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PubMed1997

Two distinct anti-allergic drugs, amlexanox and cromolyn, bind to the same kinds of calcium binding proteins, except calmodulin, in bovine lung extract.

In order to explore candidates for proteins required in exocytosis, we used two anti-allergic drugs, amlexanox and cromolyn, which inhibit IgE mediated degranulation of mast cells and basophils, as molecular probes in affinity chromatography. These two drugs chiefly bound to the same kinds of calcium binding proteins in bovine lung. These proteins were as follows: bovine calgranulin C homolog, an 8-kDa unknown protein, S-100L, calgranulin B, calcyphosine, and annexins I-V. The homologous affinity of the two drugs to these proteins is in accord with the similar anti-allergic property of both drugs. From these findings it is presumed that these drugs interact with these proteins and affect pharmacologically the degranulation.

Amino Acid Sequence↗
PubMed2004

Human myometrial gene expression before and during parturition.

Identification of temporal and spatial changes in myometrial gene expression during parturition may further the understanding of the coordinated regulation of myometrial contractions during parturition. The objective of this study was to compare the gene expression profiles of human fundal myometrium from pregnant women before and after the onset of labor using a functional genomics approach, and to further characterize the spatial and temporal expression patterns of three genes believed to be important in parturition. Fundal myometrial mRNA was isolated from five women in labor and five women not in labor, and analyzed using human UniGEM-V microarrays with 9182 cDNA elements. Real-time polymerase chain reaction using myometrial RNA from pregnant women in labor or not in labor was used to examine mRNA levels for three of the genes; namely, prostaglandin-endoperoxide synthase 2 (PTGS2), calgranulin B (S100A9), and oxytocin receptor (OXTR). The spatial expression pattern of these genes throughout the pregnant uterus before and after labor was also determined. Immunolocalization of cyclooxygenase-2 (also known as PTGS2) and S100A9 within the uterine cervix and myometrium were analyzed by immunohistochemistry. Few genes were differentially expressed in fundal myometrial tissues at term with the onset of labor. However, there appears to be a subset of genes important in the parturition cascade. The cellular properties of S100A9, its spatial localization, and dramatic increase in cervix and myometrium of women in labor suggest that this protein may be very important in the initiation or propagation of human labor.

Calgranulin B↗
PubMed2006

Warm ischemia-induced alterations in oxidative and inflammatory proteins in hepatic Kupffer cells in rats.

The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells. Lean Zucker rats (n = 6 each group) were randomized to 75 min of warm ischemia or sham operation. After reperfusion for 8 h, Kupffer cells were isolated by enzymatic perfusion and density gradient centrifugation. Proteins were tryptically digested into peptides and differentially labeled with iTRAQ (isobaric tags for relative and absolute quantitation) reagent. After fractionation by cation exchange chromatography, peptides were identified by mass spectrometry (ESI-LC-MS/MS). Spectra were interrogated against the Swiss-Prot database and quantified using ProteinProspector. The results for heat shock protein 70 and myeloperoxidase were validated by ELISA. Quantitative information for more than 1559 proteins was obtained. In the ischemia group proteins involved in inflammation were significantly up-regulated. The ratio for calgranulin B in the ischemia/sham group was 1.81 +/- 0.97 (p < 0.0001), for complement C3 the ratio was 1.81 +/- 0.49 (p < 0.0001), and for myeloperoxidase the ratio was 1.30 +/- 0.32. Myeloperoxidase was only recently documented in Kupffer cells. The antioxidative proteins Cu,Zn-superoxide dismutase (1.34 +/- 0.19; p < 0.001) and catalase (1.23 +/- 0.43; p < 0.001) were also elevated. In conclusion, ischemia/reperfusion injury induces alterations in the Kupffer cell proteome. Isotope ratio mass spectrometry is a powerful tool to investigate these reactions. The ability to simultaneously monitor several pathways involved in reperfusion stress may result in important mechanistic insight and possibly new treatment options.

Animals↗
PubMed2004

A technical triade for proteomic identification and characterization of cancer biomarkers.

