BACKGROUND: Human colostrum and milk contain components that influence development. Our aim was to use a protein array to determine the cytokine profile of human lacteal secretions and changes that occur during the early postpartum period. METHODS: We collected 17 samples of colostrum during the first 2 days postpartum and a 2nd group of 5 sets of 2 to 3 sequential colostrum or milk samples (at 20- to 30-h intervals). We analyzed the samples with array membranes consisting of 42 or 79 antibodies directed against cytokines. RESULTS: In most samples, we detected the previously described cytokines interleukin-8 (IL-8)/CXCL8, epidermal growth factor (EGF), growth-related oncoprotein (GRO)/CXCL1-3, angiogenin, transforming growth factor beta-2, and monocyte chemotactic protein 1 (MCP-1/CCL2). In addition, we found 32 cytokines that have not been described before in colostrum. Cytokine concentrations differed among mothers, and the spectrum of cytokines changed with time after delivery. A significant decrease occurred in IL-12 and macrophage inflammatory protein-1delta/CCL15 and a significant increase in MCP-1/CCL2. The production of angiogenin, vascular endothelial growth factor, GRO/CXCL1-3, EGF, and IL-8/CXCL8 remained high throughout. The concentrations of 2 selected cytokines measured with the array technique and ELISA showed moderate to strong correlation (r = 0.63 for EGF and r = 0.84 for IL-8/CXCL8). CONCLUSION: Despite the lack of precise quantification, the protein array might be suitable for cytokine screening. It allows simultaneous detection of a broad spectrum of cytokines (including those not described before) in lacteal secretions.
- MeSH
- Time Factors MeSH
- Chemokines analysis MeSH
- Protein Array Analysis MeSH
- Cytokines analysis MeSH
- Adult MeSH
- Enzyme-Linked Immunosorbent Assay MeSH
- Financing, Organized MeSH
- Colostrum chemistry MeSH
- Humans MeSH
- Milk, Human chemistry MeSH
- Intercellular Signaling Peptides and Proteins analysis MeSH
- Postpartum Period MeSH
- Proteomics MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Comparative Study MeSH
Jumonji (JMJ, Jarid2), a prototypical member of the jumonji domain-containing protein family, plays a major role in embryonic cardiac development, but its role in the developed heart is unclear. Cardiomyocytes from neonatal mouse heart were treated in culture with NO donor SIN-1, 500 microM, for 2, 4, and 20 h. SIN-1 treatment was associated with a significant and 6.9 +/- 2.5 fold increase in jmj gene expression over all time points. The expression of jmj increased markedly and significantly 4.2 +/- 1.1 fold, 16.6 +/- 4.1 fold, and 2.7 +/- 0.3 fold, respectively, at time points 2 h, 4 h, and 20 h after treatment. The ability of the increase in gene expression to translate into an increase in cellular protein expression was ascertained by Western blotting, which showed an increase in the JMJ protein in whole-cell lysates. Because of the relationship of JMJ to Rb and ANP in the heart, gene expression of these proteins was also examined. SIN-1 produced a small but significant increase in Rb2, but not Rb1 or Rb-binding proteins 4, 6, or 7. In contrast, SIN-1 produced a marked and significant reduction in natriuretic peptide precursor type B but not type C to 0.24 +/- 0.09 fold of the control. These data suggest that JMJ may be a critical, previously unrecognized factor that mediates some of the cellular effects of NO, that NO may be able to increase JMJ in diseases associated with reduced JMJ expression.
- MeSH
- Nitric Oxide Donors pharmacology MeSH
- Myocytes, Cardiac metabolism drug effects MeSH
- Cells, Cultured MeSH
- Molsidomine analogs & derivatives pharmacology MeSH
- Mice MeSH
- Natriuretic Peptide, Brain genetics metabolism MeSH
- Nitric Oxide metabolism MeSH
- Retinoblastoma-Like Protein p130 genetics MeSH
- Nerve Tissue Proteins genetics metabolism MeSH
- Gene Expression Regulation MeSH
- Oligonucleotide Array Sequence Analysis MeSH
- Heart MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
Overexpression of procathepsin D (pCD) is reported to occur in numerous types of cancer and is associated with increased growth and metastasis. It has been established that pCD affects multiple stages of tumor progression including proliferation, angiogenesis and metastasis. Previously, we showed that the mitogenic effect of pCD on cancer cells is mediated by interaction of its activation peptide (AP) with yet unidentified cell surface receptor. In this investigation, gene expression profiles were compared between AP-treated and control human breast cancer ZR-75-1 cells to elucidate the mechanism of AP mitogenicity. Several differentially expressed genes involved in signal transduction, regulation of cell cycle, apoptosis, tumor invasion and metastasis were identified using microarray technology. These findings, including overexpression of NF-kappaB2, were confirmed in breast cancer cell lines by reverse-transcriptase PCR (RT-PCR). Understanding the mechanism of pCDs effect on breast cancer cells could extend possibilities of breast cancer treatment in the future.
