CDP840. A prototype of a novel class of orally active anti-inflammatory phosphodiesterase 4 inhibitors

The discovery, synthesis and biological activity of a series of triarylethane phosphodiesterase 4 inhibitors is described. Structure-activity relationship studies are presented for CDP840 (29), a potent, chiral, selective inhibitor of PDE 4 (IC(50) 4nM). CDP840 is non-emetic in the ferret at 30mgkg(-1) (po), active in models of inflammation and reverses ozone-induced bronchial hyperreactivity in the guinea pig.     

Interactions of copper with glycated proteins: possible involvement in the etiology of diabetic neuropathy

Humans and animals with diabetes frequently develop peripheral vascular dysfunction and peripheral neuropathies. There is accumulating evidence that impaired peripheral nerve function may derive from diminished endoneural blood flow. The decrements in nerve blood flow may, in turn, be due to diminished endothelium-dependent vasodilation. Although a number of possible causes of this defective vasodilation have been suggested, none has been definitely proven. Regardless of the precise cause, the impaired vasodilatory activity may reflect diminished availability of endothelium-derived relaxing factor (EDRF), variously thought to be nitric oxide or thiol adducts of nitric oxide. Other investigators have reported that administration of transition metal chelators to diabetic rats corrects EDRF-mediated arterial relaxation and restores both neural blood flow and nerve conduction velocity, suggesting the involvement of transition metals. Our investigations center about the hypothesis that glycated proteins bind transition metals such as copper and iron, and that such 'glycochelates' accumulate within the vasculature in diabetes and catalytically inactivate EDRF. In partial support of this hypothesis: (1) Glycated albumin binds approximately 3-fold greater amounts of both copper and iron. (2) Copper bound to glycated albumin remains redox active (e.g. capable of supporting the oxidation of ascorbic acid). (3) Copper and copper-containing glycochelates cause the rapid decomposition of one putative form of EDRF, nitrosocysteine. (4) The amount of exchangeable (i.e. chelatable) copper in the plasma of diabetic rats is approximately twice that in normal rat plasma. (5) Similarly, tail tendons of diabetic animals have about twice as much bound copper as do tendons of normal rats. (6) Implants bearing adsorbed glycated albumin placed in the peritonea of normal mice for 48 h accumulate approximately 5 times as much bound copper as do implants coated with control albumin. Overall, these observations support--but do not conclusively prove - the hypothesis that transition metals such as copper, bound to glycated proteins, may blunt normal EDRF-dependent relaxation of diabetic arteries and provide a rationale for the use of transition metal chelators in the therapy of diabetic vasculopathy and neuropathy.     

S-adenosyl-L-homocysteine hydrolase is necessary for aldosterone-induced activity of epithelial Na(+) channels

The A6 cell line was used to study the role ofS-adenosyl-L-homocysteine hydrolase (SAHHase) inthe aldosterone-induced activation of the epithelial Na⁺channel (ENaC). Because aldosterone increases methylation of severaldifferent molecules, and because this methylation is associated withincreased Na⁺ reabsorption, we tested the hypothesis thataldosterone increases the expression and activity of SAHHase protein.The rationale for this work is that general methylation may be promotedby activation of SAHHase, the only enzyme known to metabolize SAH, apotent end-product inhibitor of methylation. Although aldosteroneincreased SAHHase activity, steroid did not affect SAHHase expression.Antisense SAHHase oligonucleotide decreased SAHHaseexpression and activity. Moreover, this oligonucleotide, as well as apharmacological inhibitor of SAHHase, decreased aldosterone-inducedactivity of ENaC via a decrease in ENaC open probability. The kineticsof ENaC in cells treated with antisense plus aldosterone were similarto those reported previously for the channel in the absence of steroid. This is the first report showing that active SAHHase, in part, increases ENaC open probability by reducing the transition rate fromopen states in response to aldosterone. Thus aldosterone-induced SAHHase activity plays a critical role in shifting ENaC from a gatingmode with short open and closed times to one with longer open andclosed times.

     

First isolation of an aquatic birnavirus from farmed and wild fish species in Australia

