Capillary network characteristics are invaluable for diagnostics of muscle diseases. Biopsy material is limited in size and mostly not accessible for intensive research. Therefore, especially in human tissue, studies are performed on autopsy material. To approach the problem whether it is reliable to deduce hypotheses from autopsy material to explain physiological and pathological processes, we studied capillarity in pig soleus muscle 1 and 24 hr after death. Capillaries and muscle fibers were immunofluorescently marked, and images were acquired with a confocal microscope. Characteristics of the capillary network were estimated by image analysis methods using several plugins of the Ellipse program. Twenty-four hours after death, the measured characteristics of the capillary network differ by up to 50% when compared with samples excised 1 hr after death. Muscle fiber diameter, the measured capillary length, and tortuosity were reduced, and capillary network became more anisotropic. The main postmortem change that affects capillaries is evidently geometric deformation of muscle tissue. In conclusion, when comparing results from biopsy samples with those from autopsy samples, the effect of postmortem changes on the measured parameters must be carefully considered.
- MeSH
- kapiláry patologie ultrastruktura MeSH
- konfokální mikroskopie metody MeSH
- kosterní svaly krevní zásobení patologie MeSH
- počítačové zpracování obrazu metody MeSH
- posmrtné změny MeSH
- prasata MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Caspases have functions particularly in apoptosis and inflammation. Increasing evidence indicates novel roles of these proteases in cell differentiation, including those involved in osteogenesis. This investigation provides a complex screening of osteogenic markers affected by pan caspase inhibition in micromass cultures derived from mouse forelimbs. PCR Array analysis showed significant alterations in expression of 49 osteogenic genes after 7 days of inhibition. The largest change was a decrease in CD36 expression, which was confirmed at organ level by caspase inhibition in cultured mouse ulnae followed by CD36 immunohistochemical analysis. So far, available data point to osteogenic potential of pro-apoptotic caspases. Therefore, the expression of pro-apoptotic caspases (-3, -6, -7, -8, -9) within the growth plate of mouse forelimbs at the stage where the individual zones are clearly apparent was studied. Caspase-9 was reported in the growth plate for the first time as well as caspase-6 and -7 in the resting zone, caspase-7 in the proliferation, and caspase-6 and -8 in the ossification zone. For all caspases, there was a gradient increase in activation toward the ossification zone. The distribution of staining varied significantly from that of apoptotic cells, and thus, the results further support non-apoptotic participation of caspases in osteogenesis.
- MeSH
- antigeny CD36 analýza genetika MeSH
- imunohistochemie MeSH
- inhibitory kaspas farmakologie MeSH
- kaspasy metabolismus MeSH
- kultivované buňky MeSH
- myši MeSH
- orgánové kultury - kultivační techniky MeSH
- osteogeneze * účinky léků MeSH
- přední končetina růst a vývoj metabolismus MeSH
- vývojová regulace genové exprese účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In glioblastoma, a fraction of malignant cells consists of therapy-resistant glioblastoma stem cells (GSCs) residing in protective niches that recapitulate hematopoietic stem cell (HSC) niches in bone marrow. We have previously shown that HSC niche proteins stromal cell-derived factor-1α (SDF-1α), C-X-C chemokine receptor type 4 (CXCR4), osteopontin (OPN), and cathepsin K (CatK) are expressed in hypoxic GSC niches around arterioles in five human glioblastoma samples. In HSC niches, HSCs are retained by binding of SDF-1α and OPN to their receptors CXCR4 and CD44, respectively. Protease CatK cleaves SDF-1α to release HSCs out of niches. The aim of the present study was to reproduce the immunohistochemical localization of these GSC markers in 16 human glioblastoma samples with the addition of three novel markers. Furthermore, we assessed the type of blood vessels associated with GSC niches. In total, we found seven GSC niches containing CD133-positive and nestin-positive GSCs as a single-cell layer exclusively around the tunica adventitia of 2% of the CD31-positive and SMA-positive arterioles and not around capillaries and venules. Niches expressed SDF-1α, CXCR4, CatK, OPN, CD44, hypoxia-inducible factor-1α, and vascular endothelial growth factor. In conclusion, we show that GSC niches are present around arterioles and express bone marrow HSC niche proteins.