Biomarkers are needed to elucidate the biological background and to improve the detection of cancer. Therefore, we have analyzed laser-microdissected cryostat sections from head and neck tumors and adjacent mucosa on ProteinChip arrays. Two differentially expressed proteins (P = 3.34 x 10(-5) and 4.6 x 10(-5)) were isolated by two-dimensional gel electrophoresis and identified as S100A8 (calgranulin A) and S100A9 (calgranulin B) by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immunodepletion assay. The relevance of these single marker proteins was evaluated by immunohistochemistry. Positive tissue areas were reanalyzed on ProteinChip arrays to confirm the identity of these proteins. As a control, a peak with low P was identified as calgizzarin (S100A11) and characterized in the same way. This technical triade of tissue microdissection, ProteinChip technology, and immunohistochemistry opens up the possibility to find, identify, and characterize tumor relevant biomarkers, which will allow the movement toward the clonal heterogeneity of malignant tumors. Taking this approach, proteins were identified that might be responsible for invasion and metastasis.

Biomarkers, Tumor↗
PubMed2004

Diagnosis of intra-amniotic infection by proteomic profiling and identification of novel biomarkers.

CONTEXT: Intra-amniotic infection (IAI) is commonly associated with preterm birth and adverse neonatal sequelae. Early diagnosis of IAI, however, has been hindered by insensitive or nonspecific tests. OBJECTIVE: To identify unique protein signatures in rhesus monkeys with experimental IAI, a proteomics-based analysis of amniotic fluid was used to develop diagnostic biomarkers for subclinical IAI in amniotic fluid and blood of women with preterm labor. DESIGN, SETTING, AND PARTICIPANTS: Surface-enhanced laser desorption-ionization/time-of-flight mass spectrometry, gel electrophoresis, and tandem mass spectrometry were used to characterize amniotic fluid peptides in 19 chronically instrumented pregnant rhesus monkeys before and after experimental IAI. Candidate biomarkers were determined by liquid chromatography-tandem mass spectrometry. Polyclonal antibodies were generated from synthetic peptides for validation of biomarkers of IAI. Amniotic fluid peptide profiles identified in experimental IAI were subsequently tested in a cohort of 33 women admitted to Seattle, Wash, hospitals between June 25, 1991, and June 30, 1997, with preterm delivery at 35 weeks or earlier associated with subclinical IAI (n = 11), preterm delivery at 35 weeks or earlier without IAI (n = 11), and preterm contractions with subsequent term delivery at later than 35 weeks (n = 11). MAIN OUTCOME MEASURES: Identification of peptide biomarkers for occult IAI. RESULTS: Protein expression profiles in amniotic fluid showed unique signatures of overexpression of polypeptides in the 3- to 5-kDa and 10- to 12-kDa molecular weight ranges in all animals after infection and in no animal prior to infection. In women, the 10- to 12-kDa signature was identified in all 11 patients with subclinical IAI, in 2 of 11 with preterm delivery without IAI, and in 0 of 11 with preterm labor and term delivery without infection (P<.001). Peptide fragment analysis of the diagnostic peak in amniotic fluid identified calgranulin B and a unique fragment of insulinlike growth factor binding protein 1, which were also expressed in maternal serum. Mapping of other amniotic fluid proteins differentially expressed in IAI identified several immunoregulators not previously described in amniotic fluid. CONCLUSIONS: This proteomics-based characterization of the differential expression of amniotic fluid proteins in IAI identified a distinct proteomic profile in an experimental primate chorioamnionitis model that detected subclinical IAI in a human cohort with preterm labor. These diagnostic protein expression signatures, complemented by immunodetection of specific biomarkers in amniotic fluid and in maternal serum, might have application in the early detection of IAI.

Adult↗
PubMed2006

Urine protein markers distinguish stone-forming from non-stone-forming relatives of calcium stone formers.

