Mometasone furoate is a less specific glucocorticoid than fluticasone propionate.

Fluticasone propionate (FP) and mometasone furoate (MF) are potent synthetic corticosteroids that are widely used as anti-inflammatory agents to treat respiratory diseases. As part of the assessment of the potential for side-effects associated with their use, their activities, not only at the glucocorticoid receptor (GR) but also at the other members of the steroid nuclear receptor family, have been compared. Cell-based functional systems were established to measure different aspects of GR function, as well as the activity at all the other steroid nuclear receptors. The effects of MF and FP on the GR were potent and indistinguishable. Neither corticosteroid showed any activity at the oestrogen receptor, while both were weak antagonists of the androgen receptor. FP was a relatively weak agonist of the progesterone receptor but MF was a very potent agonist of the progesterone receptor, giving activity at similar concentrations to those that stimulate the GR (concentration generating 50% maximal effect (EC50)=50 pM). Moreover, while FP was a weak antagonist of the mineralocorticoid receptor (concentration generating 50% maximal inhibitory effect=80 nM), MF displayed potent partial agonist activity (EC50=3 nM, 30%). Mometasone furoate is considerably less specific for the glucocorticoid receptor than fluticasone propionate, showing significant activity at other nuclear steroid receptors.

Glucocorticoid-induced osteopenia in the mouse as assessed by histomorphometry, microcomputed tomography, and biochemical markers.

Glucocorticoids are potent anti-inflammatory molecules used in the treatment of asthma, rheumatoid arthritis, inflammatory bowel disease, and other inflammatory and dermatological diseases, as well as in posttransplantation immunotherapy. Although glucocorticoids have been prescribed for many years, their potential side effects, when administered orally, can prevent their long-term use. The most serious side effect observed in the clinic is glucocorticoid-induced osteoporosis (GIOP). To develop a small animal model to characterize glucocorticoid-induced bone loss, we carried out a series of experiments using BALB/c mice given daily intraperitoneal doses of the synthetic glucocorticoid, dexamethasone. Following dexamethasone treatment, the mice became osteopenic, with highly significant decreases in bone formation rate and mineral apposition rate, as assessed by standard histomorphometry. Moreover, 3 week treatment with dexamethasone resulted in a decrease in trabecular thickness and trabecular number with an increase in surface-to-volume ratio of trabeculae in the distal femur, as measured using microcomputed tomography (micro-CT). The serum bone formation marker, osteocalcin, was dose-dependently decreased in all mice treated with dexamethasone and showed a parallel extent of regulation to the bone formation rate changes. In addition, serum levels of leptin, recently identified as playing a role in the regulation of bone mass, increased following dexamethasone treatment. BALB/c mice therefore represent a useful model system in which the detrimental effects of glucocorticoids on bone can be studied.

Exploiting the utility of yeast in the context of programmed cell death.

Many researchers have explored the extent to which yeast can be used to dissect the mechanisms of programmed cell death in higher cells. Yeast has been used as a system to analyze protein-protein interactions and structure-function relationships, and as a cloning tool to identify novel higher eukaryote regulators of apoptosis. In addition, classic genetic strategies in yeast have been used to analyze the mechanisms of action of core pathway members. The purpose of this chapter is to describe the strategies pursued and act as a source for the technical details necessary to exploit the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe in the context of programmed cell death.

A genome-based approach for the identification of essential bacterial genes.

