to sham (S), laparotomy (Lap), and cecal ligation and puncture (CLP) under anesthesia. Splenic macrophages were isolated 72 h after the initial injury and were stimulated with endotoxin (LPS) in the presence and absence of epinephrine. Compared with S and Lap, splenic macrophages from the CLP group produced significantly less TNF- and IL-6 and more IL-10 when stimulated with LPS. Macrophage cultures from CLP animals incubated with either epinephrine or IL-10 for 2 h had significantly reduced TNF- and IL-6 release in response to LPS. However, similar cultures pretreated with IL-10 antibody before the addition of exogenous epinephrine failed to reverse the attenuation of LPS-stimulated cytokines. Pretreatment of macrophage cultures with ₂- (ICI-118551) but not ₁-adrenergic (atenolol) receptor antagonists reversed the epinephrine-mediated cytokine attenuation following LPS treatment. Data are also presented that demonstrate the involvement of protein kinase A activation with adrenergic agonist but not with IL-10 stimulation. Taken together, these findings suggest that adrenergic mechanisms may influence peripheral tissue macrophage inflammatory cytokine response following trauma and sepsis, independent of the effects of IL-10. catecholamines; sympathetic; cecal ligation and puncture; bacterial endotoxin; -antagonist     

Efficient insertional mutagenesis in rice using the maize En/Spm elements

We have developed a novel system for insertional mutagenesis in rice (Oryza sativa) based on the maize (Zea mays) enhancer/suppressor mutator (En/Spm) element. In this system, a single T-DNA construct with Spm-transposase and the non-autonomous defective suppressor mutator (dSpm) element is used in conjunction with green fluorescent protein (GFP) and Discosoma sp. Red Fluorescence Protein (DsRed) fluorescent markers to select unlinked stable transpositions of dSpm. Using this system, we could demonstrate high frequencies of unlinked germinal transposition of dSpm in rice. Analysis of dSpm flanking sequences from 353 stable insertion lines revealed that the dSpm insertions appear to be widely distributed on rice chromosomes with a preference for genic regions (70%). The dSpm insertions appear to differ from Activator-Dissociation (Ac-Ds) elements in genomic distribution and exhibit a greater fraction of unlinked transpositions when compared with Ds elements. The results obtained in this study demonstrate that the maize En/Spm element can be used as an effective tool for functional genomics in rice and can complement efforts using other insertional mutagens. Further, the efficacy of the non-invasive fluorescence-based selection system is promising for its application to other crops.     

1-Amino-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid as a Tic mimetic: application in the synthesis of potent human melanocortin-4 receptor selective agonists

The discovery of 1-amino-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid analogs as potent human melanocortin-4 selective agonists is described.     

Synthesis and antidepressant activity of some 1,3,5-triphenyl-2-pyrazolines and 3-(2''-hydroxy naphthalen-1''-yl)-1,5-diphenyl-2-pyrazolines

Five new 1,3,5-triphenyl-2-pyrazolines were synthesised by reacting 1,3-diphenyl-2-propene-1-one with phenyl hydrazine hydrochloride and another five new 3-(2'-hydroxy naphthalen-1'-yl)-1,5-diphenyl-2-pyrazolines were synthesised by reacting 1-(2'-hydroxynaphthyl)-3-phenyl-2-propene-1-one with phenyl hydrazine hydrochloride. The structures of the compounds were proved by means of their IR, (1)H NMR spectroscopic data, and microanalyses. The antidepressant activity of these compounds was evaluated by the 'Porsolt behavioural despair test' on Swiss-Webster mice.1-Phenyl-3-(2'-hydroxyphenyl)-5-(4'-dimethylaminophenyl)-2-pyrazoline, 5-(4'-dimethylaminophenyl)-1,3-diphenyl-2-pyrazoline, 1-phenyl-3-(2'-hydroxynaphthalen-1'-yl)-5-(3',4',5'-trimethoxyphenyl)-2-pyrazoline, 1-phenyl-3-(4'-methylphenyl)-5-(4'-dimethylaminophenyl)-2-pyrazoline and 1-phenyl-3-(4'-bromophenyl)-5-(4'-dimethyl amino phenyl)-2-pyrazoline reduced immobility times 25.63-59.25% at 100mg/kg dose level. In addition, it was found that the compounds possessing electron-releasing groups such as dimethyl amino, methoxy and hydroxyl substituents, on both the aromatic rings at positions 3 and 5 of pyrazolines, considerably enhanced the antidepressant activity when compared to the pyrazolines having no substituents on the phenyl rings.     

