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1.  Systematic investigation of lycopene effects in LNCaP cells by use of novel large-scale proteomic analysis software 
Lycopene, the red pigment of tomatoes, is a carotenoid with potent antioxidant properties. Although lycopene might function as a prostate cancer chemoprevention agent, little is known about its effects at the cellular level. To define general changes induced by treatment of cells with lycopene, and to gain insights into the possible chemoprevention properties of lycopene, we investigated changes in protein expression after lycopene treatment in human LNCaP cells. The high throughput proteomics data were then visualized and analyzed by novel biological protein pathway modeling software. Differentially expressed proteins were identified, and the data were analyzed by protein pathway simulation software without need for specialized programming by importing pathway models from a number of sources or creating their own. One notable outcome was the identification of a group of upregulated proteins involved in detoxification of reactive oxygen species. This finding suggests that a possible mechanism of lycopene chemoprevention is the stimulation of detoxification enzymes associated with the antioxidant response element. Novel biological pathway modeling software enhances analysis of large proteomics data. When applied to the analysis of proteins differentially expressed in prostate cancer cells upon treatment with lycopene, the upregulation of detoxification enzymes was identified.
doi:10.1002/prca.200600511
PMCID: PMC2926987  PMID: 20740054
detoxification enzymes; ICAT; LNCaP; lycopene; teranode
2.  Phosphorylation of Slx4 by Mec1 and Tel1 Regulates the Single-Strand Annealing Mode of DNA Repair in Budding Yeast▿  
Molecular and Cellular Biology  2007;27(18):6433-6445.
Budding yeast (Saccharomyces cerevisiae) Slx4 is essential for cell viability in the absence of the Sgs1 helicase and for recovery from DNA damage. Here we report that cells lacking Slx4 have difficulties in completing DNA synthesis during recovery from replisome stalling induced by the DNA alkylating agent methyl methanesulfonate (MMS). Although DNA synthesis restarts during recovery, cells are left with unreplicated gaps in the genome despite an increase in translesion synthesis. In this light, epistasis experiments show that SLX4 interacts with genes involved in error-free bypass of DNA lesions. Slx4 associates physically, in a mutually exclusive manner, with two structure-specific endonucleases, Rad1 and Slx1, but neither of these enzymes is required for Slx4 to promote resistance to MMS. However, Rad1-dependent DNA repair by single-strand annealing (SSA) requires Slx4. Strikingly, phosphorylation of Slx4 by the Mec1 and Tel1 kinases appears to be essential for SSA but not for cell viability in the absence of Sgs1 or for cellular resistance to MMS. These results indicate that Slx4 has multiple functions in responding to DNA damage and that a subset of these are regulated by Mec1/Tel1-dependent phosphorylation.
doi:10.1128/MCB.00135-07
PMCID: PMC2099619  PMID: 17636031
3.  The 2′-O-Ribose Methyltransferase for Cap 1 of Spliced Leader RNA and U1 Small Nuclear RNA in Trypanosoma brucei▿ †  
Molecular and Cellular Biology  2007;27(17):6084-6092.
mRNA cap 1 2′-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2′-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2′-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2′-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2′-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3′-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.
doi:10.1128/MCB.00647-07
PMCID: PMC1952150  PMID: 17606627
4.  The urologic epithelial stem cell database (UESC) – a web tool for cell type-specific gene expression and immunohistochemistry images of the prostate and bladder 
BMC Urology  2007;7:19.
Background
Public databases are crucial for analysis of high-dimensional gene and protein expression data. The Urologic Epithelial Stem Cells (UESC) database is a public database that contains gene and protein information for the major cell types of the prostate, prostate cancer cell lines, and a cancer cell type isolated from a primary tumor. Similarly, such information is available for urinary bladder cell types.
Description
Two major data types were archived in the database, protein abundance localization data from immunohistochemistry images, and transcript abundance data principally from DNA microarray analysis. Data results were organized in modules that were made to operate independently but built upon a core functionality. Gene array data and immunostaining images for human and mouse prostate and bladder were made available for interrogation. Data analysis capabilities include: (1) CD (cluster designation) cell surface protein data. For each cluster designation molecule, a data summary allows easy retrieval of images (at multiple magnifications). (2) Microarray data. Single gene or batch search can be initiated with Affymetrix Probeset ID, Gene Name, or Accession Number together with options of coalescing probesets and/or replicates.
