Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.     

Synthesis and electrophysiological studies of a novel epibatidine analogue

The new epibatidine analogue exo-2-(2-pyridyl)-7-azabicyclo[2.2.1]heptane (2PABH) was synthesised. Separation of enantiomers was performed on chiral HPLC chromatography in polar-organic phase mode at 0 degree C. Enantiomeric purity was greater than 99.8%ee for the (-)- and 90.5%ee for the (+)-enantiomer respectively. Optical rotation was determined to be [alpha]23D = +/- 13 degrees. Electrophysiological studies of 2PABH were carried out on alpha 4 beta 2, alpha 3 beta 4 and alpha 7 nAChR subtypes cloned from rat and reconstituted in Xenopus oocytes. Both enantiomers could not significantly activate the heteromeric subtypes. The homomeric alpha 7 nAChR displays a high sensitivity only towards (-)-2PABH. The EC50 for (-)-2PABH and ACh were determined (32.5 +/- 9.5 microM, 137.3 +/- 16.5 microM). (-)-2PABH was shown to be a partial agonist (80% of ACh). Thus the efficacy of 2PABH differs markedly from that of epibatidine. The intramolecular N-N-distance and the spatial pyridine nitrogen orientation play a central role in nAChR recognition.     

POLLINATION BIOLOGY OF PLATANTHERA STRICTA (ORCHIDACEAE) IN OLYMPIC NATIONAL PARK,WASHINGTON

Platanthera striata Lindley is entomophilous and can produce seed via facultative self-pollination and intraracemic and interracemic pollination. Capsule production is pollinator-limited and seed set may be pollen-limited. In experimental plants capsules produced via self- and intraracemic pollination contained fewer seeds with normally developed embryos than did capsules produced via interracemic pollination. The inflorescence of Platanthera stricta is fragrant and is attractive to a wide array of anthophilous insects. It is pollinated by a diverse assemblage of short-tongued insects. The primary pollinators are Eustroma fasciata B. and McD. (Lepidoptera: Geometridae), Bombus flavifrons Cresson and B. melanopygus Nylander (Hymenoptera: Apidae), an undescribed species of Greya (Lepidoptera: Prodixidae), and several species of Empis, Rhamphomyia, and Anthepiscopus longipalpis Melander (Diptera: Empididae). Small amounts of glucose are present on the raceme. The extrafloral glucose may retain small pollinators on the inflorescence until they locate the floral spur aperture.     

Mitochondrial gene order is not conserved in arthropods: prostriate and metastriate tick mitochondrial genomes

The entire mitochondrial genome was sequenced in a prostriate tick, Ixodes hexagonus, and a metastriate tick, Rhipicephalus sanguineus. Both genomes encode 22 tRNAs, 13 proteins, and two ribosomal RNAs. Prostriate ticks are basal members of Ixodidae and have the same gene order as Limulus polyphemus. In contrast, in R. sanguineus, a block of genes encoding NADH dehydrogenase subunit 1 (ND1), tRNA(Leu)(UUR), tRNA(Leu)(CUN), 16S rDNA, tRNA(Val), 12S rDNA, the control region, and the tRNA(Ile) and tRNA(Gln) have translocated to a position between the tRNA(Glu) and tRNA(Phe) genes. The tRNA(Cys) gene has translocated between the control region and the tRNA(Met) gene, and the tRNA(Leu)(CUN) gene has translocated between the tRNA(Ser)(UCN) gene and the control region. Furthermore, the control region is duplicated, and both copies undergo concerted evolution. Primers that flank these rearrangements confirm that this gene order is conserved in all metastriate ticks examined. Correspondence analysis of amino acid and codon use in the two ticks and in nine other arthropod mitochondrial genomes indicate a strong bias in R. sanguineus towards amino acids encoded by AT-rich codons.      

360 MHz PMR studies on the involvement of the Y-nucleoside in the conformation of 2''-OMeGpApApYpAppsi from torula yeast tRNAphe.

360 MHz measurements of chemical shifts, 3J1'-2', and T1 as a function of temperature for various protons of the hexanucleotide 2'-OMeGpApApYpAppsi from torula yeast tRNAphe have revealed a unique involvement of the Yt base in the structure and conformation of this oligonucleotide. Whereas the adenosine residues in the anticodon triplet are relatively stable to temperature increase, the Yt readily undergoes destacking and a change in ribose conformation. The destacking most likely involves a torsional displacement of the Yt base occasioned by a rotation of the phosphate-ribose backbone. The possible relevance of this unusual behavior to the influence of the Yt residue in tRNA function in protein biosynthesis is discussed.     

Oxidation of methane in the absence of oxygen in lake water samples.

