dnaA suppressor (dasF) mutants of Escherichia coli are stable DNA replication (sdrA/rnh) mutants

Summary The possible allelic relationship between dasF (dnaA suppressor) and sdrA/rnh (stable DNA replication/RNase H) mutations was examined. dasF mutations could not only suppress various dnaA(ts) mutations, but also the insertional inactivation of the dnaA gene or deletion of the oriC sequence, as could sdrA mutations. dasF mutants were found to exhibit the stable DNA replication phenotype, and the sensitivity to rich media, of sdrA mutants. The dasF and sdrA mutations were mapped very closely between metD and proA on the E. coli genetic map. The mutations were recessive to the wild-type allele for all the above phenotypes. It was concluded that dasF is allelic to sdrA/mh.     

Discovery and optimization of potent broad-spectrum arenavirus inhibitors derived from benzimidazole

A chemically diverse library of about 400,000 small molecules was screened for antiviral activity against lentiviral pseudotypes with the Lassa virus envelope glycoprotein (LASV GP) gene incorporated. High-throughput screening resulted in discovery of a hit compound (ST-37) possessing a benzimidazole core which led to a potent compound series. Herein, we report SAR studies which involved structural modifications to the phenyl rings and methylamino linker portion attached to the benzimidazole core. Many analogs in this study possessed single digit nanomolar potency against LASV pseudotypes. Compounds in this benzimidazole series also exhibited nanomolar antiviral activity against pseudotypes generated from other arenavirus envelopes indicating the potential for development of a broad-spectrum inhibitor. Ultimately, lead compound ST-193 was identified and later found to be efficacious in a lethal LASV guinea pig model showing superior protection compared to ribavirin treatment.     

Mutation of a critical arginine in microsomal prostaglandin E synthase-1 shifts the isomerase activity to a reductase activity that converts prostaglandin H2 into prostaglandin F2alpha

Microsomal prostaglandin E synthase type 1 (mPGES-1) converts prostaglandin endoperoxides, generated from arachidonic acid by cyclooxygenases, into prostaglandin E2. This enzyme belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral membrane proteins, and because of its link to inflammatory conditions and preferential coupling to cyclooxygenase 2, it has received considerable attention as a drug target. Based on the high resolution crystal structure of human leukotriene C4 synthase, a model of mPGES-1 has been constructed in which the tripeptide co-substrate glutathione is bound in a horseshoe-shaped conformation with its thiol group positioned in close proximity to Arg-126. Mutation of Arg-126 into an Ala or Gln strongly reduces the enzyme's prostaglandin E synthase activity (85-95%), whereas mutation of a neighboring Arg-122 does not have any significant effect. Interestingly, R126A and R126Q mPGES-1 exhibit a novel, glutathione-dependent, reductase activity, which allows conversion of prostaglandin H2 into prostaglandin F2alpha. Our data show that Arg-126 is a catalytic residue in mPGES-1 and suggest that MAPEG enzymes share significant structural components of their active sites.     

The potential for selection on pollen colour dimorphisms in Nigella degenii: morph-specific differences in pollinator visitation, fertilisation success and siring ability

Two subspecies of Nigella degenii (Ranunculaceae) possess a dimorphism in pollen colour and vary extensively in frequency of the two morphs in natural populations. Here we investigate the role of selection on pollen colour during the pollination phase in the two subspecies and its potential contribution to the maintenance of this colour variation. In a combination of common garden experiments and field observations, we obtained data on pollinator visitation rates and explored the effect of pollen colour on fertilisation success and siring ability under conditions of low vs. high pollen competition. In experimental gardens, naïve pollinators responded differently to plants with different pollen colour, but the favoured morph varied between dates and locations, and colour morphs were not visited in a frequency-dependent manner. Donor plants with dark pollen had a reproductive advantage (higher seed set) in single-donor pollinations, but the realised siring ability (measured by progeny morph ratio) was highly variable between different two-donor crosses with no general bias towards the light or dark morph. Therefore, although the dark pollen type appears to have a general selective advantage in terms of fertilisation success, our data are also consistent with a scenario involving the maintenance of both colour morphs, particularly under conditions of high pollen competition, a variable genetic background and/or spatial or temporal variation in the pollinator fauna.     

Variability in synovial inflammation in rheumatoid arthritis investigated by microarray technology

In recent years microarray technology has been used increasingly to acquire knowledge about the pathogenic processes involved in rheumatoid arthritis. The present study investigated variations in gene expression in synovial tissues within and between patients with rheumatoid arthritis. This was done by applying microarray technology on multiple synovial biopsies obtained from the same knee joints. In this way the relative levels of intra-patient and inter-patient variation could be assessed. The biopsies were obtained from 13 different patients: 7 by orthopedic surgery and 6 by rheumatic arthroscopy. The data show that levels of heterogeneity varied substantially between the biopsies, because the number of genes found to be differentially expressed between pairs of biopsies from the same knee ranged from 6 to 2,133. Both arthroscopic and orthopedic biopsies were examined, allowing us to compare the two sampling methods. We found that the average number of differentially expressed genes between biopsies from the same patient was about three times larger in orthopedic than in arthroscopic biopsies. Using a parallel analysis of the tissues by immunohistochemistry, we also identified orthopedic biopsies that were unsuitable for gene expression analysis of synovial inflammation due to sampling of non-inflamed parts of the tissue. Removing these biopsies reduced the average number of differentially expressed genes between the orthopedic biopsies from 455 to 171, in comparison with 143 for the arthroscopic biopsies. Hierarchical clustering analysis showed that the remaining orthopedic and arthroscopic biopsies had gene expression signatures that were unique for each patient, apparently reflecting patient variation rather than tissue heterogeneity. Subsets of genes found to vary between biopsies were investigated for overrepresentation of biological processes by using gene ontology. This revealed representative 'themes' likely to vary between synovial biopsies affected by inflammatory disease.     