- MeSH
- antigeny CD44 analýza MeSH
- arterioly patologie MeSH
- barvení a značení metody MeSH
- chemokin CXCL12 analýza MeSH
- dospělí MeSH
- glioblastom krevní zásobení patologie MeSH
- hematopoetické kmenové buňky patologie MeSH
- imunohistochemie metody MeSH
- kathepsin K analýza MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádorové kmenové buňky patologie MeSH
- nádory mozku krevní zásobení patologie MeSH
- nika kmenových buněk * MeSH
- osteopontin analýza MeSH
- receptory CXCR4 analýza MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA damage response (DDR) in ribosomal genes and mechanisms of DNA repair in embryonic stem cells (ESCs) are less explored nuclear events. DDR in ESCs should be unique due to their high proliferation rate, expression of pluripotency factors, and specific chromatin signature. Given short population doubling time and fast progress through G1 phase, ESCs require a sustained production of rRNA, which leads to the formation of large and prominent nucleoli. Although transcription of rRNA in the nucleolus is relatively well understood, little is known about DDR in this nuclear compartment. Here, we directed formation of double-strand breaks in rRNA genes with I- PpoI endonuclease, and we studied nucleolar morphology, DDR, and chromatin modifications. We observed a pronounced formation of I- PpoI-induced nucleolar caps, positive on BRCA1, NBS1, MDC1, γH2AX, and UBF1 proteins. We showed interaction of nucleolar protein TCOF1 with HDAC1 and TCOF1 with CARM1 after DNA injury. Moreover, H3R17me2a modification mediated by CARM1 was found in I- PpoI-induced nucleolar caps. Finally, we report that heterochromatin protein 1 is not involved in DNA repair of nucleolar caps.
- MeSH
- acetylace MeSH
- arginin metabolismus MeSH
- buněčné jadérko genetika ultrastruktura MeSH
- buněčné linie MeSH
- dvouřetězcové zlomy DNA * MeSH
- embryonální kmenové buňky metabolismus ultrastruktura MeSH
- fosfoproteiny metabolismus MeSH
- geny rRNA MeSH
- histondeacetylasa 1 metabolismus MeSH
- histony metabolismus MeSH
- jaderné proteiny metabolismus MeSH
- metylace MeSH
- myši MeSH
- oprava DNA MeSH
- proteinarginin-N-methyltransferasy metabolismus MeSH
- RNA ribozomální genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Wnt signaling comprises a group of pathways emanating from the extracellular environment through cell-surface receptors into the intracellular milieu. Wnt signaling cascades can be divided into two main branches, the canonical/β-catenin pathway and the non-canonical pathways containing the Wnt/planar cell polarity and Wnt/calcium signaling. Syndecans are type I transmembrane proteoglycans with a long evolutionary history, being expressed in all Bilateria and in almost all cell types. Both Wnt pathways have been extensively studied over the past 30 years and shown to have roles during development and in a multitude of diseases. Although the first evidence for interactions between syndecans and Wnts dates back to 1997, the number of studies connecting these pathways is low, and many open questions remained unanswered. In this review, syndecan's involvement in Wnt signaling pathways as well as some of the pathologies resulting from dysregulation of the components of these pathways are summarized.