We have investigated urine protein inhibitors of calcium oxalate crystallization to determine whether variations in these proteins are associated with kidney stone disease and whether protein measurements improve the identification of stone formers compared with conventional risk factors (RF). Using Western blotting, we studied variations in the electrophoretic mobility patterns and relative abundances of crystallization-inhibitory proteins in urine from 50 stone-forming (SF) and 50 non-stone-forming (NS) first-degree relatives of calcium SF patients, matched by gender and age. Standard urine chemistry stone risk measurements were also made. Multivariate discriminant analysis was used to test the association of these proteins with nephrolithiasis. Differences in form and abundance of several urine proteins including inter-alpha-trypsin inhibitor (ITI), prothrombin fragment 1 (PF1), CD59, and calgranulin B (calB) were found to be associated with stone formation. By multivariate discriminant analysis, measurements of forms of PF1, ITI, and calB in men and ITI and CD59 in women, classified 84% of men and 76% of women correctly by stone status. In contrast, standard urine chemistry RF identified only 70% of men correctly and failed to distinguish female SF from NS. Thus a small subset of protein measurements distinguished SF from NS far better than conventional RF in a population of relatives of calcium SF, illustrating the significant association of these proteins with stone disease. Variations in these proteins may serve as markers of stone disease activity or vulnerability to recurrence and may provide new insights into mechanisms of stone formation.

Alpha-Globulins↗
PubMed2006

IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis.

IL-22 is an IFN-IL-10 cytokine family member, which is produced by activated Th1 and NK cells and acts primarily on epithelial cells. Here we demonstrate that IL-22, in contrast to its relative IFN-gamma, regulates the expression of only a few genes in keratinocytes. This is due to varied signal transduction. Gene expressions regulated by IL-22 should enhance antimicrobial defense [psoriasin (S100A7), calgranulin A (S100A8), calgranulin B (S100A9)], inhibit cellular differentiation (e.g., profilaggrin, keratins 1 and 10, kallikrein 7), and increase cellular mobility [e.g., matrix metalloproteinease 1 (MMP1, collagenase 1), MMP3 (stromelysin 1), desmocollin 1]. In contrast, IFN-gamma favored the expression of MHC pathway molecules, adhesion molecules, cytokines, chemokines, and their receptors. The IL-22 effects were transcriptional and either independent of protein synthesis and secretion, or mediated by a secreted protein. Inflammatory conditions, but not keratinocyte differentiation, amplified the IL-22 effects. IL-22 application in mice enhanced cutaneous S100A9 and MMP1 expression. High IL-22 levels in psoriatic skin were associated with strongly up-regulated cutaneous S100A7, S100A8, S100A9, and MMP1 expression. Psoriatic patients showed strongly elevated IL-22 plasma levels, which correlated with the disease severity. Expression of IL-22 and IL-22-regulated genes was reduced by anti-psoriatic therapy. In summary, despite similarities, IFN-gamma primarily amplifies inflammation, while IL-22 may be important in the innate immunity and reorganization of epithelia.

Animals↗
PubMed1992

Subunit structure of calgranulins A and B obtained from sputum, plasma, granulocytes and cultured epithelial cells.

The calcium-binding proteins calgranulins A and B co-purified with an elastase-specific inhibitor after the affinity and cation-exchange chromatography of the perchloric acid-soluble fraction of pooled sputum collected from patients with chronic obstructive pulmonary disease (Sallenave, J.-M. and Ryle, A.P. (1991) Biol. Chem. Hoppe-Seyler 372, 13-21). The calgranulins were separated from the inhibitor by reverse-phase FPLC. Protein blot analysis of the calgranulin fraction in the absence of reducing agent revealed a band of 25 kDa corresponding to the disulphide-bonded heterodimerization of the two monomer components. Similar results were obtained from the immunoprecipitation and protein blot analysis of plasma, granulocytes and cultured epithelial cells. This implies that the calgranulins exist in the heterodimeric form in secretions in vivo. Their association with pancreatic elastase during the affinity chromatography stage of purification implicates them in the tissue destruction elicited by the inflammatory response in chronic obstructive pulmonary diseases.

Calcium↗
PubMed2005

Proteomic study of human bronchoalveolar lavage fluids from smokers with chronic obstructive pulmonary disease by combining surface-enhanced laser desorption/ionization-mass spectrometry profiling with mass spectrometric protein identification.

