CiteULike: jyuh's Wan

Proteomic identification of palmitoylated proteins.

AF Roth — 2008-05-11T14:44:32-00:00

Methods (San Diego, Calif.), Vol. 40, No. 2. (October 2006), pp. 135-142.

A proteomic method that purifies and identifies palmitoylated proteins from complex protein extracts is described. Using the fatty acid exchange labeling chemistry (described in the preceding report), palmitoyl modifications are exchanged for biotinylated compounds, allowing the subset of palmitoyl-proteins to be affinity-purified and then identified by mass spectroscopic protein identification technologies. The advantages and pitfalls of this new technology are discussed within the context of the recent application of this method in the yeast Saccharomyces cerevisiae.

Urinary biomarkers in septic acute kidney injury

Bagshaw — 2007-08-29T21:07:07-00:00

Intensive Care Medicine, Vol. 33, No. 7. (July 2007), pp. 1285-1296.

Immediate-early inducible function in upstream region of junB gene.

H Wan — 2008-01-25T14:55:30-00:00

Biomed Environ Sci, Vol. 19, No. 3. (June 2006), pp. 210-213.

OBJECTIVE: To analyze the upstream region of radiation-induced junB gene. METHODS: Four plasmids containing 250 bp, 590 bp, 900 bp and 1650 bp, and CAT reporter gene were constructed separately and introduced to L8704 cells. The cells were irradiated with 2 Gy X-rays and incubated at different intervals. Total RNA was extracted from the cells and fluctuation of the CAT mRNA level was assessed by the RNA ratio of CAT/beta-actin measured by quantitative Northern blot hybridization. RESULTS: CAT mRNA expression containing 900 bp and 1560 bp junB promoter remarkably and rapidly increased, and reached its peak 30 min after 2 Gy X-ray irradiation. CONCLUSIONS: 590-900 bp fragments located in the upstream region of junB gene play an important role in the early process of cells against radiation.

The Cholesterol Paradox Is Flawed; Cholesterol Must Be Lowered in Dialysis Patients

Wan — 2007-11-10T19:22:42-00:00

Seminars in Dialysis, Vol. 20, No. 6. (December 2007), pp. 504-509.

High-throughput plasmid cDNA library screening.

KH Wan — 2007-11-02T03:38:57-00:00

Nat Protoc, Vol. 1, No. 2. (2006), pp. 624-632.

Libraries of cDNA clones are valuable resources for analyzing the expression, structure and regulation of genes, and for studying protein functions and interactions. Full-length cDNA clones provide information about intron and exon structures, splice junctions, and 5' and 3' untranslated regions (UTRs). Open reading frames (ORFs) derived from cDNA clones can be used to generate constructs allowing the expression of both wild-type proteins and proteins tagged at their amino or carboxy terminus. Thus, obtaining full-length cDNA clones and sequences for most or all genes in an organism is essential for understanding genome functions. EST sequencing samples cDNA libraries at random, an approach that is most useful at the beginning of large-scale screening projects. As projects progress towards completion, however, the probability of identifying unique cDNAs by EST sequencing diminishes, resulting in poor recovery of rare transcripts. Here we describe an adapted, high-throughput protocol intended for the recovery of specific, full-length clones from plasmid cDNA libraries in 5 d.

EGFR-mediated expression of aquaporin-3 is involved in human skin fibroblast migration.

C Cao — 2007-10-23T08:07:16-00:00

Biochem J, Vol. 400, No. 2. (1 December 2006), pp. 225-234.