Signal pathways involved in the regulation of prostaglandin E synthase-1 in human gingival fibroblasts

Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme regulating the synthesis of prostaglandin E2 (PGE2) in inflammatory conditions. In this study we investigated the regulation of mPGES-1 in gingival fibroblasts stimulated with the inflammatory mediators interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNFalpha). The results showed that IL-1beta and TNFalpha induce the expression of mPGES-1 without inducing the expression of early growth response factor-1 (Egr-1). Treatment of the cells with the PLA2 inhibitor 4-bromophenacyl bromide (BPB) decreased the cytokine-induced mPGES-1 expression accompanied by decreased PGE2 production whereas the addition of arachidonic acid (AA) upregulated mPGES-1 expression and PGE2 production. The protein kinase C (PKC) activator PMA did not upregulate the expression of mPGES-1 in contrast to COX-2 expression and PGE2 production. In addition, inhibitors of PKC, tyrosine and p38 MAP kinase markedly decreased the cytokine-induced PGE2 production but not mPGES-1 expression. Moreover, the prostaglandin metabolites PGE2 and PGF2alpha induced mPGES-1 expression as well as upregulated the cytokine-induced mPGES-1 expression indicating positive feedback regulation of mPGES-1 by prostaglandin metabolites. The peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), decreased mPGES-1 expression but not COX-2 expression or PGE2 production. The results indicate that the inflammatory-induced mPGES-1 expression is regulated by PLA2 and 15d-PGJ2 but not by PKC, tyrosine kinase or p38 MAP kinase providing new insights into the regulation of mPGES-1.     

Tunable pharmacokinetics: modifying the in vivo half-life of antibodies by directed mutagenesis of the Fc fragment

Immunoglobulins (Igs) are large proteins of 150 kDa with prolonged residence time in blood. Their half-life is controlled by their ability to interact with the protective neonatal Fc receptor (FcRn, Brambell receptor) present on endothelial cells. Here, we describe a protocol using site-specific mutagenesis of individual residues responsible for this interaction, resulting in engineered antibodies with distinct half-lives. The method is a powerful tool that enables manipulation of half-lives and is applicable to all antibodies and Fc fusion proteins for the development of agents with controlled pharmacokinetic properties. Moreover, the protocol is applicable to any situation where the structure and/or function of engineered proteins are to be studied. The protocol begins with the mutagenesis reaction at the DNA level and proceeds to describe mammalian expression and purification of recombinant proteins, radiolabeling and evaluation in vivo. The time frame for completing the procedure is about 6 months, provided that no complications are encountered.     

Progesterone and levonorgestrel regulate expression of 17βHSD-enzymes in progesterone receptor positive breast cancer cell line T47D

The use of combined hormone replacement therapy (HRT) with oestrogens and progestins in postmenopausal women has been associated with an increased risk for developing breast cancer. The reasons are not fully understood, but influence of HRT on endogenous conversion of female sex hormones may be involved. The expression of 17β hydroxysteroid dehydrogenases (17βHSD), which are enzymes catalysing the conversion between more or less potent oestrogens, may partly be regulated by progestins. The breast cancer cell lines T47D, MCF7 and ZR75-1 were treated with progesterone, medroxyprogesterone acetate (MPA) or levonorgestrel for 48 and 72 h at 10(-7) and 10(-9)M to investigate influence on 17βHSD1, 17βHSD2 and 17βHSD5 mRNA expression measured by real time PCR. The expression of 17βHSD1 increased in progesterone and levonorgestrel treated T47D cells (48 h 10(-7)M P=0.002; P<0.001) and 17βHSD5 increased after progesterone treatment (48 h 10(-7)M P=0.003), whereas the expression of 17βHSD2 decreased after the (48 h 10(-7)M P=0.003; P<0.001). Similar, but less prominent effects were seen in MCF7 and ZR75-1. The progestin effects on 17βHSD-expression were lost when T47D cells were co-treated with progestins and the progesterone receptor (PgR) inhibitor mifprestone. We show that both reductive (17βHSD1 and 17βHSD5) and oxidative (17βHSD2) members of the 17βHSD-family are under control of progesterone and progestins in breast cancer cell lines. This is most clear in T47D cells which have high PgR expression. 17βHSD-enzymes are important players in the regulation of sex steroids locally in breast tumours and tumoural expression of various 17βHSD-enzymes have prognostic and treatment predictive relevance. We propose a mechanism for increased breast cancer risk after HRT in which hormone replacement affects the expression of 17βHSD-enzymes, favouring the expression of reductive enzymes, which in turn could increase levels of bioactive and mitogenic estrogens in local tissue, e.g. breast tissue.