Conclusion
Databases are invaluable for biomedical research, and their utility depends on data quality and user friendliness. UESC provides for database queries and tools to examine cell type-specific gene expression (normal vs. cancer), whereas most other databases contain only whole tissue expression datasets. The UESC database provides a valuable tool in the analysis of differential gene expression in prostate cancer genes in cancer progression.
doi:10.1186/1471-2490-7-19
PMCID: PMC2231381  PMID: 18072977
5.  PhosphoPep—a phosphoproteome resource for systems biology research in Drosophila Kc167 cells 
The ability to analyze and understand the mechanisms by which cells process information is a key question of systems biology research. Such mechanisms critically depend on reversible phosphorylation of cellular proteins, a process that is catalyzed by protein kinases and phosphatases. Here, we present PhosphoPep, a database containing more than 10 000 unique high-confidence phosphorylation sites mapping to nearly 3500 gene models and 4600 distinct phosphoproteins of the Drosophila melanogaster Kc167 cell line. This constitutes the most comprehensive phosphorylation map of any single source to date. To enhance the utility of PhosphoPep, we also provide an array of software tools that allow users to browse through phosphorylation sites on single proteins or pathways, to easily integrate the data with other, external data types such as protein–protein interactions and to search the database via spectral matching. Finally, all data can be readily exported, for example, for targeted proteomics approaches and the data thus generated can be again validated using PhosphoPep, supporting iterative cycles of experimentation and analysis that are typical for systems biology research.
doi:10.1038/msb4100182
PMCID: PMC2063582  PMID: 17940529
data integration; Drosophila; interactive database; phosphoproteomics; systems biology
6.  Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress 
The Journal of Cell Biology  2007;176(1):89-100.
Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine–rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane–coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress.
doi:10.1083/jcb.200605093
PMCID: PMC2063630  PMID: 17190791
7.  The promoter and transcribed regions of the Leishmania tarentolae spliced leader RNA gene array are devoid of nucleosomes 
BMC Microbiology  2007;7:44.
Background
The spliced leader (SL) RNA provides the 5' m7G cap and first 39 nt for all nuclear mRNAs in kinetoplastids. This small nuclear RNA is transcribed by RNA polymerase II from individual promoters. In Leishmania tarentolae the SL RNA genes reside in two multi-copy tandem arrays designated MINA and MINB. The transcript accumulation from the SL promoter on the drug-selected, episomal SL RNA gene cassette pX-tSL is ~10% that of the genomic array in uncloned L. tarentolae transfectants. This disparity is neither sequence- nor copy-number related, and thus may be due to interference of SL promoter function by epigenetic factors. To explore these possibilities we examined the nucleoplasmic localization of the SL RNA genes as well as their nucleosomal architecture.
Results
The genomic SL RNA genes and the episome did not co-localize within the nucleus. Each genomic repeat contains one nucleosome regularly positioned within the non-transcribed intergenic region. The 363-bp MINA array was resistant to micrococcal nuclease digestion between the -258 and -72 positions relative to the transcription start point due to nucleosome association, leaving the promoter elements and the entire transcribed region exposed for protein interactions. A pattern of ~164-bp protected segments was observed, corresponding to the amount of DNA typically bound by a nucleosome. By contrast, nucleosomes on the pX-tSL episome were randomly distributed over the episomal SL cassette, reducing transcription factor access to the episomal promoter by approximately 74%. Cloning of the episome transfectants revealed a range of transcriptional activities, implicating a mechanism of epigenetic heredity.
Conclusion
The disorganized nucleosomes on the pX episome are in a permissive conformation for transcription of the SL RNA cassette approximately 25% of the time within a given parasite. Nucleosome interference is likely the major factor in the apparent transcriptional repression of the SL RNA gene cassette. Coupled with the requirement for run-around transcription that drives expression of the selectable drug marker, transcription of the episomal SL may be reduced even further due to sub-optimal nucleoplasmic localization and initiation complex disruption.
doi:10.1186/1471-2180-7-44
PMCID: PMC1888695  PMID: 17517143
8.  The skiers knee without swelling or instability, a difficult diagnosis: a case report 
Skiing as a recreational activity has increased exponentially in the last twenty-years. Similar to any sporting activity, participants can sustain various types of injury, which provides the emergency departments with a continuous supply of patients. The injury pattern from the slopes has also changed over this time period, due to alterations and improvements in ski equipment. An increased diversity in alpine skiing techniques, as well as snowboarding and cross-terrain disciplines has also influenced this change.
We present a multi-media experience of a high-speed ski fall that caused a valgus-external rotation injury to the right knee that precluded the patient from further ski activity. There was no bruising, swelling or instability demonstrated and the patient returned to ski activities 24-hours post-injury. Although this injury appeared clinically benign initially, the patient complained of persistent pain around the right knee which was causing occupational difficulties. Following normal clinical assessment, the patient returned to work but continued to complain of persistent pain at the lateral aspect of the right knee. Magnetic Resonance Imaging (MRI) demonstrated extensive bone marrow oedema (BMO), a mild depression of the articular cortex compression with a small focus of articular cartilage disruption and microfractures of the lateral tibial plateau. The patient was treated conservatively and remains well with avoidance of impact exercises 14-months post-injury.
In the presence of any high speed injury, we would stress that regardless of initial normal investigations, clinical suspicion should remain paramount and not deter the physician from further investigation in the presence of continuing symptomatology.
doi:10.1186/1752-1947-1-11
PMCID: PMC1865549  PMID: 17448236

Results 1-8 (8)