Toll/interleukin receptor (TIR) domain-containing proteins encoded in the Arabidopsis (Arabidopsis thaliana) genome include the TIR-nucleotide binding site (TN) and TIR-unknown site/domain (TX) families. We investigated the function of these proteins. Transient overexpression of five TX and TN genes in tobacco (Nicotiana benthamiana) induced chlorosis. This induced chlorosis was dependent on ENHANCED DISEASE RESISTANCE1, a dependency conserved in both tobacco and Arabidopsis. Stable overexpression transgenic lines of TX and TN genes in Arabidopsis produced a variety of phenotypes associated with basal innate immune responses; these were correlated with elevated levels of salicylic acid. The TN protein AtTN10 interacted with the chloroplastic protein phosphoglycerate dehydrogenase in a yeast (Saccharomyces cerevisiae) two-hybrid screen; other TX and TN proteins interacted with nucleotide binding-leucine-rich repeat proteins and effector proteins, suggesting that TN proteins might act in guard complexes monitoring pathogen effectors.

Renal cell carcinoma (RCC) accounts for 11,000 deaths per year in the United States. When detected early, generally serendipitously by imaging conducted for other reasons, long term survival is generally excellent. When detected with symptoms, prognosis is poor. Under these circumstances, a screening biomarker has the potential for substantial public health benefit. The purpose of this study was to evaluate the utility of urine metabolomics analysis for metabolomic profiling, identification of biomarkers, and ultimately for devising a urine screening test for RCC. Fifty urine samples were obtained from RCC and control patients from two institutions, and in a separate study, urine samples were taken from 13 normal individuals. Hydrophilic interaction chromatography-mass spectrometry was performed to identify small molecule metabolites present in each sample. Cluster analysis, principal components analysis, linear discriminant analysis, differential analysis, and variance component analysis were used to analyze the data. Previous work is extended to confirm the effectiveness of urine metabolomics analysis using a larger and more diverse patient cohort. It is now shown that the utility of this technique is dependent on the site of urine collection and that there exist substantial sources of variation of the urinary metabolomic profile, although group variation is sufficient to yield viable biomarkers. Surprisingly there is a small degree of variation in the urinary metabolomic profile in normal patients due to time since the last meal, and there is little difference in the urinary metabolomic profile in a cohort of pre-and postnephrectomy (partial or radical) renal cell carcinoma patients, suggesting that metabolic changes associated with RCC persist after removal of the primary tumor. After further investigations relating to the discovery and identity of individual biomarkers and attenuation of residual sources of variation, our work shows that urine metabolomics analysis has potential to lead to a diagnostic assay for RCC. The study of all endogenously produced metabolites, known as metabolomics (or metabonomics), is the youngest of the omics sciences. It is becoming increasingly clear that, of all of the omics techniques, metabolomics has the greatest potential for biomarker discovery because this technique defines the signature of the actual processes that are occurring within the body rather than examining compounds (such as untranscribed DNA or pre-or post-translationally modified proteins) that may be superfluous to these processes (1). In addition, there is a relatively small number of metabolites to examine (with the notable exception of plants, which produce a plethora of secondary metabolites) as compared with genes, transcripts, and proteins in their respective omics fields, and therefore the data germane to metabolomics are more easily handled and analyzed. Proponents of metabolomics provide convincing justification that this technique offers more immediate translational benefit than the other omics fi...

