Biomedical subjects

Radislav Sedlacek

Publications and source records attributed to Radislav Sedlacek.

8 recordsLinked to original sources

PubMed2026

Searching for New Genes That Cause Usher Syndrome.

PURPOSE: The purpose of this project was to identify novel Usher syndrome (USH) candidate genes from phenotyping data of 9139 knockout (KO) mouse lines. METHODS: We evaluated phenotype data for concurrent retinopathy and hearing abnormalities in single-gene KO mice generated by the International Mouse Phenotyping Consortium (IMPC). A search was performed to determine whether each gene had been previously associated with retinopathy and/or deafness in humans. Bioinformatic tools were used to predict protein interactions, molecular functions, signaling pathways, and the expression of human orthologues of candidate genes in the retina and inner ear. RESULTS: We identified 18 single-gene KO lines exhibiting hearing abnormality and retinopathy after ear and eye examinations, respectively, and/or by histopathology. The molecular functions and signaling pathways of the human orthologues of the 18 candidate genes partially overlapped with those of USH genes. Particularly, FER and DYRK1B proteins were predicted to interact with proteins encoded by known ciliopathy genes. ADIPOR1, ATP8B1, and MPDZ were associated with retinal degeneration in humans. CHSY1 and IDUA may be pathogenic causes of hearing impairment in people. Furthermore, CHSY1, CSTB, and SPRED1 were located adjacent to unsolved genetic loci related to USH. CONCLUSIONS: A screen of 9139 KO mouse lines revealed 18 candidate genes exhibiting both retinal and inner ear abnormalities consistent with the principal clinical features associated with USH. As the observed phenotypes are attributed to gene deletion in mice, these genes warrant further study to determine the causation of retinal degeneration and hearing loss in patients.

Animals↗

PubMed2025

A consensus guide to preclinical indirect calorimetry experiments.

Understanding the complex factors influencing mammalian metabolism and body weight homeostasis is a long-standing challenge requiring knowledge of energy intake, absorption and expenditure. Using measurements of respiratory gas exchange, indirect calorimetry can provide non-invasive estimates of whole-body energy expenditure. However, inconsistent measurement units and flawed data normalization methods have slowed progress in this field. This guide aims to establish consensus standards to unify indirect calorimetry experiments and their analysis for more consistent, meaningful and reproducible results. By establishing community-driven standards, we hope to facilitate data comparison across research datasets. This advance will allow the creation of an in-depth, machine-readable data repository built on shared standards. This overdue initiative stands to markedly improve the accuracy and depth of efforts to interrogate mammalian metabolism. Data sharing according to established best practices will also accelerate the translation of basic findings into clinical applications for metabolic diseases afflicting global populations.

Calorimetry, Indirect↗

PubMed2006

Matrix metalloproteinase-19 expression in keratinocytes is repressed by transcription factors Tst-1 and Skn-1a: implications for keratinocyte differentiation.

Matrix metalloproteinase-19 (MMP-19), unlike other members of the MMP family, is expressed in basal keratinocytes of intact epidermis whereas keratinocytes in suprabasal and higher epidermal layers express this enzyme only during cutaneous disorders. As the activity of MMP-19 effects proliferation, migration, and adhesion of keratinocytes we examined whether transcription factors involved in keratinocyte differentiation repress the expression of MMP-19. Using luciferase reporter assays, POU transcription factors Tst-1 (Oct-6) and Skn-1a (Oct-11) markedly downregulated the activity of MMP-19 promoter in COS-7 cells and HaCaT keratinocytes. Tst-1 alone was able to inhibit 85% of the promoter activity. Skn-1a exhibited a weak inhibitory effect although it synergistically increased effects of Tst-1. HaCaT cells stably transfected with Tst-1 showed a strong decrease of activity of MMP-19 promoter that correlated with suppression of MMP-19, cytokeratin 14 and 5, decreased cell proliferation, and altered expression of involucrin and loricrin. The expression of MMP-9 was also significantly reduced in Tst-1 expressing keratinocytes. MMP-2 was substantially affected during its activation whereas the expression of MMP-28 was unchanged. Our results suggest that Tst-1 and Skn-1a regulate expression of MMPs in keratinocytes and effect both the expression and activation of these proteolytic enzymes.

