Biomedical subjects

R Reid Townsend

Publications and source records attributed to R Reid Townsend.

12 recordsLinked to original sources

PubMed2025

How Does Tendon Region, Donor, and the Presence of Disease Affect Protein Composition of the Achilles Tendon?

BACKGROUND: Response to treatment for tendinopathy is variable, which may reflect variability in underlying etiology and capacity for the tendon to respond to treatment. Understanding variability in tendon protein composition may help improve our understanding of the mechanistic underpinnings of painful tendon degeneration and inform treatment targets. QUESTIONS/PURPOSES: (1) What factors (tendon region, individual characteristics, presence of disease) contribute to protein compositional (proteomic) and structural variation in human Achilles tendons? (2) What compositional changes characterize tendinopathy, and what protein interactions might contribute to tendon degeneration? (3) How does diabetes influence tendon composition, and what mechanisms might underlie tendon dysfunction in individuals with diabetes? METHODS: In this exploratory, cross-sectional study, human Achilles tendon specimens were obtained from individuals with (diabetes group, n = 5) or without diabetes (control group, n = 5) undergoing lower extremity amputation and from individuals undergoing tendon debridement surgeries for tendinopathy (tendinopathy group, n = 8). Specimens were collected between 2019 and 2023. Protein abundances were quantified and analyzed using mass spectrometry, hierarchical clustering, and principal component analysis. To evaluate the role of tendon region and donor on tendon protein compositional variability, we assessed proteomic differences between three regions in nontendinopathic tendons from three individuals. To identify the contribution of disease (that is, presence of tendinopathy or diabetes) on protein composition, we compared tendons from the tendinopathy (n = 8 [2 males, 6 females], mean ± SD age 48 ± 11 years), diabetes (n = 5 [3 males, 2 females], age 54 ± 9 years), and control (n = 5 [3 males, 2 females], age 42 ± 12 years) groups. Proteomic differences associated with tendinopathy and diabetes were further examined using functional enrichment and protein-protein interaction network analysis. RESULTS: Variability in tendon protein composition was primarily from presence of disease, followed by donor and then tendon region. Protein composition distinguished tendons with tendinopathy from controls, with 311 proteins differentially expressed (152 overexpressed and 159 underexpressed; fold change ≥ 1.5, p < 0.05) and higher Bonar scores indicating greater degeneration (mean ± SD Bonar score tendinopathy group 8.6 ± 1.2 versus control group 2.1 ± 0.7; p = 0.01). Pathway analysis identified dysregulation in extracellular matrix remodeling (TIMP1, MMP3, MMP10), inflammatory response (TNF-α, EGFR1), and metabolic reprogramming. Tendons from individuals with diabetes exhibited minimal proteomic changes compared with the control group, with 66 differentially expressed proteins (31 overexpressed and 35 underexpressed; fold change ≥ 1.5, p < 0.05) with no histopathologic differences between diabetes and control group tendons (mean ± SD Bonar score diabetes group 3.4 ± 1.0 versus control group 2.1 ± 0.7; p = 0.19). Tendons in the diabetes group showed reductions in Type I collagen, enrichment of pathways associated with fibrosis and metabolic dysfunction, and inflammatory pathways associated with α 6 β 4 integrin. CONCLUSION: Our findings indicate that Achilles tendon composition primarily differs based on disease etiology, with tendinopathy showing extensive extracellular matrix disruption and inflammatory activity, whereas tendons from individuals with diabetes exhibit more subtle compositional changes. This distinction suggests that tendinopathy may require targeted interventions addressing tissue remodeling and inflammation, whereas diabetes may predispose tendons to injury but not directly result in degeneration. Understanding these protein compositional variations can help refine hypotheses about disease progression, treatment response, and potential therapeutic targets. CLINICAL RELEVANCE: While proteomic analysis is not currently a part of routine clinical assessment, these findings provide a framework for identifying protein markers that may aid in early diagnosis or patient stratification to improve treatment alignment. Future studies could determine whether these proteomic changes correlate with treatment response and further inform our understanding of early-stage degeneration from chronic disease. By bridging molecular findings with clinical presentation, this study lays the groundwork for future research on precision medicine approaches for tendon disorders, with the long-term goal of tailoring treatment based on both biological and symptomatic characteristics.

