Duplication-induced mutation of a new Neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product.

A cloned Neurospora crassa genomic sequence, selected as preferentially transcribed when acetate was the sole carbon source, was introduced in extra copies at ectopic loci by transformation. Sexual crossing of transformants yielded acetate nonutilizing mutants with methylation and restriction site changes within both the ectopic DNA and the normally located gene. Such changes are typical of the duplication-induced premeiotic disruption (the RIP effect) first described by Selker et al. (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987). The mutants had the unusual phenotype of growth on ethanol but not on acetate as the carbon source. In a cross to the wild type of a mutant strain in which the original ectopic gene sequence had been removed by segregation, the acetate nonutilizing phenotype invariably segregated together with a RIP-induced EcoRI site at the normal locus. This mutant was transformed to the ability to use acetate by the cloned sequence. The locus of the mutation, designated acu-8, was mapped between trp-3 and un-15 on linkage group 2. The transcribed portion of the clone, identified by probing with cDNA, was sequenced, and a putative 525-codon open reading frame with two introns was identified. The codon usage was found to be strongly biased in a way typical of most Neurospora genes sequenced so far. The predicted amino acid sequence shows no significant resemblance to anything previously recorded. These results provide a first example of the use of the RIP effect to obtain a mutant phenotype for a gene previously known only as a transcribed wild-type DNA sequence.     

Discovery and SAR of 2-amino-5-(thioaryl)thiazoles as potent and selective Itk inhibitors

A series of structurally novel aminothiazole based small molecule inhibitors of Itk were prepared to elucidate their structure-activity relationships (SARs), selectivity, and cell activity in inhibiting IL-2 secretion in a Jurkat T-cell assay. Compound 3 is identified as a potent and selective Itk inhibitor which inhibits anti-TCR antibody induced IL-2 production in mice in vivo and was previously reported to reduce lung inflammation in a mouse model of ovalbumin induced allergy/asthma.     

Arginase I induction by modified lipoproteins in macrophages: a peroxisome proliferator-activated receptor-gamma/delta-mediated effect that links lipid metabolism and immunity

Macrophages are phagocytic cells that play essential roles in innate immunity and lipid homeostasis. The uptake of modified lipoproteins is an important early event in the development of atherosclerosis. We analyzed the ability of modified low-density lipoprotein (LDL) (oxidized and acetylated) to alter the expression and activity of arginases (ArgI and ArgII) in macrophages. We show that ArgI expression is potently induced by both oxidized and acetylated LDL in macrophages. We further show that this effect is mediated by peroxisome proliferator-activated receptors (PPAR). ArgI expression is highly responsive to agonists for PPARgamma and PPARdelta but not PPARalpha. Moreover, the induction of ArgI by both PPAR agonists and IL-4 is blocked in macrophages from PPARgamma- and PPARdelta-deficient mice. Functionally, PPAR activity induces macrophage activation toward a more Th2 immune phenotype in a model of Leishmania major infection. We show that PPARgamma and -delta ligands promote intracellular amastigote growth in infected macrophages, and this effect is dependent on both PPAR expression and Arg activity. Collectively, our results strongly suggest that ArgI is a key marker of the alternative program triggered by PPAR in macrophages.     

Construction of the model for the Genetic Analysis Workshop 14 simulated data: genotype-phenotype relationships,gene interaction,linkage, association,disequilibrium, and ascertainment effects for a complex phenotype

Background

In order to confirm a previous finding of linkage to alcoholism on chromosome 1 we have carried out a genetic linkage study.

Methods

DNA from eighteen families, densely affected by alcoholism, was used to genotype a set of polymorphic microsatellite markers at loci approximately 10 centimorgans apart spanning the short arm and part of the long arm of chromosome 1. Linkage analyses were performed using the classical lod score and a model-free method. Three different definitions of affection status were defined, these were 1. Heavy Drinking (HD) where affected subjects drank more than the Royal College of Psychiatrists recommended weekly amount. 2. The Research Diagnostic Criteria for alcoholism (RDCA) 3. Alcohol Dependence Syndrome (ADS) as defined by Edwards and Gross (1976) and now incorporated into ICD10 and DSMIV.