Animals↗

PubMed2005

Structural characterization and binding properties of the hemopexin-like domain of the matrixmetalloproteinase-19.

The matrixmetalloproteinase-19 (MMP-19) belongs to the superfamily of the zinc-dependent endopeptidases, which are secreted by cells and are involved in the remodeling of the extracellular matrix. The full-length protein consists of a signal peptide, a propeptide, a catalytic domain and a C-terminal hemopexin-like domain. For other members of this superfamily, the hemopexin-like domain has been described to be involved in substrate recognition. In this study, the hemoxpexin domain of MMP-19 was expressed in Escherichia coli, refolded, and purified. For structural characterization, circular dichroism and NMR spectroscopy were used. We show that the hemopexin-like domain of MMP-19 is able to bind calcium and this binding induces a conformational change and an increase in the thermal stability of the domain. MMP-19 promotes proliferation of keratinocytes by cleaving the insulin-like-growth factor binding protein-3, thereby causing the release of IGF-1, which is a potent growth factor for these cells. By plasmon resonance experiments, we show that the isolated hemopexin-like domain is able to bind to the insulin-like-growth factor binding protein-3. These results provide a basis for further structural investigations that could be used for the rational design of potential agonists and antagonists.

Cell Proliferation↗

PubMed2005

Screening for protease substrate by polyvalent phage display.

Proteases are key regulators of many physiological and pathological processes [1,2], and are recognized as important and tractable drug candidates. Consequently, knowledge of protease substrate recognition and specificity promotes identification of biologically relevant substrates, helps elucidating a protease's biological function, and the design of specific inhibitors. Traditional methods for establishing substrate recognition profiles involve the identification of the scissile bond within a given protein substrate by proteomic methods such as Edman degradation. Then, synthetic peptide variants of this sequence can be screened in an iterative fashion to arrive at more optimized substrates. Even though it can be fruitful, this iterative strategy is biased toward the original substrate sequence and it is also tremendously cumbersome. Furthermore, it is not amenable to high throughput analysis. In 1993, Matthew & Wells presented a method for the use of monovalent "substrate phage" libraries for discovering peptide substrates for proteases, in which more than 10(7) potential substrates can be tested concurrently [3]. A library of fusion proteins was constructed containing randomized substrate sequences placed between a binding domain and the gene III coat protein of the filamentous phage, M13, which displays the fusion protein and packages the gene coding for it inside. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). This method allows one to rapidly survey the substrate recognition and specificity of individual or closely related members of proteases. Over the past decade, substrate phage screening has shown terrific utility in rapidly determining protease specificity and characterization of substrate recognition profile of proteases. In some cases, the structural insights of the catalytic domain were obtained from comparison of substrate specificity among closely related family of proteases [4-6]. The number of proteases (from various classes) characterized by this approach testifies to its power. Since the initial development of substrate phage library, different versions of the substrate phage cloning vectors have been constructed to further improve the utility of substrate phage display. This review will provide an overview of the construction of substrate phage display libraries, screening of substrate phage libraries, examples of application, summary and future directions.

Antibody Affinity↗

PubMed2003

Matrix metalloproteinase 19 regulates insulin-like growth factor-mediated proliferation, migration, and adhesion in human keratinocytes through proteolysis of insulin-like growth factor binding protein-3.