We have used comparative genomics to identify 26 Escherichia coli open reading frames that are both of unknown function (hypothetical open reading frames or y-genes) and conserved in the compact genome of Mycoplasma genitalium. Not surprisingly, these genes are broadly conserved in the bacterial world. We used a markerless knockout strategy to screen for essential E. coli genes. To verify this phenotype, we constructed conditional mutants in genes for which no null mutants could be obtained. In total we identified six genes that are essential for E. coli (yhbZ, ygjD, ycfB, yfil, yihA, and yjeQ). The respective orthologs of the genes yhbZ, ygjD, ycfB, yjeQ, and yihA are also essential in Bacillus subtilis. This low number of essential genes was unexpected and might be due to a characteristic of the versatile genomes of E. coli and B. subtilis that is comparable to the phenomenon of nonorthologous gene displacement. The gene ygjD, encoding a sialoglycoprotease, was eliminated from a minimal genome computationally derived from a comparison of the Haemophilus influenzae and M. genitalium genomes. We show that ygjD and its ortholog ydiE are essential in E. coli and B. subtilis, respectively. Thus, we include this gene in a minimal genome. This study systematically integrates comparative genomics and targeted gene disruptions to identify broadly conserved bacterial genes of unknown function required for survival on complex media.

Substitutions in the pheromone-responsive Gbeta protein of Saccharomyces cerevisiae confer a defect in recovery from pheromone treatment.

The pheromone-responsive Galpha protein of Saccharomyces cerevisiae, Gpa1p, stimulates an adaptive mechanism that downregulates the mating signal. In a genetic screen designed to identify signaling elements required for Gpa1p-mediated adaptation, a large collection of adaptive-defective (Adp-) mutants were recovered. Of the 49 mutants characterized thus far, approximately three-quarters exhibit a dominant defect in the negative regulation of the pheromone response. Eight of the dominant Adp- mutations showed tight linkage to the gene encoding the pheromone-responsive Gbeta, STE4. Sequence analysis of the STE4 locus in the relevant mutant strains revealed seven novel STE4 alleles, each of which was shown to disrupt proper regulation of the pheromone response. Although the STE4 mutations had only minor effects on basal mating pathway activity, the mutant forms of Gbeta dramatically affected the ability of the cell to turn off the mating response after exposure to pheromone. Moreover, the signaling activity of the aberrant Gbetagamma subunits was suppressed by G322E, a mutant form of Gpa1p that blocks the pheromone response by sequestering Gbetagamma, but not by E364K, a hyperadaptive form of Gpa1p. On the basis of these observations, we propose that Gpa1p-mediated adaptation involves the binding of an unknown negative regulator to Gbetagamma.

Phospholipases in yeast.

The central role that different phospholipases play in many signal transduction pathways has been intensively studied by classical biochemical and molecular approaches. One approach not extensively pursued, has been the use of yeast as a model system for functional analysis of different aspects of phospholipase signalling. This review concentrates on attempts to establish yeast in this role and aims to demonstrate the potential offered by this approach.

Expression of bak in S. pombe results in a lethality mediated through interaction with the calnexin homologue Cnx1.

Expression studies in the yeast S. pombe have been utilised to establish the basis for a genetic analysis designed to identify the lethal partners of the pro-apoptotic proteins bak and bax. Bak expression in S. pombe is lethal and this lethality is rescued by co-expression of bcl-2 or bcl-x(L). S. pombe cells expressing bak have a terminal phenotype in which the majority of cells are blocked in the G1 phase of the cell cycle while the remainder of cells, unable to complete M-phase, mis-coordinate the timing of subsequent events in the cell cycle. Although bax expression in S. pombe gives rise to a slow growth phenotype, not a lethality, bax expressing cells display the same cell cycle phenotypes described for bak. Electron microscopy of cells expressing bak reveals a dramatic accumulation of large vesicular structures. A two-hybrid screen designed to identify S. pombe proteins which interact with bak, isolated the S. pombe calnexin homologue cnx1. Genetic analysis demonstrates that the Cnx1 domain which binds to bak in two-hybrid experiments, is necessary for bak lethality in S. pombe. This report identifies a lethal interacting partner for bak and the observations suggest a model for bak mediated lethality which can be tested in higher cells.

The formation of professional and consumer solutions: ethics in the general practice setting.