Humans↗

PubMed2006

Accurate mass-driven analysis for the characterization of protein phosphorylation. Study of the human Chk2 protein kinase.

We describe the data-dependent analysis of protein phosphorylation using rapid-acquisition nano-LC-linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry (nano-LC-FTMS). The accurate m/z values of singly, doubly, and triply charged species calculated from the theoretical protonated masses of peptides phosphorylated at all Ser, Thr, or Tyr residues of the human checkpoint 2 (Chk2) protein kinase were used for selected ion extraction and chromatographic analysis. Using a kinase-inactive Chk2 mutant as a control, accurate mass measurements from FTMS and collision-induced dissociation spectra, 11 novel Chk2 autophosphorylation sites were assigned. Additionally, the presence of additional Chk2 phosphorylation sites in two unique peptides was deduced from accurate mass measurements. Selected ion chromatograms of all Chk2 phosphopeptides gave single peaks except in three cases in which two closely eluting species were observed. These pairs of phosphopeptides were determined to be positional isomers from MS/MS analysis. In this study, it was also found that ions due to the neutral loss of phosphoric acid from the parent peptide ion were not prominent in 18 of 36 MS/MS spectra of O-linked Chk2 phosphopeptides. Thus, accurate mass-driven analysis and rapid parallel MS/MS acquisition is a useful method for the discovery of new phosphorylation sites that is independent of the signature losses from phosphorylated amino acid residues.

Amino Acid Sequence↗

PubMed2006

Physiologic genomics.

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Diagnosis, Differential↗

PubMed2006

Proteins associated with disease and clinical course in pancreas cancer: a proteomic analysis of plasma in surgical patients.

New biomarkers for pancreas cancer are needed to improve its detection and management. We surveyed the plasma of patients undergoing surgical resection to identify proteins which change in abundance after complete resection of tumor. Using longitudinally collected specimens from surgical patients, we control for normal inter-individual variation which can confound cross-sectional analysis. Recent refinements in two-dimensional gel electrophoresis allowed us to quantify changes in low abundance plasma proteins with precision. To circumvent the traditional limitations of image analysis in comparing two-dimensional gels, we used fluorometric two-dimensional difference gel electrophoresis to resolve the proteins from pre- and post-surgical plasma from each patient on one physical gel. Furthermore, we increased the ability of our assay to detect low-abundance proteins by depleting the plasma of 12 high-abundance proteins with a multi-affinity column. Informative protein spots from 20 plasma samples across 10 patients were submitted for identification with mass-spectrometry. We identified a group of proteins which change consistently in plasma following complete resection of pancreas tumor. Furthermore, we identified proteins which correlate with post-surgical rapid recurrence of disease. With further identification and validation, the candidate biomarkers which we identify in this study may prove to be useful in the diagnosis, management and prognostication of patients with pancreas cancer.

Biomarkers↗

PubMed2005

Comparative proteomic analysis of intra- and interindividual variation in human cerebrospinal fluid.

Cerebrospinal fluid (CSF) is a potential source of biomarkers for many disorders of the central nervous system, including Alzheimer disease (AD). Prior to comparing CSF samples between individuals to identify patterns of disease-associated proteins, it is important to examine variation within individuals over a short period of time so that one can better interpret potential changes in CSF between individuals as well as changes within a given individual over a longer time span. In this study, we analyzed 12 CSF samples, composed of pairs of samples from six individuals, obtained 2 weeks apart. Multiaffinity depletion, two-dimensional DIGE, and tandem mass spectrometry were used. A number of proteins whose abundance varied between the two time points was identified for each individual. Some of these proteins were commonly identified in multiple individuals. More importantly, despite the intraindividual variations, hierarchical clustering and multidimensional scaling analysis of the proteomic profiles revealed that two CSF samples from the same individual cluster the closest together and that the between-subject variability is much larger than the within-subject variability. Among the six subjects, comparison between the four cognitively normal and the two very mildly demented subjects also yielded some proteins that have been identified in previous AD biomarker studies. These results validate our method of identifying differences in proteomic profiles of CSF samples and have important implications for the design of CSF biomarker studies for AD and other central nervous system disorders.