Bronchoalveolar lavage fluid (BALF) is an important diagnostic source to investigate cellular and molecular changes in the course of lung disorders. The pattern of soluble proteins in BALF obtained from patients at different stages of respiratory disorders may provide deeper insights in the molecular mechanisms of the disease. We used surface-enhanced laser desorption/ionization mass spectrometry (MS) for differential protein display combined with reversed-phase chromatography and subsequent matrix-assisted laser desorption/ionization-MS or nanoliquid chromatography MS/MS analysis for protein identification to compare the protein pattern of BALF samples obtained from ten smokers suffering from chronic obstructive pulmonary disease (COPD), eight clinically asymptomatic smokers, and eight nonsmokers without pulmonary disease. In this context, we were able to identify small proteins and peptides, either differentially expressed or secreted in the course of COPD or in a direct response to cigarette smoke. The concentrations of neutrophil defensins 1 and 2, S100A8 (calgranulin A), and S100A9 (calgranulin B) were elevated in BALFs of smokers with COPD when compared to asymptomatic smokers. Increased concentrations in S100A8 (Calgranulin A), salivary proline-rich peptide P-C, and lysozyme C were detected in BALFs of asymptomatic smokers when compared to nonsmokers, whereas salivary proline-rich peptide P-D and Clara cell phospholipid-binding protein (CC10) were reduced in their concentration. The identified proteins and peptides might be useful in the future as diagnostic markers for smoke-induced lung irritations and COPD.

Bronchoalveolar Lavage Fluid↗
PubMed1995

[Expression of calgranulin A and B in middle ear cholesteatoma].

Cholesteatoma epithelium is characterised by a keratinocyte dysregulation with a hyperproliferative growth and altered differentiation. Keratinocytes with a reduced turnover time show an increase of calcium binding proteins like calgranulin A and B. These proteins are highly linked with the modulation of cell growth and differentiation. The expression of calgranulin A and B was studied immunohistochemically using the monoclonal antibody F12 on cryosections of cholesteatoma biopsies. Normal skin served as control. The staining in normal skin was confined to the highly proliferative activated follicular keratinocytes, whereas most cholesteatomas showed a staining of all cell layers of the epithelium. Our findings show that the keratinocyte dysregulation of the cholesteatoma epithelium is reflected by variations of the expression of calcium binding proteins.

Biopsy↗
PubMed1992

Differential expression of calgranulin A and B in various epithelial cell lines and reconstructed epidermis.

The monoclonal antibody F12, raised against epidermal cells from a psoriatic lesion, decorated antigens highly expressed in psoriatic epidermis and in cultured normal human keratinocytes. In normal human skin, F12 reacted only with follicular keratinocytes. Characterization of the immunoprecipitated antigens by two-dimensional gel electrophoresis revealed their identity with calgranulin A and B. A semiquantitative study with various established epithelial cell lines demonstrated that the expression of calgranulin A and B in hyperproliferative keratinocytes correlates with their potential to undergo terminal differentiation. In epidermis reconstructed in vitro, the antigen expression was stimulated by retinoids and suppressed under vitamin A starvation.

Antibodies, Monoclonal↗
PubMed2003

Identification of 9 genes differentially expressed in head and neck squamous cell carcinoma.

BACKGROUND: Current treatment modalities in squamous cell carcinoma of the head and neck have failed to improve survival. Advances in the discovery of novel biomarkers and targets for therapy are necessary. DESIGN: Differential display and microarray analysis were used to identify differences in gene expression between squamous carcinoma and matched nonmalignant biopsy specimens. Differences in gene expression found in vivo were also tested in vitro by comparing primary cultured normal oral epithelium with head and neck squamous cell carcinoma (HNSCC) cell lines. Results were confirmed by relative reverse transcriptase-polymerase chain reaction and immunohistochemical analysis. RESULTS: In tumors, microarray analysis showed down-regulation of calgranulin B (CAGB), CD24, lymphoepithelial Kazal-type-related inhibitor (LEKTI), zinc finger protein (ZNF-185), transglutaminase-3 (TGM3), and the ETS homologous factor (EHF). In addition, differential display revealed down-regulation of headpin. In contrast, periostin and the human homologue of the Drosophila white gene (ABCG1) were found to be up-regulated by microarray analysis and differential display, respectively. In HNSCC cell lines, LEKTI, ZNF-185, TGM3, headpin, and ABCG1 showed an expression pattern similar to that observed in tumor specimens. Periostin showed an opposite expression pattern in cell lines compared with that of tumor specimens. No consistent pattern of expression was found for CAGB, CD24, and EHF in cell lines. Immunohistochemical analysis revealed that the expression of headpin in nonmalignant mucosa was undetectable in tumors. CONCLUSION: Using differential display and microarray analysis, we have identified and confirmed the differential expression of 9 genes in HNSCC. Work is in progress to determine the biological significance of these genes and their potential as biomarkers or targets for therapy.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed1999

