The fibroblast growth factor receptors (FGFRs) are important oncogenes promoting tumor progression in many types of cancer, such as breast, bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma. However, little is known about how these receptors are internalized and down-regulated in cells. We have here applied proximity biotin labeling to identify proteins involved in FGFR4 signaling and trafficking. For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed in U2OS cells. Upon addition of biotin to these cells, proteins in proximity to the FGFR4-BirA* fusion protein became biotinylated and could be isolated and identified by quantitative mass spectrometry. We identified in total 291 proteins, including 80 proteins that were enriched in samples where the receptor was activated by the ligand (FGF1), among them several proteins previously found to be involved in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly, many of the identified proteins were implicated in endosomal transport, and by precise annotation we were able to trace the intracellular pathways of activated FGFR4. Validating the data by confocal and three-dimensional structured illumination microscopy analysis, we concluded that FGFR4 uses clathrin-mediated endocytosis for internalization and is further sorted from early endosomes to the recycling compartment and the trans-Golgi network. Depletion of cells for clathrin heavy chain led to accumulation of FGFR4 at the cell surface and increased levels of active FGFR4 and PLCγ, while AKT and ERK signaling was diminished, demonstrating that functional clathrin-mediated endocytosis is required for proper FGFR4 signaling. Thus, this study reveals proteins and pathways involved in FGFR4 transport and signaling that provide possible targets and opportunities for therapeutic intervention in FGFR4 aberrant cancer.
- MeSH
- Staining and Labeling MeSH
- Biotinylation MeSH
- Endocytosis MeSH
- Endosomes metabolism MeSH
- Clathrin metabolism MeSH
- Humans MeSH
- Microscopy methods MeSH
- Cell Line, Tumor MeSH
- Receptor, Fibroblast Growth Factor, Type 4 metabolism MeSH
- Signal Transduction MeSH
- trans-Golgi Network metabolism MeSH
- Protein Transport MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
PURPOSE OF THE STUDY Treatment of comminuted three- and four-part displaced proximal humerus fractures continues to be discussed in daily trauma practice. In fractures with metaphyseal comminuted fractures the anatomical reconstruction is often technically unfeasible. For cases of comminuted metaphyseal proximal humerus fractures we proposed the so called non-anatomical reconstruction with simple osteosynthesis. Even today, when nailing and plating are commonly used in osteosynthesis, the non-anatomical reconstruction plays an irreplaceable role. Its application is conditioned by at least partially preserved vascular blood supply of the head fragment. This paper describes our original technique to manage these fractures and provides an evaluation of results of the group of patients in whom this procedure was performed. MATERIAL AND METHODS Our group included a total of 72 patients (who underwent surgery in the period from 1 January 1989 to 22 March 2016), of whom 57 were clinically assessed (8 patients died, 7 patients failed to be traced back). The mean age at the time of procedure was 53.61 years (range 19-81 years). The mean follow-up was 14.3 years (range 0.3-26.3 years) after the surgery. The method consists in removing the comminution zone, impacted modified diaphyseal fragment to head spongiosis and osteosynthesis of greater and lesser tubercle or their remainders to diaphyseal fragment using tensile cerclage. RESULTS The mean post-operative Constant score was 81.4 (range 30-100 points). The mean abduction was 120.4 degrees (range 60-165 degrees) and ventral flexion was 129.2 degrees (range 70-170 degrees). Excellent clinical outcome according to the Constant score was achieved in 19 patients, good outcome in 23 patients, fair in 8 patients and poor in 7 patients. DISCUSSION We have been using our original method for 27 years. Compared to osteosynthesis by locking plates, minimally invasive procedures and trauma shoulder joint replacement, our method helps achieve very good clinical outcomes. Its main advantage, however, is the fact that by this technique the specific type of fractures can be treated, otherwise manageable exclusively by arthroplasty. CONCLUSIONS At our clinic, the non-anatomical reconstruction belongs to irreplaceable methods for treating certain proximal humerus fractures. The clinical outcomes of this method can be described as very good. The method of non-anatomical reconstruction eliminates the disadvantages and risks of arthroplasty. Nonetheless, it shall be stressed that this method can be successful exclusively when applied to precisely indicated types of fractures and when performed with technical precision. Its another advantage are the minimal financial requirements. Key words: non-anatomical reconstruction, osteosynthesis, proximal humerus, cerclage.
