A RAC-GEF network critical for early intestinal tumourigenesis.

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2-/- Vav3-/- Tiam1-/-), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.

A fluorescence polarization assay for inhibitors of Hsp90.

Hsp90 encodes a ubiquitous molecular chaperone protein conserved among species which acts on multiple substrates, many of which are important cell-signaling proteins. Inhibition of Hsp90 function has been promoted as a mechanism to degrade client proteins involved in tumorigenesis and disease progression. Several assays to monitor inhibition of Hsp90 function currently exist but are limited in their use for a drug discovery campaign. Using data from the crystal structure of an initial hit compound, we have developed a fluorescence polarization assay to monitor binding of compounds to the ATP-binding site of Hsp90. This assay is very robust (Z' > 0.9) and can detect affinity of compounds with IC50s to 40 nM. We have used this assay in conjunction with cocrystal structures of small molecules to drive a structure-based design program aimed at the discovery and optimization of a novel class of potent Hsp90 inhibitors.

The planning of rural health research: rurality and rural population issues.

Rurality and rural population issues require special consideration when planning both qualitative and quantitative health research in rural areas. The objective of this article was to explore the issues that require attention when planning the research. This is the first of two articles and focus on issues that require consideration when undertaking rural health research. The diversity of study populations, the feasibility of a research topic, the selection of a research team, and the cultural traditions of Indigenous communities, are all aspects of rural health research planning that require attention. Procedures such as identifying the characteristics of the population, the selection of measures of rurality appropriate for the research topic, the use of local liaison persons, decisions on the use of 'insider' or 'outsider' researchers, and the identification of skills resources available, increase the quality of the research outcomes. These issues are relevant to both qualitative and quantitative research. Procedures are available to address issues of particular concern in developing appropriate methods for rural health research. While we have concentrated on Australian issues and solutions, rural localities in other countries may face similar issues. Attention to rurality and rural situations when planning rural health research, results in studies that support the continued improvement of health in rural communities.

The conducting and reporting of rural health research: rurality and rural population issues.

Rurality and rural population issues require consideration when conducting and reporting on rural health research. A first article focused on the planning stage of the research. The objective of this article is to explore conducting and reporting issues that require attention when undertaking rural health research. The privacy of participants, the collection of data, the cultural traditions of Indigenous communities, the dissemination of results, and giving something back to the community, are all aspects of conducting and reporting rural health research that require attention. Procedures such as identifying the characteristics of the population, attention to safety issues when collecting data, the use of local liaison persons and acknowledging the ownership of intellectual property, increase the quality of the research outcomes. They are issues that are relevant to both qualitative and quantitative research methods. Procedures are available to address issues of particular concern in developing appropriate methods for rural health research. While we have concentrated on Australian issues, and possible solutions, rural localities in many other countries may face similar issues. In any rural setting, paying attention to issues that may affect the conducting and reporting of rural health research will hopefully result in studies that support the continued improvement of health in rural communities.

The detection of non-O157 E. coli in food by immunomagnetic separation.

AIMS: To compare immunomagnetic separation (IMS) protocols (enrichment media and temperature) for the isolation of Escherichia coli serotypes O26 and O111 from four different foods. METHODS AND RESULTS: Foods (minced beef, cheese, apple juice and pepperoni) spiked with low numbers (<100 g(-1)) of stressed nalidixic mutant E. coli serotypes O26 and O111 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at temperatures of 37 and 42 degrees C to optimize the IMS technique. BPW enrichments gave increased recoveries of both serotypes compared with tryptone soya and EC broths. Elevated temperatures of incubation at 42 degrees C were superior to 37 degrees C. CONCLUSIONS: Positive detection of low numbers of stressed target pathogens in all replicate tests was only possible using BPW enrichments. The majority of tests from alternative enrichments resulted in zero or single colonies recovered post-IMS. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum IMS protocol would improve isolation rates of E. coli O26 and O111 from foods and lead to increased safety for the consumer. Sub-optimal IMS protocols could lead to foods being incorrectly labelled free from these pathogens.

The carbenoid approach to peptide synthesis.