During routine sampling and testing, as part of a systematic surveillance program (the Tasmanian Salmonid Health Surveillance Program), an aquatic birnavirus was isolated from 'pin-head' (fish exhibiting deficient acclimatisation on transfer to saltwater) Atlantic salmon Salmo salar, approximately 18 mo old, farmed in net-pens located in Macquarie Harbour on the west coast of Tasmania, Australia. The isolate grows readily in a range of fish cell lines including CHSE-214, RTG-2 and BF-2 and is neutralised by a pan-specific rabbit antiserum raised against infectious pancreatic necrosis virus (IPNV) Ab strain and by a commercial pan-specific IPNV-neutralising monoclonal antibody. Presence of the virus was not associated with gross clinical signs. Histopathological examination revealed a range of lesions particularly in pancreatic tissue. The virus was localised in pancreas sections by immunoperoxidase staining using the polyclonal antiserum and by electron microscopy. Examination by electron microscopy demonstrated that the virus isolated in cell culture (1) belongs to the family Birnaviridae, genus Aquabirnaviridae; (2) was ultrastructurally and antigenically similar to virus identified in the index fish; (3) is related to IPNV. Western blot analysis using the polyclonal rabbit antiserum confirmed the cross-reactions between various aquatic birnavirus isolates. In addition, PCR analysis of isolated viral nucleic acid from the index case indicated that the virus is more closely related to IPNV fr21 and N1 isolates than to other birnavirus isolates available for comparison. Sampling of other fish species within Macquarie Harbour has demonstrated that the virus is present in several other species of fish including farmed rainbow trout Oncorhynchus mykiss, wild flounder Rhombosolea tapirina, cod Pseudophycis sp., spiked dogfish Squalus megalops and ling Genypterus blacodes.     

Study of the complex formation of daunomycin with deoxytetranucleotides with bases of differing sequence in an aqueous solution by 1H-NMR spectroscopy

The complex formation of the antibiotic daunomycin with deoxytetranucleotides of different base sequence in the chain, 5'-d(GpCpGpC), 5'-d(CpGpCpG), and 5'-d(TpGpCpA) in aqueous salt solution was studied by 1D and 2D (2M-TOCSY and 2M-NOESY) 1H-NMR spectroscopy. Concentration and temperature dependences of proton chemical shifts of molecules were measured. Based on these dependences, reaction equilibrium constants, relative content of various complexes depending on concentration and temperature, limiting values of chemical shifts of protons of daunomycin incorporated in various complexes, and the thermodynamic parameters delta H and delta S of complex formation were calculated. The analysis of the results enables the conclusion that the sites of predominant intercalation of daunomycin are triplet nucleotide sequences, the binding sites of the antibiotic with three consecutive GC pairs in the tetranucleotide duplex being more preferential. Daunomycin exhibits no sequence specificity upon binding to the single-stranded deoxynucleotide sequence. From the calculated values of induced chemical shifts of daunomycin protons and 2M-NOE data, the most probable spatial structures of complexes (1:2) of the antibiotic with deoxytetranucleotides were constructed. The binding of the second daunomycin molecule to both the single-stranded and duplex form of tetramers is of pronounced anticooperative mode, which is explained by the presence in the antibiotic of a positively charged amino sugar residue, which poses considerable steric constraints for the insertion of the second antibiotic molecule into the short tetranucleotide sequence. The results were compared with the data obtained under identical experimental conditions for typical intercalators proflavine and ethidium bromide.     

Synthesis of N,N-dialkylaniline-2'-deoxyuridine conjugates for DNA-mediated electron transfer studies

Syntheses of two analogs of deoxyuridine with N,N-dialkylaniline chromophores are reported. 5-[3-(N-methylphenylamino)propanoyl]-2'-deoxyuridine (1) and 5-[2-(4-N,N-dimethylaminophenyl)ethyl)]-2'-deoxyuridine (2) are prepared by palladium-mediated coupling. Preparation of 2 was facilitated by in situ transient O4-trimethylsilyl protection during alkynylation which suppressed secondary cyclization of the coupling adduct.     

The effect of rapamycin on single ENaC channel activity and phosphorylation in A6 cells

Rapamycin and FK-506 are immunosuppressive drugs thatbind a ubiquitous immunophilin, FKBP12, but immunosuppressivemechanisms and side effects appear to be different. Rapamycin bindsrenal FKBP12 to change renal transport. We used cell-attached patch clamp to examine rapamycin's effect on Na⁺ channels in A6cells. Channel NP_o was 0.5 ± 0.08 (n = 6)during the first 5 min but fell close to zero after 20 min. Application of 1 µM rapamycin reactivated Na⁺ channels(NP_o = 0.47 ± 0.1; n=6), but 1 µMFK-506 did not. Also, GF-109203X, a protein kinase C (PKC) inhibitor,mimicked the rapamycin-induced reactivation in a nonadditive manner.However, rapamycin did not reactivate Na⁺ channels if cellswere exposed to 1 µM FK-506 before rapamycin. In PKC assays,rapamycin was as effective as the PKC inhibitor; however, epithelialNa⁺ channel (ENaC) phosphorylation was low under baselineconditions and was not altered by PKC inhibitors or activators. Theseresults suggest that rapamycin activates Na⁺ channels bybinding FKBP12 and inhibiting PKC, and, in renal cells, despite bindingthe same immunophilin, rapamycin and FK-506 activate differentintracellular signaling pathways.