- MeSH
- Shoulder Fractures * diagnosis physiopathology surgery MeSH
- Middle Aged MeSH
- Humans MeSH
- Follow-Up Studies MeSH
- Postoperative Complications diagnosis MeSH
- Shoulder Injuries * diagnostic imaging surgery MeSH
- Shoulder Joint physiopathology surgery MeSH
- Range of Motion, Articular MeSH
- Fractures, Comminuted MeSH
- Fracture Fixation, Internal * adverse effects instrumentation methods MeSH
- Treatment Outcome MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
- Keywords
- proximity ligation assay,
- MeSH
- Fluorescent Antibody Technique MeSH
- Genes, p53 * MeSH
- Protein Interaction Mapping * MeSH
- Tumor Suppressor Proteins * MeSH
- Tumor Suppressor Protein p53 MeSH
- Neoplasms pathology MeSH
- Oligonucleotide Probes MeSH
- Oligonucleotides MeSH
- Protein Isoforms MeSH
- Antibodies MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
In organized tissues, the precise geometry and the overall shape are critical for the specialized functions that the cells carry out. Odontoblasts are major matrix-producing cells of the tooth and have also been suggested to participate in sensory transmission. However, refined morphologic data on these important cells are limited, which hampers the analysis and understanding of their cellular functions. We took advantage of fluorescent color-coding genetic tracing to visualize and reconstruct in 3 dimensions single odontoblasts, pulp cells, and their assemblages. Our results show distinct structural features and compartments of odontoblasts at different stages of maturation, with regard to overall cellular shape, formation of the main process, orientation, and matrix deposition. We demonstrate previously unanticipated contacts between the processes of pulp cells and odontoblasts. All reported data are related to mouse incisor tooth. We also show that odontoblasts express TRPM5 and Piezo2 ion channels. Piezo2 is expressed ubiquitously, while TRPM5 is asymmetrically distributed with distinct localization to regions proximal to and within odontoblast processes.
- MeSH
- Ameloblasts cytology ultrastructure MeSH
- Cell Nucleus ultrastructure MeSH
- Cell Surface Extensions ultrastructure MeSH
- Dentin ultrastructure MeSH
- Extracellular Matrix ultrastructure MeSH
- Fluorescent Antibody Technique MeSH
- Ion Channels ultrastructure MeSH
- TRPM Cation Channels ultrastructure MeSH
- Cell Compartmentation MeSH
- Mesenchymal Stem Cells cytology ultrastructure MeSH
- Microscopy, Electron, Scanning methods MeSH
- Mice, Transgenic MeSH
- Mice MeSH
- Odontoblasts cytology ultrastructure MeSH
- Incisor cytology ultrastructure MeSH
- Cell Shape MeSH
- Imaging, Three-Dimensional methods MeSH
- Dental Pulp cytology ultrastructure MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The mechanism used by mouse polyomavirus (MPyV) overcomes the crowded cytosol to reach the nucleus has not been fully elucidated. Here, we investigated the involvement of importin α/β1 mediated transport in the delivery of MPyV genomes into the nucleus. Interactions of the virus with importin β1 were studied by co-immunoprecipitation and proximity ligation assay. For infectivity and nucleus delivery assays, the virus and its capsid proteins mutated in the nuclear localization signals (NLSs) were prepared and produced. We found that at early times post infection, virions bound importin β1 in a time dependent manner with a peak of interactions at 6 h post infection. Mutation analysis revealed that only when the NLSs of both VP1 and VP2/3 were disrupted, virus did not bind efficiently to importin β1 and its infectivity remarkably decreased (by 80%). Nuclear targeting of capsid proteins was improved when VP1 and VP2 were co-expressed. VP1 and VP2 were effectively delivered into the nucleus, even when one of the NLS, either VP1 or VP2, was disrupted. Altogether, our results showed that MPyV virions can use VP1 and/or VP2/VP3 NLSs in concert or individually to bind importins to deliver their genomes into the cell nucleus.