The anorexigenic neuromodulator α-melanocyte-stimulating hormone (α-MSH; referred to here as α-MSH 1-13 ) undergoes extensive posttranslational processing, and its in vivo activity is short lived due to rapid inactivation. The enzymatic control of α-MSH 1-13 maturation and inactivation is incompletely understood. Here we have provided insight into α-MSH 1-13 inactivation through the generation and analysis of a subcongenic mouse strain with reduced body fat compared with controls. Using positional cloning, we identified a maximum of 6 coding genes, including that encoding prolylcarboxypeptidase (PRCP), in the donor region. Realtime PCR revealed a marked genotype effect on Prcp mRNA expression in brain tissue. Biochemical studies using recombinant PRCP demonstrated that PRCP removes the C-terminal amino acid of α-MSH 1-13 , producing α-MSH 1-12 , which is not neuroactive. We found that Prcp was expressed in the hypothalamus in neuronal populations that send efferents to areas where α-MSH 1-13 is released from axon terminals. The inhibition of PRCP activity by small molecule protease inhibitors administered peripherally or centrally decreased food intake in both wild-type and obese mice. Furthermore, Prcp-null mice had elevated levels of α-MSH 1-13 in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet; they were also resistant to high-fat diet-induced obesity. Our results suggest that PRCP is an important component of melanocortin signaling and weight maintenance via control of active α-MSH 1-13 levels.

Kidney cancer is frequently metastatic on presentation at which point the disease is associated with a 95% mortality. Assessment of tumor grade on pathological examination is the most powerful means for prognostication as well as for stratification of patients into those who might respond to conventional or targeted therapy. Although there exist several grading systems in common use, all suffer from significant disparity among observers. In an attempt to objectify this process as well as to acquire grade-specific mechanistic information, we performed LC-MS/MS-based proteomics analysis on 50 clear cell kidney cancers equally distributed among normal tissues and Fuhrman grades 1-4. Initial experiments confirmed the utility of using archived formalinfixed paraffin-embedded samples for LC-MS/MS-based proteomics analysis, and the LC-MS/MS findings were validated by extensive immunoblotting. We now show that changes among many biochemical processes and pathways are strongly grade-dependent with the glycolytic and amino acid synthetic pathways highly represented. In addition, proteins relating to acute phase and xenobiotic metabolism signaling are highly represented. Self-organized mapping of proteins with similar patterns of expression led to the creation of a heat map that will be useful in grade characterization as well as in future research relating to oncogenic mechanisms and targeted therapies for kidney cancer. Molecular & Cellular Proteomics 8:971-985, 2009. Kidney cancer (or renal cell carcinoma (RCC)1 ) is the seventh most common malignancy, the 10th most common cause of cancer death in men, and the ninth most common cancer in women. In 2009, an estimated 13,000 deaths (8,100 men and 4,900 women) will occur in the United States. The disease is frequently asymptomatic; a third of cases are diagnosed when the disease is already metastatic at which time it has 95% mortality (1).Assessment of tumor grade is the most powerful available means to date of determining tumor prognosis; thus objective criteria for assessing grade are essential such that prognostication is unambiguous. In addition, grade criteria are useful in stratifying patients into those most likely to respond to conventional as well as new targeted therapies. There exist several systems for assigning tumor grade in RCC, although most pathologists utilize the Fuhrman grading system. As is evidenced by the abundance of extant grading systems (2), there appears to be a general lack of consensus and thus considerable variability in assigning tumor grades. Objective criteria for grade assignment utilizing specific protein markers will be useful in objectifying this process and thereby allowing for more accurate prognostication. Furthermore assessment of the biological basis of the differences among grades, as evidenced by diverse biochemical pathways altered in a grade-specific fashion, will lead to the development of novel diagnostic assays as well as therapeutic interventions.Once objective grading criteria are put forth, molecular mechanisms by which ...

Background: Renal cell carcinoma (RCC) is the sixth leading cause of cancer death and is responsible for 11,000 deaths per year in the US. Approximately one-third of patients present with disease which is already metastatic and for which there is currently no adequate treatment, and no biofluid screening tests exist for RCC. In this study, we have undertaken a comprehensive proteomic analysis and subsequently a pathway and network approach to identify biological processes involved in clear cell RCC (ccRCC). We have used these data to investigate urinary markers of RCC which could be applied to high-risk patients, or to those being followed for recurrence, for early diagnosis and treatment, thereby substantially reducing mortality of this disease.