Proteomics in human disease: cancer, heart and infectious diseases.

In recent years, genomics has increased the understanding of many diseases. Proteomics is a rapidly growing research area that encompasses both genetic and environmental factors. The protein composition represents the functional status of a biological compartment. The five approaches presented here resulted in the detection of disease-associated proteins. Calgranulin B was upregulated in colorectal cancer, and hepatoma-derived aldose reductase-like protein was reexpressed in a rat model during hepatocarcinogenesis. In these two investigations, attention was focused on one protein, obviously differing in amount, directly after two-dimensional electrophoresis (2-DE). Additional methods, such as enzyme activity measurements and immunohistochemistry, confirmed the disease association of the two candidates resulting from 2-DE subtractive analysis. The following three investigations take advantage of the holistic potential of the 2-DE approach. The comparison of 2-DE patterns from dilated cardiomyopathy patients with those of controls revealed 25 statistically significant intensity differences, from which 12 were identified by amino acid analysis, Edman degradation or matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). A human myocardial 2-DE database was constructed, containing 3300 protein spots and 150 identified protein species. The number of identified proteins was limited by the capacity of our group, rather than by the principle of feasibility. Another field where proteomics proves to be a valuable tool in identifying proteins of importance for diagnosis is proteome analysis of pathogenic microorganisms such as Borrelia burgdorferi (Lyme disease) and Toxoplasma gondii (toxoplasmosis). Sera from patients with early or late symptoms of Lyme borreliosis contained antibodies of various classes against about 80 antigens each, containing the already described antigens OspA, B and C, flagellin, p83/100, and p39. Similarly, antibody reactivity to seven different marker antigens of T. gondii allowed differentiation between acute and latent toxoplasmosis, an important diagnostic tool in both pregnancy and immunosuppressed patients.

Animals↗
PubMed2001

Identification of a new potential airway irritation marker, palate lung nasal epithelial clone protein, in human nasal lavage fluid with two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight.

We have analyzed protein patterns of human nasal lavage fluid (NLF) with two-dimensional gel electrophoresis (2-DE) and identified several proteins (such as transthyretin, Clara Cell protein 16, lipocalin-1, cystatin S, cystatin SN, immunoglobulin binding factor, statherin, calgranulin B, prolactin-inducible protein, and zinc-alpha2-glycoprotein) by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizationtime of flight (MALDI-TOF) mass spectrometry. To investigate whether airway irritation causes alterations in NLF 2-DE patterns, we compared epoxy workers with airway irritation (n=8) and healthy controls (n=6) before and after 2 h exposure to the epoxy chemical, dimethylbenzylamine (DMBA, 100 microg/m3) in an exposure chamber. A 25 kDa protein with pI 5.5 was found to be altered in the NLF 2-DE patterns; a trypsin digest of the 2-DE spot analyzed by MALDI-TOF and expressed sequence tags (ESTs) determined after post-source decay (PSD) identified the protein as palate lung and nasal epithelial clone (PLUNC). In controls, the levels of NLF-PLUNC were generally lower after 2 h exposure, whereas in epoxy workers, the levels were increased three- to twentyfold after exposure. The human gene sequence for PLUNC was just recently reported and so far no biofunctional data are available. Our results suggest that PLUNC is involved in the airway inflammatory response after exposure to irritants.

Amino Acid Sequence↗
PubMed2001

Profiling proteins from azoxymethane-induced colon tumors at the molecular level by matrix-assisted laser desorption/ionization mass spectrometry.