Unlike most other matrix metalloproteinases (MMPs) MMP-19 is expressed in undifferentiated basal keratinocytes of healthy human skin. The human keratinocyte cell line HaCaT, which like basal keratinocytes constitutively expresses MMP-19, down-regulated the expression of MMP-19 at high calcium concentrations. Calcium-regulation occurred through E-cadherin mediated cell-cell contacts because neutralizing anti-E-cadherin antibodies restored MMP-19 expression in high calcium. Overexpression of MMP-19 in HaCaT cells (HaCaT-WT) increased cellular proliferation, as well as migration and adhesion on type I collagen. This was due to proteolysis of the insulin-like growth factor (IGF) binding protein-3 by MMP-19, which augmented signaling through the IGF-I receptor, as evidenced by its increased autophosphorylation. Conversely, these effects were not observed in cells transfected with MMP-2 or a catalytically inactive MMP-19 mutant. As further proof that increased IGF-signaling promoted adhesion and migration in HaCaT-WT cells, we reproduced these effects by treating parental HaCaT with IGF-I. We observed dephosphorylation of the focal adhesion kinase in HaCaT-WT as well as IGF-I-treated HaCaT cells, suggesting that inactivating focal adhesion kinase is a mechanism by which IGF-I enhances adhesion. Furthermore, IGF-I-triggered motility on type I collagen was mediated by MMP activity, which, however, was distinct from MMP-19. Considering the coexpression of IGFBP-3 and MMP-19 in the skin, we conclude that MMP-19 is a likely candidate to be the major IGFBP-3 degrading MMP in the quiescent epidermis. This activity might have widespread consequences for the behavior of epidermal keratinocytes.

Cadherins↗

PubMed2003

Matrix metalloproteinase-19 expression in normal and diseased skin: dysregulation by epidermal proliferation.

Most of the matrix metalloproteinases (MMP) are not expressed in normal intact skin but they are upregulated in inflamed or diseased skin. The recently cloned MMP-19 is one of the few MMP members that are also expressed in healthy epidermis. In this study, we found that MMP-19 is generally coexpressed with cytokeratin 14 that is confined to keratinocytes of the stratum basale. MMP-19 was also detected in hair follicles, sebaceous glands, and eccrine sweat glands. Its expression, however, changed in cutaneous diseases exhibiting increased alternations of epidermal proliferation, such as psoriasis, eczema, and tinea. In the affected area, MMP-19 was also found in suprabasal and spinous epidermal layers. We also studied the regulation of MMP-19 expression at the protein level, as well as by using a promoter assay. The constitutive expression of MMP-19 was upregulated with phorbol myristate acetate and downregulated with retinoic acid and dexamethasone. Tumor necrosis factor-alpha, interleukin (IL)-6, TGF-beta, IL-15, IL-8, and RANTES as well as the bacterial compounds lipopolysaccharide and lipoteichoic acid did not show any profound effect in HaCaT cells. In contrast, type IV and type I collagens upregulated MMP-19 significantly. The dysregulation of MMP-19 expression in epidermis suggests its possible involvement in the perpetuation of cutaneous infections and proliferative disorders such as psoriasis.

Blood Vessels↗

PubMed2002

Matrix metalloproteinase-19 is expressed in myeloid cells in an adhesion-dependent manner and associates with the cell surface.

We have previously reported the isolation of the human matrix metalloproteinase (MMP)-19 (also referred to as RASI) from a synovium of a patient suffering from rheumatoid arthritis and its expression at the cell surface of activated PBMC. In this study, we have analyzed the regulation and cell surface expression of human MMP-19 in several human cell lines and blood-derived cells. Among the cell lines analyzed, MMP-19 is largely expressed by lung fibroblasts as well as by myeloid cell lines THP-1 and HL-60. After fractionating PBMC into CD14- and CD14+ populations we found that only the latter one expresses MMP-19. Although the myeloid cell lines as well as CD14+ cells express MMP-19 without stimulation, its production can be up-regulated by phorbol esters (PMA) or by adhesion. The adhesion-dependent expression was down-regulated or even abrogated by blockade of adhesion or interfering with adhesion-controlling signaling using alpha-tocopherol. We have shown that MMP-19 associates with the cell surface of myeloid cells. This cell surface association was not affected by phospholipase C. However, acidic treatment of the THP-1-derived cell membranes abolished the immunoprecipitation of MMP-19 thereof. Moreover, a high salt treatment of THP-1 cells diminished the MMP-19 detection on the cell surface. This implicates a noncovalent attachment of MMP-19 to the cell surface. Because a truncated form of the MMP-19, in which the hemopexin-like domain was deleted (Delta(hp)MMP-19), does not associate with the surface, the hemopexin-like domain appears to be critical for the cell surface attachment of human MMP-19.

Amino Acid Sequence↗