A general practice research project on ethics is underway at the University of New South Wales, funded by GPEP (General Practice Evaluation Program, Commonwealth Department of Human Services and Health, GPEP 386). Ethical issues, as defined and explored by general practitioners and consumers, are being examined across four areas of Sydney. So far, telephone interviews have been conducted (64% response rate) with a random sample of general practitioners (GPs). Face-to-face interviews have been conducted with 107 consumers, randomly sampled using ABS collection district information. Focus groups have been formed to discuss acceptable solutions to GP and consumer identified ethical issues. This report will report on some preliminary findings to date and will explore professional and consumer roles in the formation of ethical solutions.

A pathway in the yeast cell division cycle linking protein kinase C (Pkc1) to activation of Cdc28 at START.

In an effort to study further the mechanism of Cdc28 function and cell cycle commitment, we describe here a genetic approach to identify components of pathways downstream of the Cdc28 kinase at START by screening for mutations that decrease the effectiveness of signaling by Cdc28. The first locus to be characterized in detail using this approach was PKC1 which encodes a homolog of the Ca(2+)-dependent isozymes of the mammalian protein kinase C (PKC) superfamily (Levin et al., 1990). By several genetic criteria, we show a functional interaction between CDC28 and PKC1 with PKC1 apparently functioning with respect to bud emergence downstream of START. Consistent with this, activity of the MAP kinase homolog Mpk1 (a putative Pkc1 effector) is stimulated by activation of Cdc28. Furthermore, we demonstrate a cell cycle-dependent hydrolysis of phosphatidylcholine to diacylglycerol (a PKC activator) and choline phosphate at START. Diacylglycerol production is stimulated by Cdc28 in cycling cells and is closely associated with Cdc28 activation at START. These results imply that the activation of Pkc1, which is known to be necessary during bud morphogenesis, is mediated via the CDC28-dependent stimulation of PC-PLC activity in a novel cell cycle-regulated signaling pathway.

A second gene product of the inositol-phospholipid-specific phospholipase C delta subclass.

Sequence analysis of a inositol-phospholipid-specific phospholipase C (PtdIns-PLC) purified from bovine brain has led to the isolation of a novel cDNA that encodes this protein. While this cDNA contains two introns, these appear to be removed upon transfection of the cDNA into COS-1 cells. The protein transiently expressed in COS-1 cells shows phosphatidylinositol 4,5-bisphosphate hydrolysing activity which distributes preferentially into the particulate fraction. Comparison of the predicted amino acid sequence of this PtdIns-PLC with other known PtdIns-PLCs reveals a high degree of similarity, throughout all of its sequence, with PtdIns-PLC delta. Thus, we believe that the identification of this cDNA represents evidence for multiple functional-gene products within the delta subclass of PtdIns-PLCs.

A novel inositol-phospholipid-specific phospholipase C. Rapid purification and characterization.

A novel bovine brain inositol-phospholipid-specific phospholipase C has been identified on the basis of chromatographic behaviour and purified to apparent homogeneity by a rapid three-step procedure. The purified enzyme has a molecular mass of 85 kDa on SDS/polyacrylamide gel electrophoresis and a specific activity of 24 mumol.min-1.mg-1. The enzyme is dependent on Ca2+ and shows a marked preference for inositol phospholipid substrates. The unique nature of this polypeptide was confirmed through partial protein sequence analysis.

Determination of the primary structure of PLC-154 demonstrates diversity of phosphoinositide-specific phospholipase C activities.

Protein sequence analysis of a bovine brain phosphoinositide-specific phospholipase C (PI-PLC; PLC-154) has permitted the isolation of a cDNA that appears to code for this protein. Transient expression of this cDNA in COS-1 cells demonstrates that the cDNA encodes a functional phospholipase C that migrates at approximately 150,000 daltons. A transcript of approximately 7 kb is observed in RNA derived from bovine brain and a related transcript of the same size is present in certain human cell lines. Southern blot analysis indicates that one or possibly two genes hybridize with a PLC-154 probe. Regions of homology between PLC-154 and the previously described PLC-148 allow the assignment of a putative catalytic domain to the central region of PLC-154.