Results

Linkage analyses with the markers D1S1588, D1S2134, D1S1675 covering the cytogenetic region 1p22.1-11.2 all gave positive two point and multipoint lods with a maximum lod of 1.8 at D1S1588 (1p22.1) for the RDCA definition of alcoholism. Another lod of 1.8 was found with D1S1653 in the region 1q21.3-24.2 using the HD affection model.

Conclusion

These results both support the presence of linkage in the 1p22.1-11.2 region which was previously implicated by the USA Collaborative Study of the Genetics of Alcoholism (COGA) study and also suggest the presence of another susceptibility locus at 1q21.3-24.2.     

Ultraviolet B radiation generates platelet-activating factor-like phospholipids underlying cutaneous damage

Ultraviolet B light (UVB) causes cutaneous inflammation and cell death, but the agents responsible are not defined. These studies examined the role of the platelet-activating factor (PAF) signaling system in UVB-mediated effects. Expression of the PAF receptor in the PAF receptor-negative epidermoid cell line KB augmented apoptosis in response to UVB irradiation. Overexpression of the PAF receptor in primary human keratinocytes also enhanced UVB-mediated apoptosis in vitro, and it enhanced apoptosis in an in vivo model of human keratinocytes grafted onto severe combined immune-deficient (SCID) mice. To define the mechanism by which UVB activates the PAF receptor, we used mass spectrometry to demonstrate significant amounts of the C4 PAF analogs 1-alkyl-2-(butanoyl and butenoyl)-sn-glycero-3-phosphocholine, as well as native PAF in an epidermal cell line after UVB irradiation. Supplementing the cells with the precursor phospholipid 1-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (HAPC) increased the amount of C4 PAF analogs recovered after UVB exposure. We irradiated HAPC directly and found, even in the absence of a photosensitizer, fragmentation to C4-PAF receptor ligands. We conclude UVB photo-oxidizes cellular phospholipids, creating PAF analogs that stimulate the PAF receptor to induce further PAF synthesis and apoptosis. PAF signaling may participate in the cutaneous inflammation that occurs during photo-aggravated dermatoses.     

Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus

The tobacco N gene confers resistance to tobacco mosaic virus (TMV) and encodes a Toll-interleukin-1 receptor/nucleotide binding site/leucine-rich repeat (TIR-NBS-LRR) class protein. We have developed and used a tobacco rattle virus (TRV) based virus induced gene silencing (VIGS) system to investigate the role of tobacco candidate genes in the N-mediated signalling pathway. To accomplish this we generated transgenic Nicotiana benthamiana containing the tobacco N gene. The transgenic lines exhibit hypersensitive response (HR) to TMV and restrict virus spread to the inoculated site. This demonstrates that the tobacco N gene can confer resistance to TMV in heterologous N. benthamiana. We have used this line to study the role of tobacco Rar1-, EDS1-, and NPR1/NIM1- like genes in N-mediated resistance to TMV using a TRV based VIGS approach. Our VIGS analysis suggests that these genes are required for N function. EDS1-like gene requirement for the N function suggests that EDS1 could be a common component of bacterial, fungal and viral resistance signalling mediated by the TIR-NBS-LRR class of resistance proteins. Requirement of Rar1- like gene for N-mediated resistance to TMV and some powdery mildew resistance genes in barley provide the first example of converging points in the disease resistance signalling pathways mediated by TIR-NBS-LRR and CC-NBS-LRR proteins. The TRV based VIGS approach as described here to study N-mediated resistance signalling will be useful for the analysis of not only disease resistance signalling pathways but also of other signalling pathways in genetically intractable plant systems.