AQP3 (aquaporin-3), known as an integral membrane channel in epidermal keratinocytes, facilitates water and glycerol movement into and out of the skin. Here, we demonstrate that AQP3 is also expressed in cultured human skin fibroblasts, which under normal wound healing processes migrate from surrounding tissues to close the wound. EGF (epidermal growth factor), which induced fibroblast migration, also induced AQP3 expression in a time- and dose-dependent manner. CuSO4 and NiCl2, previously known as AQP3 water transport inhibitors, as well as two other bivalent heavy metals Mn2+ and Co2+, inhibited EGF-induced cell migration in human skin fibroblasts. AQP3 knockdown by small interfering RNA inhibited EGF-induced AQP3 expression and cell migration. Furthermore, an EGFR (EGF receptor) kinase inhibitor, PD153035, blocked EGF-induced AQP3 expression and cell migration. MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK inhibitor U0126 and PI3K (phosphoinositide 3-kinase) inhibitor LY294002 also inhibited EGF-induced AQP3 expression and cell migration. Collectively, our findings show for the first time that AQP3 is expressed in human skin fibroblasts and that EGF induces AQP3 expression via EGFR, PI3K and ERK signal transduction pathways. We have provided evidence for a novel role of AQP3 in human skin fibroblast cell migration, which occurs during normal wound healing.

Phosphoprotein pathway mapping: Akt/mammalian target of rapamycin activation is negatively associated with childhood rhabdomyosarcoma survival.

EF Petricoin — 2007-10-16T02:13:09-00:00

Cancer Res, Vol. 67, No. 7. (1 April 2007), pp. 3431-3440.

Mapping of protein signaling networks within tumors can identify new targets for therapy and provide a means to stratify patients for individualized therapy. Despite advances in combination chemotherapy, the overall survival for childhood rhabdomyosarcoma remains approximately 60%. A critical goal is to identify functionally important protein signaling defects associated with treatment failure for the 40% nonresponder cohort. Here, we show, by phosphoproteomic network analysis of microdissected tumor cells, that interlinked components of the Akt/mammalian target of rapamycin (mTOR) pathway exhibited increased levels of phosphorylation for tumors of patients with short-term survival. Specimens (n = 59) were obtained from the Children's Oncology Group Intergroup Rhabdomyosarcoma Study (IRS) IV, D9502 and D9803, with 12-year follow-up. High phosphorylation levels were associated with poor overall and poor disease-free survival: Akt Ser(473) (overall survival P < 0.001, recurrence-free survival P < 0.0009), 4EBP1 Thr(37/46) (overall survival P < 0.0110, recurrence-free survival P < 0.0106), eIF4G Ser(1108) (overall survival P < 0.0017, recurrence-free survival P < 0.0072), and p70S6 Thr(389) (overall survival P < 0.0085, recurrence-free survival P < 0.0296). Moreover, the findings support an altered interrelationship between the insulin receptor substrate (IRS-1) and Akt/mTOR pathway proteins (P < 0.0027) for tumors from patients with poor survival. The functional significance of this pathway was tested using CCI-779 in a mouse xenograft model. CCI-779 suppressed phosphorylation of mTOR downstream proteins and greatly reduced the growth of two different rhabdomyosarcoma (RD embryonal P = 0.00008; Rh30 alveolar P = 0.0002) cell lines compared with controls. These results suggest that phosphoprotein mapping of the Akt/mTOR pathway should be studied further as a means to select patients to receive mTOR/IRS pathway inhibitors before administration of chemotherapy.

Established risk factors account for most of the racial differences in cardiovascular disease mortality.

SO Henderson — 2007-10-08T04:16:36-00:00

PLoS ONE, Vol. 2, No. 4. (2007)