- MeSH
- Financing, Organized MeSH
- Cathepsin D pharmacology MeSH
- Humans MeSH
- Tumor Cells, Cultured MeSH
- Neoplasm Proteins genetics MeSH
- Breast Neoplasms genetics MeSH
- NF-kappa B p52 Subunit genetics metabolism MeSH
- Peptide Fragments pharmacology MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Enzyme Precursors pharmacology MeSH
- Gene Expression Regulation, Neoplastic drug effects MeSH
- Oligonucleotide Array Sequence Analysis MeSH
- Signal Transduction MeSH
- Gene Expression Profiling MeSH
- Check Tag
- Humans MeSH
Intrinsically disordered regions (IDRs) are protein regions that lack persistent secondary or tertiary structure under native conditions. IDRs represent >40% of the eukaryotic proteome and play a crucial role in protein-protein interactions. The classical approach for identification of these interaction interfaces is based on mutagenesis combined with biochemical techniques such as coimmunoprecipitation or yeast two-hybrid screening. This approach either provides information of low resolution (large deletions) or very laboriously tries to precisely define the binding epitope via single amino acid substitutions. Here, we report the use of a peptide microarray based on the human scaffold protein AXIN1 for high-throughput and -resolution mapping of binding sites for several AXIN1 interaction partners in vitro For each of the AXIN1-binding partners tested, i.e. casein kinase 1 ϵ (CK1ϵ); c-Myc; peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1); and p53, we found at least three different epitopes, predominantly in the central IDR of AXIN1. We functionally validated the specific AXIN1-CK1ϵ interaction identified here with epitope-mimicking peptides and with AXIN1 variants having deletions of short binding epitopes. On the basis of these results, we propose a model in which AXIN1 competes with dishevelled (DVL) for CK1ϵ and regulates CK1ϵ-induced phosphorylation of DVL and activation of Wnt/β-catenin signaling.
- MeSH
- Axin Protein metabolism MeSH
- beta Catenin metabolism MeSH
- Protein Array Analysis methods MeSH
- Phosphorylation MeSH
- Protein Interaction Domains and Motifs * MeSH
- Casein Kinase 1 epsilon metabolism MeSH
- Binding, Competitive MeSH
- Humans MeSH
- Peptides metabolism MeSH
- Dishevelled Proteins metabolism MeSH
- Wnt Proteins metabolism MeSH
- Wnt Signaling Pathway MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
AIM: Evaluation of serum levels of 17 cytokines and 5 adhesion molecules in patients with newly diagnosed acute myeloid leukemia (AML) and in healthy subjects using biochip array technology. METHODS: A total of 15 AML patients and 15 healthy subjects (blood donors) were studied. Serum samples were analyzed by biochip based immunoassays on the Evidence Investigator analyzer. This approach allows multi-analytical determination from a single sample. T-tests were used for statistical analysis. RESULTS: In newly diagnosed AML patients, we found significant increase (p < 0.01) in serum VCAM-1, ICAM-1, E-selectin, L-selectin, and significant increase (p < 0.05) in serum IL-6, IL-8. No significant differences were found in the levels of other evaluated cytokines and adhesion molecules. CONCLUSION: Our results indicate that serum levels of specific cytokines and adhesion molecules (VCAM-1, ICAM-1, E-selectin, L-selectin, IL-6, IL-8) are significantly altered in patients with newly diagnosed AML, showing activity of the disease. Whether these alterations could serve as a prognostic marker for AML is not known. Further studies will be needed to define the potential role of these and additional markers in the risk stratification of AML.
- MeSH
- Leukemia, Myeloid, Acute blood genetics pathology MeSH
- Vascular Cell Adhesion Molecule-1 blood MeSH
- Protein Array Analysis methods MeSH
- Cytokines blood MeSH
- Adult MeSH
- E-Selectin blood MeSH
- Interleukin-6 blood MeSH
- Interleukin-8 blood MeSH
- L-Selectin blood MeSH
- Middle Aged MeSH
- Humans MeSH
- Intercellular Adhesion Molecule-1 blood MeSH
- Cell Adhesion Molecules blood MeSH
- Biomarkers, Tumor blood MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Mass spectrometry proteomics data are typically evaluated against publicly available annotated sequences, but the proteogenomics approach is a useful alternative. A single genome is commonly utilized in custom proteomic and proteogenomic data analysis. We pose the question of whether utilizing numerous different genome assemblies in a search database would be beneficial. We reanalyzed raw data from the exoprotein fraction of four reference Enterobacterial Repetitive Intergenic Consensus (ERIC) I-IV genotypes of the honey bee bacterial pathogen Paenibacillus larvae and evaluated them against three reference databases (from NCBI-protein, RefSeq, and UniProt) together with an array of protein sequences generated by six-frame direct translation of 15 genome assemblies from GenBank. The wide search yielded 453 protein hits/groups, which UpSet analysis categorized into 50 groups based on the success of protein identification by the 18 database components. Nine hits that were not identified by a unique peptide were not considered for marker selection, which discarded the only protein that was not identified by the reference databases. We propose that the variability in successful identifications between genome assemblies is useful for marker mining. The results suggest that various strains of P. larvae can exhibit specific traits that set them apart from the established genotypes ERIC I-V.