Exogenous glycine betaine highly stimulates the growth rate of various members of the Enterobacteriaceae, including Escherichia coli, in media with high salt concentrations (D. Le Rudulier and L. Bouillard, Appi. Environ. Microbiol. 46:152-159, 1983). In a nitrogen-and carbon-free medium, glycine betaine did not support the growth of E. coli either on low-salt or high-salt media. This molecule was taken up by the cells but was not catabolized. High levels of glycine betaine transport occurred when the cells were grown in media of elevated osmotic strength, whereas relatively low activity was found when the cells were grown in minimal medium. A variety of electrolytes, such as NaCl, KCI, NaH2PO4, K2HPO4, K2SO4, and nonelectrolytes like sucrose, raffinose, and inositol triggered the uptake of glycine betaine. Furthermore, in ceUs subjected to a sudden osmotic upshock, glycine betaine uptake showed a sixfold stimulation 30 min after the addition of NaCl. Part of this stimulation might be a consequence of protein synthesis. The transport of glycine betaine was energy dependent and occurred against a concentration gradient. 2,4-Dinitrophenol almost totally abolished the glycine betaine uptake. Azide and arsenate exerted only a small inhibition. In addition, N,N'dicyclohexylcarbodiimide had a very low inhibitory effect at 1 mM. These results indicated that glycine betaine transport is driven by the electrochemical proton gradient. The kinetics of glycine betaine entry followed the Michaelis-Menten relationship, yielding a K. of 35 ,uM and a V,.,. of 42 nmol min-' mg of protein-'. Glycine betaine transport showed considerable structural specificity. The only potent competitor was proline betaine when added to the assay mixtures at 20-fold the glycine betaine concentration. From these results, it is proposed that E. coli possesses an active and specific glycine betaine transport system which is regulated by the osmotic strength of the growth medium.

A 30 kb DNA region from Azospirillum brasilense Sp7, containing the nitrogenase structural genes (nifHDK), has been cloned. The presence of nif genes, in the 20 kb located next to nifHDK, was explored by Tn5 mutagenesis after subcloning various restriction fragments in the broad-host-range suicide vehicle pSUP202. Over 25 mutations due to Tn5 random insertions were obtained in the 20 kb and each recombined into the genome of strain Sp7. Four new nif loci were identified, located at about 4, 9, 12 and 18 kb downstream from nifK respectively. Hybridization with heterologous nif probes from Klebsiella pneumoniae, Bradyrhizobium japonicum and Azorhizobium caulinodans was performed to characterize the new nif regions. The region proximal to nifK appears to contain nifE and the region distal to nifK contains genes homologous to nifUS and fixABC. nifgene(s) from the fourth locus were not identified. Mutants in this locus, which were devoid of nitrogenase activity when tested under nitrogen-free conditions, displayed a high nitrogenase activity when glutamate was added to the growth medium. This phenomenon was also observed with mutants of the fixABC homology region, but to a lesser extent. Homology between strain Sp7 total DNA and a nifB-containing probe from B. japonicum was detected, although the hybridizing region was not part of the nif cluster described above.

Several applications of high throughput genome and transcriptome sequencing would benefit from a reduction of the high-copy-number sequences in the libraries being sequenced and analyzed, particularly when applied to species with large genomes. We adapted and analyzed the consequences of a method that utilizes a thermostable duplex-specific nuclease for reducing the high-copy components in transcriptomic and genomic libraries prior to sequencing. This reduces the time, cost, and computational effort of obtaining informative transcriptomic and genomic sequence data for both fully sequenced and non-sequenced genomes. It also reduces contamination from organellar DNA in preparations of nuclear DNA. Hybridization in the presence of 3 M tetramethylammonium chloride (TMAC), which equalizes the rates of hybridization of GC and AT nucleotide pairs, reduced the bias against sequences with high GC content. Consequences of this method on the reduction of high-copy and enrichment of low-copy sequences are reported for Arabidopsis and lettuce.