BACKGROUND: Cardiovascular disease (CVD) mortality varies across racial and ethnic groups in the U.S., and the extent that known risk factors can explain the differences has not been extensively explored. METHODS: We examined the risk of dying from acute myocardial infarction (AMI) and other heart disease (OHD) among 139,406 African-American (AA), Native Hawaiian (NH), Japanese-American (JA), Latino and White men and women initially free from cardiovascular disease followed prospectively between 1993-1996 and 2003 in the Multiethnic Cohort Study (MEC). During this period, 946 deaths from AMI and 2,323 deaths from OHD were observed. Relative risks of AMI and OHD mortality were calculated accounting for established CVD risk factors: body mass index (BMI), hypertension, diabetes, smoking, alcohol consumption, amount of vigorous physical activity, educational level, diet and, for women, type and age at menopause and hormone replacement therapy (HRT) use. RESULTS: Established CVD risk factors explained much of the observed racial and ethnic differences in risk of AMI and OHD mortality. After adjustment, NH men and women had greater risks of OHD than Whites (69% excess, P<0.001 and 62% excess, P = 0.003, respectively), and AA women had greater risks of AMI (48% excess, P = 0.01) and OHD (35% excess, P = 0.007). JA men had lower risks of AMI (51% deficit, P<0.001) and OHD (27% deficit, P = 0.001), as did JA women (AMI, 37% deficit, P = 0.03; OHD, 40% deficit, P = 0.001). Latinos had underlying lower risk of AMI death (26% deficit in men and 35% in women, P = 0.03). CONCLUSION: Known risk factors explain the majority of racial and ethnic differences in mortality due to AMI and OHD. The unexplained excess in NH and AA and the deficits in JA suggest the presence of unmeasured determinants for cardiovascular mortality that are distributed unequally across these populations.

MSNovo: a dynamic programming algorithm for de novo peptide sequencing via tandem mass spectrometry.

L Mo — 2007-08-26T13:33:45-00:00

Anal Chem, Vol. 79, No. 13. (1 July 2007), pp. 4870-4878.

Tandem mass spectrometry (MS/MS) has become the experimental method of choice for high-throughput proteomics-based biological discovery. The two primary ways of analyzing MS/MS data are database search and de novo sequencing. In this paper, we present a new approach to peptide de novo sequencing, called MSNovo, which has the following advanced features. (1) It works on data generated from both LCQ and LTQ mass spectrometers and interprets singly, doubly, and triply charged ions. (2) It integrates a new probabilistic scoring function with a mass array-based dynamic programming algorithm. The simplicity of the scoring function, with only 6-10 parameters to be trained, avoids the problem of overfitting and allows MSNovo to be adopted for other machines and data sets easily. The mass array data structure explicitly encodes all possible peptides and allows the dynamic programming algorithm to find the best peptide. (3) Compared to existing programs, MSNovo predicts peptides as well as sequence tags with a higher accuracy, which is important for those applications that search protein databases using the de novo sequencing results. More specifically, we show that MSNovo outperforms other programs on various ESI ion trap data. We also show that for high-resolution data the performance of MSNovo improves significantly. Supporting Information, executable files and data sets can be found at http://msms.usc.edu/supplementary/msnovo.

Palmitoylated proteins: purification and identification.

J Wan — 2007-08-14T02:00:31-00:00

Nat Protoc, Vol. 2, No. 7. (2007), pp. 1573-1584.

This proteomic protocol purifies and identifies palmitoylated proteins (i.e., S-acylated proteins) from complex protein extracts. The method relies on an acyl-biotinyl exchange chemistry in which biotin moieties are substituted for the thioester-linked protein acyl-modifications through a sequence of three in vitro chemical steps: (i) blockade of free thiols with N-ethylmaleimide; (ii) cleavage of the Cys-palmitoyl thioester linkages with hydroxylamine; and (iii) labeling of thiols, newly exposed by the hydroxylamine, with biotin-HPDP (Biotin-HPDP-N-[6-(Biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide. The biotinylated proteins are then affinity-purified using streptavidin-agarose and identified by multi-dimensional protein identification technology (MuDPIT), a high-throughput, tandem mass spectrometry (MS/MS)-based proteomic technology. MuDPIT also affords a semi-quantitative analysis that may be used to assess the gross changes induced to the global palmitoylation profile by mutation or drugs. Typically, 2-3 weeks are required for this analysis.

Proteomic identification of palmitoylated proteins

Amy Roth — 2007-07-15T09:18:40-00:00

Methods, Vol. 40, No. 2. (October 2006), pp. 135-142.

A proteomic method that purifies and identifies palmitoylated proteins from complex protein extracts is described. Using the fatty acid exchange labeling chemistry (described in the preceding report), palmitoyl modifications are exchanged for biotinylated compounds, allowing the subset of palmitoyl-proteins to be affinity-purified and then identified by mass spectroscopic protein identification technologies. The advantages and pitfalls of this new technology are discussed within the context of the recent application of this method in the yeast Saccharomyces cerevisiae.