New developments in mass spectrometry allow for the profiling of the major proteomic content of fresh tissue sections. Briefly, fresh tissue sections are sampled and blotted onto a polyethylene membrane for protein transfer and then subsequently analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Using this technology, we have compared the protein expression of normal and cancerous mouse colon tissue obtained from the same animal. By difference, several protein signals specific to cancerous tissue were observed. A protein extract obtained from the tumors was fractionated by high-performance liquid chromatography and the individual fractions analyzed by MALDI-MS. The fractions containing the targeted proteins were subjected to trypsin digestion. The resulting tryptic peptides were sequenced by tandem mass spectrometry, and based on the recovered partial amino acid sequences, three of the tumor specific protein markers were identified as calgranulin A (S100A8), calgranulin B (S100A9) and calgizzarin (S100A11).

Animals↗
PubMed2006

Gene expression profiling of skin and draining lymph nodes of rats affected with cutaneous contact hypersensitivity.

OBJECTIVES AND DESIGN: The present study aimed at a broad genome expression analysis of rat skin and draining lymph nodes affected with allergic contact dermatitis (ACD). METHODS: ACD was elicited in sensitized SD rats with 0.5% 2,4-dinitrofluorobenzene on skin areas distant from the sensitization sites. Involved skin and lymph nodes were dissected 8 and 24 h after challenge. RNA was hybridized on Affymetrix GeneChips. Expression data were analyzed with the GeneSpring software. RESULTS: Expression of 1,882 out of 8,799 examined genes in skin or lymph nodes was significantly (p < or = 0.01) changed compared to levels in normal tissue. After an additional 2- fold filtering, four expression patterns were selected and interpreted. Prominently up-regulated genes were IL-6, CCR-5, CCL-2, CCL-3, CXCL-1, CXCL-10, TIMP-1, OX-40, calgranulin b, ST2, beta-defensin, iNOS, STAT-1, MMP-3, MMP-9, MMP-12, and MMP-13. CONCLUSIONS: In addition to previously reported transcript changes, the present findings suggest/support that plasma cells, mast cells, and a specific IFN-gamma pathway play a substantial role in the pathogenesis of ACD.

Animals↗
PubMed2002

[cDNA array approach to cytokine expression profile of aseptic loosened hip arthroplasty].

FACTS: Aseptic loosening of hip arthroplasties are the main reason for revision operations. Basic research indicates a significant relevance of the interface membrane formed between the implant an the surrounding bone. Their cellular composition and the influence of various factors on the process of aseptic loosening has attracted scientific interest. Cytokines are essential for intracellular communication. QUESTION: Is it possible to reveal differences in the expression profile of cytokines between well-fixed and failed hip arthroplasties using the cDNA array approach? AIM. Generation of a cytokine expression profile characteristic for failed hip arthroplasty. METHODS AND RESULTS: Radioactively labeled cDNA probes were synthesised from mRNA isolated from the interface membrane of six patients with aseptic loosened hip arthroplasty. Using a phosphorimager the analysis of the cDNA arrays revealed nine cytokines which were overexpressed compared with the reference tissue (Calgranulin A, Calgranulin B, IL-10, MCP-1, RANTES, TFDG1, TNFR2, RAI, THYB10). CONCLUSION: In this study four out of these nine cytokines were found to be connected with the process of aseptic loosening for the first time.

Arthroplasty, Replacement, Hip↗
PubMed2004

In vitro hydroxyapatite adsorbed salivary proteins.

In spite of the present knowledge about saliva components and their respective functions, the mechanism(s) of pellicle and dental plaque formation have hitherto remained obscure. This has prompted recent efforts on in vitro studies using hydroxyapatite (HA) as an enamel model. In the present study salivary proteins adsorbed to HA were extracted with TFA and EDTA and resolved by 2D electrophoresis over a pH range between 3 and 10, digested, and then analysed by MALDI-TOF/TOF mass spectrometry and tandem mass spectrometry. Nineteen different proteins were identified using automated MS and MS/MS data acquisition. Among them, cystatins, amylase, carbonic anhydrase, and calgranulin B, were identified.

Durapatite↗