Cerebrospinal Fluid Proteins↗

PubMed2005

Use of proteomic methods to identify serum biomarkers associated with rat liver toxicity or hypertrophy.

BACKGROUND: Our objectives were to identify serum marker proteins in rats that might serve as sensitive indicators of hepatomegaly, hepatocellular necrosis, or hepatobiliary injury and to use them to analyze data from a collaborative proteomics project. METHODS: In each of 4 studies comprising the collaborative project, rats were given 1 of 4 compounds that target the liver through different mechanisms. Sera and liver samples were collected by terminal bleeds at 1 of 3 postdose time points. Sera were depleted of major secretory proteins and then separated into protein features by 2-dimensional gel electrophoresis (2DGE). Liver specimens were also processed and subjected to 2DGE. Protein spots that significantly increased or decreased in quantity after drug treatment were recovered, digested, analyzed by mass spectroscopy, and compared with available databases for identification. Criteria for further consideration were (a) temporal expression (i.e., increase or decrease at early, fulminant, or recovery periods), (b) known biological function, (c) probable hepatic origin, and (d) any previous association with toxicity in published studies. Markers that changed significantly at the early time point were important because of their potential sensitivity for signaling minimal damage. RESULTS: Vitamin D-binding protein, paraoxonase, cellular retinol-binding protein, malate dehydrogenase, F-protein, and purine nucleoside phosphorylase were identified as empirically confirmed serum markers for hepatic effects in drug-treated rats. CONCLUSION: Proteomics can be applied for the identification and confirmation of peripheral biomarkers for altered liver function after toxicant exposure.

1-Naphthylisothiocyanate↗

PubMed2005

DAP12 (KARAP) amplifies inflammation and increases mortality from endotoxemia and septic peritonitis.

DAP12 (KARAP) is a transmembrane signaling adaptor for a family of innate immunoreceptors that have been shown to activate granulocytes and monocytes/macrophages, amplifying production of inflammatory cytokines. Contrasting with these data, recent studies suggest that DAP12 signaling has an inhibitory role in the macrophage response to microbial products (Hamerman, J.A., N.K. Tchao, C.A. Lowell, and L.L. Lanier. 2005. Nat. Immunol. 6:579-586). To determine the in vivo role for DAP12 signaling in inflammation, we measured the response of wild-type (WT) and DAP12-/- mice to septic shock. We show that DAP12-/- mice have improved survival from both endotoxemia and cecal ligation and puncture-induced septic shock. As compared with WT mice, DAP12-/- mice have decreased plasma cytokine levels and a decreased acute phase response during sepsis, but no defect in the recruitment of cells or bacterial control. In cells isolated after sepsis and stimulated ex vivo, DAP12 signaling augments lipopolysaccharide-mediated cytokine production. These data demonstrate that, during sepsis, DAP12 signaling augments the response to microbial products, amplifying inflammation and contributing to mortality.

Acute-Phase Reaction↗

PubMed2005

An integrated functional genomics and metabolomics approach for defining poor prognosis in human neuroendocrine cancers.

Human neuroendocrine (NE) cancers range from relatively indolent to highly aggressive. In this study, we combine functional genomics with metabolomics to identify features of NE cancers associated with a poor outcome. Analysis of GeneChip datasets of primary prostate tumors, as well as lymph node and liver metastases from transgenic mice with a NE cell cancer, plus derived NE cell lines yielded a signature of 446 genes whose expression is enriched in neoplastic mouse prostatic NE cells. This signature was used for in silico metabolic reconstructions of NE cell metabolism, directed liquid chromatography/tandem MS analysis of metabolites in prostatic NE tumors and cell lines, and analysis of GeneChip datasets of human NE tumors with good or poor prognoses. The results indicate that a distinguishing feature of poor-prognosis NE tumors is a glutamic acid decarboxylase-independent pathway for production of GABA and a pathway for production of imidazole-4-acetate that involves dopa decarboxylase and a membrane-associated amine oxidase, amiloride-binding protein 1. Electrophysiological studies disclosed that imidazole-4-acetate can bind and activate GABA(A) receptors expressed by transformed NE cells, thus providing a previously uncharacterized paradigm for NE tumor cell signaling. Transcriptional, metabolic, and electrophysiologic features of transformed mouse NE cells are also evident in neural progenitor cells.