- MeSH
- Bacterial Proteins * genetics metabolism MeSH
- Databases, Protein MeSH
- Virulence Factors * genetics metabolism MeSH
- Genome, Bacterial * genetics MeSH
- Paenibacillus larvae * genetics pathogenicity metabolism MeSH
- Proteogenomics * methods MeSH
- Proteomics methods MeSH
- Bees microbiology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
A combined approach to signal enhancement in fluorescence affinity biosensors and assays is reported. It is based on the compaction of specifically captured target molecules at the sensor surface followed by optical probing with a tightly confined surface plasmon (SP) field. This concept is utilized by using a thermoresponsive hydrogel (HG) binding matrix that is prepared from a terpolymer derived from poly(N-isopropylacrylamide) (pNIPAAm) and attached to a metallic sensor surface. Epi-illumination fluorescence and SP-enhanced total internal reflection fluorescence readouts of affinity binding events are performed to spatially interrogate the fluorescent signal in the direction parallel and perpendicular to the sensor surface. The pNIPAAm-based HG binding matrix is arranged in arrays of sensing spots and employed for the specific detection of human IgG antibodies against the Epstein-Barr virus (EBV). The detection is performed in diluted human plasma or with isolated human IgG by using a set of peptide ligands mapping the epitope of the EBV nuclear antigen. Alkyne-terminated peptides were covalently coupled to the pNIPAAm-based HG carrying azide moieties. Importantly, using such low-molecular-weight ligands allowed preserving the thermoresponsive properties of the pNIPAAm-based architecture, which was not possible for amine coupling of regular antibodies that have a higher molecular weight.
- MeSH
- Acrylic Resins chemistry MeSH
- Biosensing Techniques methods MeSH
- Fluorescence MeSH
- Hydrogels chemistry metabolism MeSH
- Immunoglobulin G analysis immunology MeSH
- Epstein-Barr Virus Infections diagnosis immunology metabolism virology MeSH
- Humans MeSH
- Peptide Fragments immunology metabolism MeSH
- Polymers chemistry MeSH
- Epstein-Barr Virus Nuclear Antigens immunology MeSH
- Herpesvirus 4, Human immunology isolation & purification MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The emergence of communicable and non-communicable diseases has posed a health challenge for millions of people worldwide and is a major threat to the economic and social development in the coming century. The occurrence of the recent pandemic, SARS-CoV-2, caused by lethal severe acute respiratory syndrome coronavirus 2, is one such example. Rapid research and development of drugs for the treatment and management of these diseases have become an incredibly challenging task for the pharmaceutical industry. Although, substantial attention has been paid to the discovery of therapeutic compounds from natural sources having significant medicinal potential, their synthesis has made a slow progress. Hence, the discovery of new targets by the application of the latest biotechnological and synthetic biology approaches is very much the need of the hour. Polyketides (PKs) and non-ribosomal peptides (NRPs) found in bacteria, fungi and plants are a diverse family of natural products synthesized by two classes of enzymes: polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS). These enzymes possess immense biomedical potential due to their simple architecture, catalytic capacity, as well as diversity. With the advent of the latest in-silico and in-vitro strategies, these enzymes and their related metabolic pathways, if targeted, can contribute highly towards the biosynthesis of an array of potentially natural drug leads that have antagonist effects on biopolymers associated with various human diseases. In the face of the rising threat from multidrug-resistant pathogens, this will further open new avenues for the discovery of novel and improved drugs by combining natural and synthetic approaches. This review discusses the relevance of polyketides and non-ribosomal peptides and the improvement strategies for the development of their derivatives and scaffolds, and how they will be beneficial for future bioprospecting and drug discovery.
- MeSH
- COVID-19 * MeSH
- COVID-19 Drug Treatment MeSH
- Humans MeSH
- Peptides pharmacology therapeutic use MeSH
- Polyketides * chemistry metabolism pharmacology MeSH
- SARS-CoV-2 MeSH
- Drug Development MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
xv, 469 s. : il. ; 26 cm