Unique modulation of L-type Ca2+ channels by short auxiliary beta1d subunit present in cardiac muscle

Recent studies have identified a growing diversity of splice variants of auxiliary Ca2+ channel Ca(v)beta subunits. The Ca(v)beta(1d) isoform encodes a putative protein composed of the amino-terminal half of the full-length Ca(v)beta(1) isoform and thus lacks the known high-affinity binding site that recognizes the Ca2+ channel alpha1-subunit, the alpha-binding pocket. The present study investigated whether the Ca(v)beta(1d) subunit is expressed at the protein level in heart, and whether it exhibits any of the functional properties typical of full-length Ca(v)beta subunits. On Western blots, an antibody directed against the unique carboxyl terminus of Ca(v)beta(1d) identified a protein of the predicted molecular mass of 23 kDa from canine and human hearts. Immunocytochemistry and surface-membrane biotinylation experiments in transfected HEK-293 cells revealed that the full-length Ca(v)beta(1b) subunit promoted membrane trafficking of the pore-forming alpha1C (Ca(v)1.2)-subunit to the surface membrane, whereas the Ca(v)beta(1d) subunit did not. Whole cell patch-clamp analysis of transfected HEK-293 cells demonstrated no effect of coexpression of the Ca(v)beta(1d) with the alpha1C-subunit compared with the 15-fold larger currents and leftward shift in voltage-dependent activation induced by full-length Ca(v)beta(1b) coexpression. In contrast, cell-attached patch single-channel studies demonstrated that coexpression of either Ca(v)beta(1b) or Ca(v)beta(1d) significantly increased mean open probability four- to fivefold relative to the alpha1C-channels alone, but only Ca(v)beta(1b) coexpression increased the number of channels observed per patch. In conclusion, the Ca(v)beta(1d) isoform is expressed in heart and can modulate the gating of L-type Ca2+ channels, but it does not promote membrane trafficking of the channel complex.     

The bestrophin family of anion channels: identification of prokaryotic homologues

The human disease protein, Bestrophin-1, associated with vitelliform macular dystrophy, has recently been shown to be an integral membrane anion channel-forming protein. In this study we have recovered all bestrophin homologues from the NCBI database and analyzed their sequences using bioinformatic approaches. Eukaryotic homologues were found in animals and fungi but not in plants or protozoans, and prokaryotic homologues distantly related to the eukaryotic proteins, were identified in certain Gram-negative bacterial kingdoms but not in Gram-positive bacteria or archaea. Our analyses suggest a uniform 4 TMS topology for most of these homologues with regions of conservation overlapping and preceding the odd numbered TMSs and overlapping and following the even numbered TMSs. Well-conserved motifs were identified in both the eukaryotic and the prokaryotic homologues, and these proved to overlap, suggesting common structural and functional properties. Phylogenetic analyses revealed that the eukaryotic proteins cluster according to organismal type, and that the prokaryotic proteins sometimes (but not always) do so. This suggests that eukaryotic paralogues arose exclusively by recent gene duplication events although both early and late gene duplication events occurred in prokaryotes.     

Medium-chain acyl-CoA dehydrogenase deficiency in gene-targeted mice

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid β-oxidation in humans. To better understand the pathogenesis of this disease, we developed a mouse model for MCAD deficiency (MCAD^−/−) by gene targeting in embryonic stem (ES) cells. The MCAD^−/− mice developed an organic aciduria and fatty liver, and showed profound cold intolerance at 4 °C with prior fasting. The sporadic cardiac lesions seen in MCAD^−/− mice have not been reported in human MCAD patients. There was significant neonatal mortality of MCAD^−/− pups demonstrating similarities to patterns of clinical episodes and mortality in MCAD-deficient patients. The MCAD-deficient mouse reproduced important aspects of human MCAD deficiency and is a valuable model for further analysis of the roles of fatty acid oxidation and pathogenesis of human diseases involving fatty acid oxidation.     

Design,synthesis and preclinical evaluation of 5-methyl-N4-aryl-furo[2,3-d]pyrimidines as single agents with combination chemotherapy potential

The design, synthesis and biological evaluation of 4-substituted 5-methyl-furo[2,3-d]pyrimidines is described. The Ullmann coupling of 5-methyl-furo[2,3-d]pyrimidine with aryl iodides was successfully optimized to synthesize these analogs. Compounds 6–10 showed single-digit nanomolar inhibition of EGFR kinase. Compounds 1 and 6–10 inhibited VEGFR-2 kinase better than or equal to sunitinib. Compounds 1 and 3–10 were more potent inhibitors of PDGFR-β kinase than sunitinib. In addition, compounds 4–11 had higher potency in the CAM angiogenesis assay than sunitinib. Compound 1 showed in vivo efficacy in an A498 renal xenograft model in mice. Multiple RTK and tubulin inhibitory attributes of 1, 4, 6 and 8 indicates that these compounds may be valuable preclinical single agents targeting multiple intracellular targets.     

Mechanism of Mg2+-Accompanied Product Release in Sugar Nucleotidyltransferases

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Structure of the G119S Mutant Acetylcholinesterase of the Malaria Vector Anopheles gambiae Reveals Basis of Insecticide Resistance

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