Animals↗

PubMed2005

Vascular respiratory uncoupling increases blood pressure and atherosclerosis.

The observations that atherosclerosis often occurs in non-smokers without elevated levels of low-density lipoprotein cholesterol, and that most atherosclerosis loci so far identified in mice do not affect systemic risk factors associated with atherosclerosis, suggest that as-yet-unidentified mechanisms must contribute to vascular disease. Arterial walls undergo regional disturbances of metabolism that include the uncoupling of respiration and oxidative phosphorylation, a process that occurs to some extent in all cells and may be characteristic of blood vessels being predisposed to the development of atherosclerosis. To test the hypothesis that inefficient metabolism in blood vessels promotes vascular disease, we generated mice with doxycycline-inducible expression of uncoupling protein-1 (UCP1) in the artery wall. Here we show that UCP1 expression in aortic smooth muscle cells causes hypertension and increases dietary atherosclerosis without affecting cholesterol levels. UCP1 expression also increases superoxide production and decreases the availability of nitric oxide, evidence of oxidative stress. These results provide proof of principle that inefficient metabolism in blood vessels can cause vascular disease.

Aconitate Hydratase↗

PubMed2005

Use of protease proteomics to discover granzyme B substrates.

A wide variety of proteases play important roles in immunity, including the destruction of microbes, induction of apoptosis, antigen processing, and regulation of the immune response. Characterization of these proteases requires not only an understanding of substrate specificity, but also the identification of specific protein substrates. Recent advances in proteomics technology have introduced new techniques for the study of protease function. Here, we highlight a proteomic approach used in our laboratory that employs two-dimensional gel electrophoresis coupled with mass spectrometry to identify native protease substrates. With this technique, we have successfully detected both known and novel granzyme B substrates, characterized cleavage products, and identified a granzyme B cleavage site. This approach may serve as an important discovery tool for other immunologic proteases.

Animals↗

PubMed2004

A proteomic approach for the discovery of protease substrates.

Standardized, comprehensive platforms for the discovery of protease substrates have been extremely difficult to create. Screens for protease specificity are now frequently based on the cleavage patterns of peptide substrates, which contain small recognition motifs that are required for the cleavage of the scissile bond within an active site. However, these studies do not identify in vivo substrates, nor can they lead to the definition of the macromolecular features that account for the biological specificity of proteases. To use properly folded proteins in a proteomic screen for protease substrates, we used 2D difference gel electrophoresis and tandem MS to identify substrates of an apoptosis-inducing protease, granzyme B. We confirmed the cleavage of procaspase-3, one of the key substrates of this enzyme, and identified several substrates that were previously unknown, as well as the cleavage site for one of these substrates. We were also able to observe the kinetics of substrate cleavage and cleavage product accumulation by using the 2D difference gel electrophoresis methodology. "Protease proteomics" may therefore represent an important tool for the discovery of the native substrates of a variety of proteases.

Amino Acid Sequence↗

PubMed2002

Comprehensive proteomic analysis of breast cancer cell membranes reveals unique proteins with potential roles in clinical cancer.

Proteins associated with cancer cell plasma membranes are rich in known drug and antibody targets as well as other proteins known to play key roles in the abnormal signal transduction processes required for carcinogenesis. We describe here a proteomics process that comprehensively annotates the protein content of breast tumor cell membranes and defines the clinical relevance of such proteins. Tumor-derived cell lines were used to ensure an enrichment for cancer cell-specific plasma membrane proteins because it is difficult to purify cancer cells and then obtain good membrane preparations from clinical material. Multiple cell lines with different molecular pathologies were used to represent the clinical heterogeneity of breast cancer. Peptide tandem mass spectra were searched against a comprehensive data base containing known and conceptual proteins derived from many public data bases including the draft human genome sequences. This plasma membrane-enriched proteome analysis created a data base of more than 500 breast cancer cell line proteins, 27% of which were of unknown function. The value of our approach is demonstrated by further detailed analyses of three previously uncharacterized proteins whose clinical relevance has been defined by their unique cancer expression profiles and the identification of protein-binding partners that elucidate potential functionality in cancer.

Amino Acid Sequence↗