- MeSH
- Biological Transport MeSH
- Cell Nucleus MeSH
- Cell Line MeSH
- DNA, Viral metabolism MeSH
- Fluorescent Antibody Technique MeSH
- Nuclear Localization Signals genetics MeSH
- Karyopherins metabolism MeSH
- Mutation MeSH
- Mice MeSH
- Polyomavirus Infections metabolism virology MeSH
- Polyomavirus physiology ultrastructure MeSH
- Virus Assembly MeSH
- Amino Acid Substitution MeSH
- Protein Binding MeSH
- Capsid Proteins genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Arbuscular mycorrhizal (AM) fungi play a positive role in plant water relations, and the AM symbiosis is often cited as beneficial for overcoming drought stress of host plants. Nevertheless, water uptake via mycorrhizal hyphal networks has been little addressed experimentally, especially so through isotope tracing. In a greenhouse study conducted in two-compartment rhizoboxes, Medicago truncatula was planted in the primary compartment (PC), either inoculated with Rhizophagus irregularis or left uninoculated. Plant roots were either allowed to enter the secondary compartment (SC) or were restricted to the PC by root-excluding mesh. Substrate moisture was manipulated in the PC such that the plants were grown either in high moisture (15% of gravimetric water content, GWC) or low moisture (8% GWC). Meanwhile, the SC was maintained at 15% GWC throughout and served as a water source accessible (or not) by roots and/or hyphae. Water in the SC was labeled with deuterium (D) to quantify water uptake by the plants from the SC. Significantly, increased D incorporation into plants indicated higher water uptake by mycorrhizal plants when roots had access to the D source, but this was mainly explained by generally larger mycorrhizal root systems in proximity to the D source. On the other hand, AM fungal hyphae with access to the D source increased D incorporation into plants more than twofold compared to non-mycorrhizal plants. Despite this strong effect, water transport via AM fungal hyphae was low compared to the transpiration demand of the plants.
- MeSH
- Glomeromycota * MeSH
- Hyphae MeSH
- Plant Roots MeSH
- Mycorrhizae * MeSH
- Symbiosis MeSH
- Water MeSH
- Publication type
- Journal Article MeSH
In humans, neurosecretory chromaffin cells control a number of important bodily functions, including those related to stress response. Chromaffin cells appear as a distinct cell type at the beginning of midgestation and are the main cellular source of adrenalin and noradrenalin released into the blood stream. In mammals, two different chromaffin organs emerge at a close distance to each other, the adrenal gland and Zuckerkandl organ (ZO). These two structures are found in close proximity to the kidneys and dorsal aorta, in a region where paraganglioma, pheochromocytoma and neuroblastoma originate in the majority of clinical cases. Recent studies showed that the chromaffin cells comprising the adrenal medulla are largely derived from nerve-associated multipotent Schwann cell precursors (SCPs) arriving at the adrenal anlage with the preganglionic nerve fibers, whereas the migratory neural crest cells provide only minor contribution. However, the embryonic origin of the ZO, which differs from the adrenal medulla in a number of aspects, has not been studied in detail. The ZO is composed of chromaffin cells in direct contact with the dorsal aorta and the intraperitoneal cavity and disappears through an autophagy-mediated mechanism after birth. In contrast, the adrenal medulla remains throughout the entire life and furthermore, is covered by the adrenal cortex. Using a combination of lineage tracing strategies with nerve- and cell type-specific ablations, we reveal that the ZO is largely SCP-derived and forms in synchrony with progressively increasing innervation. Moreover, the ZO develops hand-in-hand with the adjacent sympathetic ganglia that coalesce around the dorsal aorta. Finally, we were able to provide evidence for a SCP-contribution to a small but significant proportion of sympathetic neurons of the posterior paraganglia. Thus, this cellular source complements the neural crest, which acts as a main source of sympathetic neurons. Our discovery of a nerve-dependent origin of chromaffin cells and some sympathoblasts may help to understand the origin of pheochromocytoma, paraganglioma and neuroblastoma, all of which are currently thought to be derived from the neural crest or committed sympathoadrenal precursors.
- Publication type
- Journal Article MeSH
Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal-recessive disorder characterized by selective atrophy and progressive weakness of proximal girdle muscles. LGMD2A, the most prevalent form of LGMD, is caused by mutations in the CAPN3 gene that encodes the skeletal muscle-specific member of the calpain family, calpain-3 (p 94). We examined the histopathologic and molecular pathologic findings in 14 Czech LGMD2A patients. Analysis of the CAPN3 gene was performed at the mRNA level, using reverse transcription-polymerase chain reaction (RT-PCR) and sequencing, and/or DNA level, using PCR and denaturing high-performance liquid chromatography (DHPLC). Our results confirm that mutation 550 delA is the most frequent CAPN3 defect in Czech LGMD2A patients (9 alleles of 28). Furthermore, we established that, in a patient with the 550 delA/R490W genotype, mRNA carrying frameshift mutation 550 delA was not detected, probably due to its degradation by nonsense-mediated mRNA decay. In muscle biopsies of two LGMD2A patients, a neurogenic pattern simulating a neurogenic lesion was observed. Immunoblot analysis revealed the deficiency of p 94 in all genetically confirmed cases of LGMD2A, and secondary dysferlin deficiency was demonstrated on muscle membranes in 6 patients using immunofluorescence. Thus, we find a combination of DNA and mRNA mutational analysis to be useful in the diagnosis of LGMD2A. Moreover, our study expands the spectrum of calpainopathies to cases that simulate a neurogenic lesion in muscle biopsies, and the knowledge of possible secondary deficiencies of muscular proteins also contributes to a diagnosis of LGMD2A. Muscle Nerve, 2006.