A different approach to the synthesis of dipeptides is described based on the formation of the NHCHR1CONH-CHR2CO bond by carbenoid N-H insertion, rather than the formation of the peptide bond itself. Thus decomposition of triethyl diazophosphonoacetate catalysed by rhodium(II) acetate in the presence of N-protected amino acid amides 8 gives the phosphonates 9. Subsequent Wadsworth-Emmons reaction of 9 with aldehydes in the presence of DBU gives dehydro dipeptides 10. The reaction has been extended to a simple two-step procedure, without the isolation of the intermediate phosphonate, for conversion of a range of amino acid amides 11 into dehydro dipeptides 12 and to an N-methylamide 11 h, and for conversion of a dipeptide to tripeptide (13-->14). Direct conversion, by using methyl diazophenylacetate, of amino acid amides to phenylglycine-containing dipeptides 19 proceeds in good chemical yield, but with poor diastereoselectivity.

Inhibition of inducible nitric oxide synthase by acetamidine derivatives of hetero-substituted lysine and homolysine.

The synthesis and in vitro evaluation of the acetamidine derivatives of hetero-substituted lysine and homolysine analogues have identified potent inhibitors of human nitric oxide synthase enzymes, including examples with marked selectivity for the inducible isoform.

Synthesis and SAR of 4-aryl-2-hydroxy-4-oxobut-2-enoic acids and esters and 2-amino-4-aryl-4-oxobut-2-enoic acids and esters: potent inhibitors of kynurenine-3-hydroxylase as potential neuroprotective agents.

The synthesis and structure-activity relationship of a series of 4-aryl-2-hydroxy-4-oxobut-2-enoic acids and esters and 2-amino-4-aryl-4-oxobut-2-enoic acids and esters as potent inhibitors of kynurenine-3-hydroxylase are described. These compounds are the most potent inhibitors of the kynurenine-3-hydroxylase enzyme so far disclosed. Additionally methyl 4-(3-chlorophenyl)-2-hydroxy-4-oxobut-2-enoate (2d), 4-(3-chlorophenyl)-2-hydroxy-4-oxobut-2-enoic acid (3d), methyl 4-(3-fluorophenyl)-2-hydroxy-4-oxobut-2-enoate (2f), and 4-(3-fluorophenyl)-2-hydroxy-4-oxobut-2-enoic acid (3f) prevent the increase in the interferon-gamma-induced synthesis of quinolinic acid in primary cultures of cultured human peripheral blood monocyte-derived macrophages.

S-aryl cysteine S,S-dioxides as inhibitors of mammalian kynureninase.

A series of 2-amino-S-aryl cysteine S,S-dioxides have been synthesised and shown to inhibit kynureninase an important enzyme in the biosynthesis of the known excitotoxic moiety quinolinic acid. The most potent of these, 2-amino-5-methyl-S-phenyl cysteine S,S-dioxide 6d, inhibits interferon-gamma induced synthesis of quinolinic acid in human macrophages.

The Aspergillus niger carbon catabolite repressor encoding gene, creA.

In order to undertake a comparative analysis of carbon catabolite repression in two Aspergillus species, the creA gene has been isolated from A. niger by cross hybridization, using the cloned A. nidulans gene. The A. niger gene has been shown to be functional in A. nidulans by heterologous complementation of the creA204 mutation of A. nidulans. Overall, the genes show 90% sequence similarity (82% identity) at the amino acid (aa) level. There were some striking similarities between the aa sequences encoded by the two fungal creA genes and two genes involved in carbon catabolite repression in Saccharomyces cerevisiae. The zinc-finger regions showed 96% similarity (84% identity) with the zinc-finger region of the MIG1 gene of S. cerevisiae. The CREA protein contains a stretch of 42 aa that is identical in A. niger and A. nidulans, and these show 81% similarity (33% identity) with a region of the S. cerevisiae RGR1 gene.

Rationally designed "dipeptoid" analogues of CCK. Acid mimics of the potent and selective non-peptide CCK-B receptor antagonist CI-988.