Methane was oxidized to carbon dioxide in the absence of oxygen by water samples from Lake Mendota, Madison, Wis. The anaerobic oxidation of methane did not result in the assimilation of carbon from methane into material precipitable by cold 10% trichloracetic acid. Only samples taken at the suface of the sediment of Lake Mendota were capable of catalyzine the anaerobic oxidation of methane. The rate of methane oxidation in the presence of oxygen was highest in samples taken from near the thermocline. Of the radioactive methane oxidized, 30 to 60% was assimilated into material precipitable by cold 10% trichloroacetic acid during aerobic incubation of the samples. These data support the conclusion that two distinct groups of methane-oxidizing organisms occur in stratifield lakes. Enrichments with acetate and methane as the sole sources of carbon and energy and sulfate as the electron acceptor resulted in the growth of bacteria that oxidize methane. Sulfate, acetate, and methane were all required for growth of enrichments. Acetate was not oxidized to carbon dioxide but was assimilated by cells. Methane was not assimilated but was oxidized to carbon dioxide in the absence of air.     

Role of lipoxygenase in the O2-dependent activation of soluble guanylate cyclase from rat lung.

Guanylate cyclase activity in rat lung supernatant fractions is stimulated 3-4 fold by aerobic incubation at 30 degrees C for approx. 30 min ('O2-dependent activation'). This stimulation was blocked by 20 microM-eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of lipoxygenase and cyclo-oxygenase, but not by aspirin or indomethacin, which are cyclo-oxygenase inhibitors. The enzyme activator(s) is presumed to be the fatty acid hydroperoxide(s) formed by lipoxygenase. Removal of lipoxygenase from the supernatant fraction by chromatography on Amberlite XAD-4 also prevented activation, which was restored by the addition of soya-bean lipoxygenase. Bovine serum albumin prevented O2-dependent activation or activation by soya-bean lipoxygenase, through its ability to bind the unsaturated fatty acid substrate of lipoxygenase. The lipoxygenase in the supernatant fraction is inhibited by endogenous glutathione peroxidase plus reduced glutathione (GSH); removal of GSH de-inhibits lipoxygenase and activates guanylate cyclase. This was effected by autoxidation, by cumene hydroperoxide (with GSH peroxidase) and by titration with N-ethylmaleimide (NEM). Activation by NEM was inhibited by serum albumin or ETYA, as was activation by low concentrations (less than 50 microM) of cumene hydroperoxide. Activation by higher concentrations was not so inhibited; therefore, cumene hydroperoxide can also activate by a direct effect on guanylate cyclase. A hypothesis for physiological activation is proposed.     

Short-term neuropathological aspects of in vivo suicide gene transfer to the F98 rat glioblastoma using liposomal and viral vectors

To date, only few preclinical protocols on liposomal suicide gene transfer in tumors have been published, none of which directly compared viral to liposomal vectors in terms of immunoreactivity and efficacy. We thus studied the neuropathological alterations in 80 rats being treated for glioblastoma using liposomal and, for comparison, adenoviral and retroviral suicide gene transfer approaches to identify vector-associated efficacy and toxicity for further clinical studies. 62 rats served as controls. F98 tumors were established in Fisher rats and transfected in vivo with the thymidine kinase gene of herpes simplex virus (HSVtk) by a single intratumoral application and an implanted intratumoral continuous delivery system. Three days later ganciclovir was given intraperitoneally for 14 days. The animals were sacrificed 17 days post completed gene transfer. Brains were examined histologically and immunohistochemically using markers for immunocompetent cells. Ten animals showed complete tumor regression; they all belonged to the liposomal and adenoviral groups. In 6 of 10 experimental groups considerable numbers of lymphocytes along the margins of the regression cavities could be observed. Control animals of the liposomal and adenoviral groups showed only little lymphocytic infiltration, underlining the minimal immunogenicity of these carriers. In contrast, the retroviral control group featured a high lymphocyte infiltration. In summary, this study indicates that, in terms of both efficacy and immunoreaction, liposomes are as appropriate as adenoviruses in the treatment of rat glial tumors using suicide gene transfer strategies.     

Assessment of Global Coronary Heart Disease Risk in Overweight and Obese African‐American Women

Objective : To examine, with the use of national guidelines, coronary heart disease (CHD) risk with increasing BMI for primary prevention in urban African‐American women. Research Methods and Procedures : Participants were recruited for CHD risk factor screening from 20 churches as part of a larger study of nutrition and fitness (Project Joy). All participants had a demographic, smoking and medical history assessment, and the following measurements were taken: weight, height, waist circumference, blood pressure, lipid levels, and glucose. Three methods of defining risk, the Framingham Point Scoring System, a count of risk factors, and the presence of the multiple metabolic syndrome, based on the National Cholesterol Education Program Adult Treatment Panel III Report and BMI classes established by the Clinical Guidelines, were used. Results : A total of 396 women were eligible. Participants were 40 to 80 years of age and had marked excess prevalence of overweight and obesity (84%); 55% were obese. There was a linear increase in risk factors as BMI increased. Lipids did not differ significantly among BMI classifications. Seventeen percent of women had multiple metabolic syndrome. Eight percent and 16% of women in the normal and overweight BMI classes, respectively, had two or more modifiable risk factors. There was no difference in number of modifiable risk factors among the obese classes. The Framingham Point Scoring System assigned a <10% risk of a hard CHD event in 10 years to 97% of the women. Discussion : National risk assessment guidelines for primary prevention of CHD may not be adequate for overweight and obese urban African‐American women and require further study.