Absolute quantification of gene expression in biomaterials research using real-time PCR.

DT Leong — 2007-07-11T15:51:54-00:00

Biomaterials, Vol. 28, No. 2. (January 2007), pp. 203-210.

One major measurement of tissue-engineered constructs efficacy and performance is determining expression levels of genes of interest at the molecular level. This measurement is commonly carried out with reverse transcription-polymerase chain reaction (RT-PCR). In this study, we described a novel method in achieving absolute quantification of gene expression using real-time PCR (aqPCR). This novel method did not require molecular cloning steps to prepare the standards for quantification comparison. Standards were linear double-stranded DNA molecules instead of the typical gene-in-plasmid format. aqPCR could also be used to give relative quantification comparisons between samples simply by dividing the copy numbers readings of the gene of interest with that of the normalization gene. RNA was extracted from monolayer and from polycaprolactone scaffold cultures and assayed for beta-actin and osteocalcin genes. We compared our aqPCR method with end-point PCR since end-point PCR is still a common means of measuring gene expression in the biomaterials field. This study showed that aqPCR was a better method to quantify gene expression than end-point PCR. With our described linear DNA standards method, we were able to obtain not only relative quantification of osteocalcin and beta-actin expression level but also actual copy numbers of osteocalcin and beta-actin for the monolayer culture and to be 1.34 x 10(4) and 1.45 x 10(7) copies, respectively and for the scaffold cultures to be 772 and 2.83 x 10(5) copies, respectively per starting total RNA mass of 10 ng. The standards curves made from these linear DNA standards showed good linearity (R(2)=0.9964 and 0.9902 for osteocalcin and beta-actin standards graphs), ranged from 10 to 10(9) copies and of comparable accuracy to current absolute quantification real-time PCR methods (which used plasmid standards obtained through molecular cloning methods). Our method might be a viable and more user-friendly alternative to current absolute quantification real-time PCR protocols.

Serial changes in urinary proteome profile of membranous nephropathy: implications for pathophysiology and biomarker discovery.

HH Ngai — 2007-06-27T02:28:28-00:00

J Proteome Res, Vol. 5, No. 11. (November 2006), pp. 3038-3047.

Membranous nephropathy is one of the most common causes of primary glomerular diseases worldwide. The present study adopted a gel-based proteomics approach to better understand the pathophysiology and define biomarker candidates of human membranous nephropathy using an animal model of passive Heymann nephritis (PHN). Clinical characteristics of Sprague-Dawley rats injected with rabbit anti-Fx1A antiserum mimicked those of human membranous nephropathy. Serial urine samples were collected at Days 0, 10, 20, 30, 40, and 50 after the injection with anti-Fx1A (number of rats = 6; total number of gels = 36). Urinary proteome profiles were examined using 2D-PAGE and SYPRO Ruby staining. Quantitative intensity analysis and ANOVA with Tukey post-hoc multiple comparisons revealed 37 differentially expressed proteins among 6 different time-points. These altered proteins were successfully identified by MALDI-TOF MS and classified into 6 categories: (i) proteins with decreased urinary excretion during PHN; (ii) proteins with increased urinary excretion during PHN; (iii) proteins with increased urinary excretion during PHN, but which finally returned to basal levels; (iv) proteins with increased urinary excretion during PHN, but which finally declined below basal levels; (v) proteins with undetectable levels in the urine during PHN; and (vi) proteins that were detectable in the urine only during PHN. Most of these altered proteins have functional significance in signaling pathways, glomerular trafficking, and controlling the glomerular permeability. The ones in categories (v) and (vi) may serve as biomarkers for detecting or monitoring membranous nephropathy. After normalization of the data with 24-h urine creatinine excretion, changes in 34 of initially 37 differentially expressed proteins remained statistically significant. These data underscore the significant impact of urinary proteomics in unraveling disease pathophysiology and biomarker discovery.