This paper outlines the synthesis of selected acid mimics of the non-peptide CCK-B selective antagonist CI-988, 1. CCK-B and CCK-A binding affinities of these analogues are described and their CCK-B affinity and selectivity rationalized by consideration of the pK(a) values, charge distribution, and geometry of the respective acid mimics. Several of the compounds have CCK-B binding affinities similar to the parent carboxylic acid 1 (CCK-B, IC50 = 1.7 nM; pK(a) = 5.6) and span a pK(a) range of less than 1 (sulfonic acid 27) to greater than 9.5 (5-thio-1,2,4-triazole 24). Among the more active compounds synthesized are tricyclo[3.3.1.1(3,7)]dec-2-yl [R-(R*,R*)]-[2-[[2-[[(3-hydroxy-5-isoxazolyl)acetyl]-amino]-2- phenylethyl]amino]-1-(1H-indol-3-ylmethyl)-1-methyl-2-oxoethyl+ ++]carbamate (15), tricyclo[3.3.1.1(3,7)]dec-2-yl [R-(R*,R*)]-[1-(1H-indol-3-ylmethyl)-1-methyl-2-oxo-2-[[2-[(1-oxo- 3-sulfopropyl)amino]-2-phenylethyl]amino]-ethyl]carbamate, monosodium salt (27), and tricyclo[3.3.1.1(3,7)]dec-2-yl [R-(R*,R*)]-[1-(1H-indol-3-ylmethyl)-1- methyl-2-oxo-2-[[2-[[(1H-1,2,4-triazol-5-ylsulfinyl)acetyl]a mino]-2-phenylethyl]amino]ethyl]carbamic acid (34) which have CCK-B binding affinities of IC50 = 2.6, 1.3, and 1.7 nM, CCK-A/-B ratios of 650, 780, and 550 and pK(a) values of 6.5, less than 1, and 7.0, respectively.

Alcohol dehydrogenase III in Aspergillus nidulans is anaerobically induced and post-transcriptionally regulated.

An alcohol dehydrogenase was shown to be induced in Aspergillus nidulans by periods of anaerobic stress. This alcohol dehydrogenase was shown to correspond to the previously described cryptic enzyme, alcohol dehydrogenase III (McKnight et al. 1985), by analysis of a mutation in the structural gene of alcohol dehydrogenase III, alcC, created by gene disruption. Survival tests on agar plates showed that this enzyme is required for long-term survival under anaerobic conditions. Northern blot analysis and gene fusion studies showed that the expression of the alcC gene is regulated at both the transcriptional and translational levels. Thus there are mechanisms in this filamentous fungus allowing survival under anaerobic stress that are similar to those described in higher plants.

Relationship between sensitivity to dietary fat and dietary cholesterol.

A group of 56 hypercholesterolemic and normocholesterolemic men and women were given approximately 700 mg a day of egg yolk cholesterol in a double-blind, crossover study while they were on a background diet containing approximately 30% of energy as fat. Overall there was a 0.23 mmol/l rise in plasma cholesterol (3.7%, p less than 0.001) after 4 weeks, a 0.19 mmol/l rise in low density lipoprotein (LDL) cholesterol (4.9%, p = 0.002), and a 0.07 mmol/l rise in high density lipoprotein (HDL) cholesterol (5.4%, p less than 0.001). Plasma triglycerides fell by 0.07 mmol/l (5.1%). Normocholesterolemic individuals (plasma cholesterol less than 5.2 mmol/l) experienced small, nonsignificant rises of 0.06, 0.02, and 0.05 mmol/l in total, LDL, and HDL cholesterol, respectively. Hypercholesterolemic subjects were classified on the basis of their response to a low fat diet. Diet-sensitive subjects were defined by a greater than 10% fall in plasma cholesterol on a 25% fat, low cholesterol (less than 200 mg/day) diet. These individuals were found to be more responsive to the effect of dietary cholesterol than were diet-insensitive subjects; the respective changes in the two groups were rises of 0.36 mmol/l versus 0.19 mmol/l in plasma cholesterol (p = 0.06) and rises of 0.30 versus 0.15 mmol/l in LDL cholesterol (p = 0.06). In addition to elevating HDL cholesterol by 0.09 mmol/l and 0.07 mmol/l, respectively, dietary cholesterol also produced an increase in the proportion of HDL2, from 40% to 44% of HDL protein (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Endocardial pacing electrode design and rate of displacement.

In a series of 184 patients with a permanent endocardial pacing system the rate of electrode displacement was 3-0 per cent using the Devices L120SR electrode, and 23-1 per cent using the Devices S120 electrode. The technique and pacing team were unchanged during the period of study. It is suggested that the design and mechanical characteristics of the elctrodes were responsible.

The effect of tablet formulation on the haemodynamic properties of glyceryl trinitrate.

A non-invasive haemodynamic study was carried out in 13 normal subjects to compare the pharmacodynamics of glyceryl trinitrate when formulated as a moulded tablet ('Nitrostat') rather than as a generic compressed tablet. The glyceryl trinitrate moulded tablet, with a much more rapid and predictable dissolution time, produced an earlier onset of systolic blood pressure fall (p less than 0.01) and a greater maximum percentage change in peripheral blood flow (p less than 0.02). No other significant differences in effect on pulse rate, blood pressure or peripheral blood flow were noted.