Inosine-5'-monophosphate dehydrogenase catalyzes the critical step in the de novo synthesis of guanosine nucleotides: the oxidation of inosine monophosphate to xanthosine monophosphate. This reaction can be inhibited by specific inhibitors, such as ribavirin or mycophenolic acid, which are widely used in clinical treatment when required to inhibit the proliferation of viruses or cells. However, it was recently found that such an inhibition affects the cells, leading to a redistribution of IMPDH2 and the appearance of IMPDH2 inclusions in the cytoplasm. According to their shape, these inclusions have been termed "Rods and Rings" (R&R). In this work, we focused on the subcellular localization of IMPDH2 protein and the ultrastructure of R&R inclusions. Using microscopy and western blot analysis, we show the presence of nuclear IMPDH2 in human cells. We also show that the nuclear pool has an ability to form Rod structures after inhibition by ribavirin. Concerning the ultrastructure, we observed that R&R inclusions in cellulo correspond to the accumulation of fibrous material that is not surrounded by a biological membrane. The individual fibers are composed of regularly repeating subunits with a length of approximately 11 nm. Together, our findings describe the localization of IMPDH2 inside the nucleus of human cells as well as the ultrastructure of R&R inclusions.
- MeSH
- aktivní transport - buněčné jádro účinky léků MeSH
- buněčné jádro enzymologie metabolismus ultrastruktura MeSH
- cytoplazma enzymologie ultrastruktura MeSH
- HeLa buňky MeSH
- IMP-dehydrogenasa antagonisté a inhibitory chemie metabolismus ultrastruktura MeSH
- inhibitory enzymů farmakologie MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cannabinoid receptor type 2 (CB2R) plays a critical role in nociception. In contrast to cannabinoid receptor type 1 ligands, CB2R agonists do not produce undesirable central nervous system effects and thus promise to treat neuropathic pain that is often resistant to medical therapy. In the study presented here, we evaluated the bilateral distribution of the CB2R protein and messenger RNA (mRNA) in rat dorsal root ganglia (DRG) after unilateral peripheral nerve injury using immunohistochemistry, western blot, and in situ hybridization analysis. Unilateral chronic constriction injury (CCI) of the sciatic nerve induced neuropathic pain behavior and bilateral elevation of both CB2R protein and mRNA in lumbar L4-L5 as well as cervical C7-C8 DRG when compared with naive animals. CB2R protein and mRNA were increased not only in DRG neurons but also in satellite glial cells. The fact that changes appear bilaterally and (albeit at a lower level) even in the remote cervical DRG can be related to propagation of neuroinflammation alongside the neuraxis and to the neuroprotective effects of CB2R.
- MeSH
- chování zvířat MeSH
- krysa rodu rattus MeSH
- messenger RNA genetika metabolismus MeSH
- modely nemocí na zvířatech MeSH
- nervus ischiadicus zranění MeSH
- neuralgie genetika metabolismus patologie MeSH
- potkani Wistar MeSH
- receptor kanabinoidní CB2 genetika metabolismus MeSH
- regulace genové exprese * MeSH
- spinální ganglia metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The nucleolus is a nuclear compartment that plays an important role in ribosome biogenesis. Some structural features and epigenetic patterns are shared between nucleolar and non-nucleolar compartments. For example, the location of transcriptionally active mRNA on extended chromatin loop species is similar to that observed for transcriptionally active ribosomal DNA (rDNA) genes on so-called Christmas tree branches. Similarly, nucleolus organizer region-bearing chromosomes located a distance from the nucleolus extend chromatin fibers into the nucleolar compartment. Specific epigenetic events, such as histone acetylation and methylation and DNA methylation, also regulate transcription of both rRNA- and mRNA-encoding loci. Here, we review the epigenetic mechanisms and structural features that regulate transcription of ribosomal and mRNA genes. We focus on similarities in epigenetic and structural regulation of chromatin in nucleoli and the surrounding non-nucleolar region and discuss the role of proteins, such as heterochromatin protein 1, fibrillarin, nucleolin, and upstream binding factor, in rRNA synthesis and processing.