- MeSH
- Alleles MeSH
- Child MeSH
- Adult MeSH
- Financing, Organized MeSH
- Fluorescent Antibody Technique MeSH
- Genotype MeSH
- Immunohistochemistry MeSH
- Isoenzymes metabolism MeSH
- Calpain metabolism MeSH
- Muscle, Skeletal pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- Membrane Proteins immunology metabolism MeSH
- RNA, Messenger biosynthesis genetics MeSH
- Adolescent MeSH
- DNA Mutational Analysis MeSH
- NAD metabolism MeSH
- Muscular Dystrophies, Limb-Girdle genetics pathology MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Child, Preschool MeSH
- Nerve Tissue Proteins metabolism MeSH
- Muscle Proteins immunology metabolism MeSH
- Chromatography, High Pressure Liquid MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Geographicals
- Czech Republic MeSH
Cystatin C je inhibitor lyzozomálních proteáz a extracelulárních cysteinových proteáz, podílí na regulaci metabolizmu extracelulárních proteinů. Je plně filtrovatelný glomerulem a zcela se vstřebává a katabolizuje buňkami proximálního tubulu ledvin. NGAL (Neutrophil gelatinase-associated lipocalin) je protein akutní fáze, podílí se na antibakteriální imunitě a jeho důležitou vlastností je tvorba komplexu s matrix metaloproteázou 9 (MMP-9), čímž zvyšuje její aktivitu a brání její degradaci. NGAL je volně filtrován přes glomerulární membránu a v proximálním tubulu ledvin je reabsorbován endocytózou. NGAL detekovaný v moči je produkován zejména v distálním nefronu. Podle sérové hladiny cystatinu C a NGAL je možné diagnostikovat akutní poškození ledvin o 1,5–2 dny dříve než při monitoraci funkce ledvin podle sérové hladiny kreatininu. Ve srovnání s informacemi, které poskytuje kreatinin nebo odhadovaná GFR, zvýšení hladiny cystatinu C přináší u pacientů s kardiovaskulárním onemocněním navíc informaci o prognóze těchto pacientů. Zvýšená hladina NGAL byla detekována u pacientů s akutním infarktem myokardu, srdečním selháním nebo po cévní mozkové příhodě. O prognostickém významu NGAL u pacientů po infarktu myokardu nebo se srdečním selháním je zatím minimum dat, rovněž chybí jejich srovnání navzájem a porovnání s běžně stanovovanými natriuretickými peptidy.
Cystatin C is an inhibitor of lysosomal proteases and extracellular cysteine protease, it participates in the regulation of metabolism of extracellular proteins. It is fully glomerular filterable, completely absorbed and catabolised in proximal tubule cells. NGAL (neutrophil gelatinase-associated lipocalin) is an acute phase protein, participating in antibacterial immunity and his important feature is the formation of complex with metalloproteinase 9 (MMP-9), thereby increasing its activity and prevents its degradation. NGAL is freely filtered across the glomerular membrane and is reabsorbed by endocytosis in the proximal tubule. NGAL detected in urine is produced mainly in the distal nephron. The serum cystatin C and NGAL can diagnose acute renal impairment one or two days earlier in the comparison with the monitoring of renal function by serum creatinine. Moreover, compared with the information provided by creatinine or by estimated GFR, the elevated cystatin C gives, in patients with cardiovascular disease, information about worse prognosis. Increased level of NGAL was detected in patients with acute myocardial infarction, heart failure or stroke. There is a lack of data about the prognostic significance of NGAL in patients after myocardial infarction or heart failure, no data about their comparison or interaction with natriuretic peptides exists up today.
- Keywords
- NGAL, biomarker,
- MeSH
- Biomarkers blood MeSH
- Cystatin C physiology blood MeSH
- Cardiovascular Diseases diagnosis blood physiopathology MeSH
- Humans MeSH
- Lipocalins physiology blood MeSH
- Kidney Diseases diagnosis blood physiopathology MeSH
- Prognosis MeSH
- Acute-Phase Proteins physiology MeSH
- Proto-Oncogene Proteins physiology blood MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