- MeSH
- buněčné jadérko genetika metabolismus ultrastruktura MeSH
- chromatin genetika ultrastruktura MeSH
- epigeneze genetická MeSH
- genetická transkripce MeSH
- geny rRNA MeSH
- histony metabolismus MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- ribozomální DNA genetika MeSH
- ribozomy genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- srovnávací studie MeSH
The aim of this study was to determine whether capillarity in the denervated and reinnervated rat extensor digitorum longus muscle (EDL) is scaled by muscle fiber oxidative potential. We visualized capillaries adjacent to a metabolically defined fiber type and estimated capillarity of fibers with very high oxidative potential (O) vs fibers with very low oxidative potential (G). Capillaries and muscle fiber types were shown by a combined triple immunofluorescent technique and the histochemical method for NADH-tetrazolium reductase. Stacks of images were captured by a confocal microscope. Applying the Ellipse program, fibers were outlined, and the diameter, perimeter, cross-sectional area, length, surface area, and volume within the stack were calculated for both fiber types. Using the Tracer plug-in module, capillaries were traced within the three-dimensional (3D) volume, the length of capillaries adjacent to individual muscle fibers was measured, and the capillary length per fiber length (Lcap/Lfib), surface area (Lcap/Sfib), and volume (Lcap/Vfib) were calculated. Furthermore, capillaries and fibers of both types were visualized in 3D. In all experimental groups, O and G fibers significantly differed in girth, Lcap/Sfib, and Lcap/Vfib, but not in Lcap/Lfib. We conclude that capillarity in the EDL is scaled by muscle fiber size and not by muscle fiber oxidative potential.
- MeSH
- denervace svalu MeSH
- financování organizované MeSH
- histocytochemie MeSH
- kapiláry anatomie a histologie MeSH
- konfokální mikroskopie MeSH
- kosterní svalová vlákna metabolismus MeSH
- kosterní svaly inervace krevní zásobení metabolismus MeSH
- krysa rodu rattus MeSH
- oxidace-redukce MeSH
- potkani Wistar MeSH
- zobrazování trojrozměrné MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
Human prominin-1 (CD133) is expressed by various stem and progenitor cells originating from diverse sources. In addition to stem cells, its mouse ortholog is expressed in a broad range of adult epithelial cells, where it is selectively concentrated in their apical domain. The lack of detection of prominin-1 in adult human epithelia might be explained, at least in part, by the specificity of the widely used AC133 antibody, which recognizes an epitope that seems dependent on glycosylation. Here we decided to re-examine its expression in adult human tissues, particularly in glandular epithelia, using a novel monoclonal antibody (80B258) generated against the human prominin-1 polypeptide. In examined tissues, we observed 80B258 immunoreactivity at the apical or apicolateral membranes of polarized cells. For instance, we found expression in secretory serous and mucous cells as well as intercalated ducts of the large salivary and lacrimal glands. In sweat glands including the gland of Moll, 80B258 immunoreactivity was found in the secretory (eccrine and apocrine glands) and duct (eccrine glands) portion. In the liver, 80B258 immunoreactivity was identified in the canals of Hering, bile ductules, and small interlobular bile ducts. In the uterus, we detected 80B258 immunoreactivity in endometrial and cervical glands. Together these data show that the overall expression of human prominin-1 is beyond the rare primitive cells, and it seems to be a general marker of apical or apicolateral membrane of glandular epithelia. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
- MeSH
- Caco-2 buňky MeSH
- CD antigeny biosyntéza imunologie MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- epitel metabolismus MeSH
- exokrinní žlázy metabolismus MeSH
- financování organizované MeSH
- glykoproteiny biosyntéza imunologie MeSH
- imunohistochemie MeSH
- játra metabolismus MeSH
- konfokální mikroskopie MeSH
- křečci praví MeSH
- lidé MeSH
- monoklonální protilátky MeSH
- orgánová specificita MeSH
- pankreas metabolismus MeSH
- peptidy imunologie MeSH
- průtoková cytometrie MeSH
- uterus MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
