Integrins are transmembrane receptors that facilitate cell adhesion and cell-extracellular matrix communication. They are involved in the sperm maturation including capacitation and gamete interaction, resulting in successful fertilization. αV integrin belongs to the integrin glycoprotein superfamily, and it is indispensable for physiological spermiogenesis and testosterone production. We targeted the gene and protein expression of the αV integrin subunit and described its membrane localization in sperm. Firstly, in mouse, we traced αV integrin gene expression during spermatogenesis in testicular fraction separated by elutriation, and we detected gene activity in spermatogonia, spermatocytes, and round spermatids. Secondly, we specified αV integrin membrane localization in acrosome-intact and acrosome-reacted sperm and compared its pattern between mouse, pig, and human. Using immunodetection and structured illumination microscopy (SIM), the αV integrin localization was confined to the plasma membrane covering the acrosomal cap area and also to the inner acrosomal membrane of acrosome-intact sperm of all selected species. During the acrosome reaction, which was induced on capacitated sperm, the αV integrin relocated and was detected over the whole sperm head. Knowledge of the integrin pattern in mature sperm prepares the ground for further investigation into the pathologies and related fertility issues in human medicine and veterinary science.

• Klíčová slova
• human, male germ cells, mouse, pig, sperm, αV integrin,
• MeSH
• akrozomální reakce MeSH
• integrin alfaV metabolismus   MeSH
• lidé MeSH
• myši MeSH
• prasata MeSH
• spermie metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• integrin alfaV MeSH

RNase J1 is the major 5'-to-3' bacterial exoribonuclease. We demonstrate that in its absence, RNA polymerases (RNAPs) are redistributed on DNA, with increased RNAP occupancy on some genes without a parallel increase in transcriptional output. This suggests that some of these RNAPs represent stalled, non-transcribing complexes. We show that RNase J1 is able to resolve these stalled RNAP complexes by a "torpedo" mechanism, whereby RNase J1 degrades the nascent RNA and causes the transcription complex to disassemble upon collision with RNAP. A heterologous enzyme, yeast Xrn1 (5'-to-3' exonuclease), is less efficient than RNase J1 in resolving stalled Bacillus subtilis RNAP, suggesting that the effect is RNase-specific. Our results thus reveal a novel general principle, whereby an RNase can participate in genome-wide surveillance of stalled RNAP complexes, preventing potentially deleterious transcription-replication collisions.

• Klíčová slova
• RNAP, RNase J1, stalling, torpedo, transcription-replication collision,
• MeSH
• Bacillus subtilis enzymologie   genetika   MeSH
• bakteriální proteiny metabolismus   MeSH
• bakteriální RNA genetika   metabolismus   MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• exoribonukleasy metabolismus   MeSH
• genetická transkripce MeSH
• messenger RNA genetika   metabolismus   MeSH
• regulace genové exprese u bakterií MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• bakteriální RNA MeSH
• DNA řízené RNA-polymerasy MeSH
• exoribonukleasy MeSH
• messenger RNA MeSH

BACKGROUND: 'Braun' is an illegal injectable dihydrocodeinone-enriched drug mixture of semi-synthetic opioids. It is prepared by palladium-catalysed hydrogenation from codeine-containing tablets. OBJECTIVE: We aimed to characterize the dermatologic consequences of long-term abuse of 'Braun'. METHODS: Skin biopsies of two long-term 'Braun' abusers were evaluated histopathologically, immunohistochemically and ultrastructurally. Palladium skin content was assessed by X-ray fluorescence (XRF) spectrometry. RESULTS: Both patients showed generalized diffuse dark blue-grey hyperpigmentation of the skin. In both, an abnormal population of cells containing intracytoplasmic brownish granular material was identified in the papillary dermis by light microscopy. Electron microscopy revealed a dense and minimally structured material that predominantly accumulated in macrophages, fibroblasts and vascular endothelial cells. XRF analysis confirmed elevated levels of palladium in the patient's skin in comparison to healthy controls. CONCLUSION: Long-term abuse of palladium-contaminated dihydrocodeinone ('Braun') results in excessive accumulation of granular material in various dermal cell types and causes generalized diffuse skin hyperpigmentation.

• MeSH
• fluorescenční spektrometrie MeSH
• hydrokodon škodlivé účinky   MeSH
• hyperpigmentace chemicky indukované   metabolismus   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• narkotika škodlivé účinky   MeSH
• palladium škodlivé účinky   metabolismus   MeSH
• poruchy způsobené užíváním narkotik komplikace   MeSH
• syntetická léčiva škodlivé účinky   MeSH
• zakázané drogy škodlivé účinky   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Názvy látek
• hydrokodon MeSH
• narkotika MeSH
• palladium MeSH
• syntetická léčiva MeSH
• zakázané drogy MeSH

The organization of the nuclear periphery is crucial for many nuclear functions. Nuclear lamins form dense network at the nuclear periphery and play a substantial role in chromatin organization, transcription regulation and in organization of nuclear pore complexes (NPCs). Here, we show that TPR, the protein located preferentially within the nuclear baskets of NPCs, associates with lamin B1. The depletion of TPR affects the organization of lamin B1 but not lamin A/C within the nuclear lamina as shown by stimulated emission depletion microscopy. Finally, reduction of TPR affects the distribution of NPCs within the nuclear envelope and the effect can be reversed by simultaneous knock-down of lamin A/C or the overexpression of lamin B1. Our work suggests a novel role for the TPR at the nuclear periphery: the TPR contributes to the organization of the nuclear lamina and in cooperation with lamins guards the interphase assembly of nuclear pore complexes.

• Klíčová slova
• Image analysis, Lamina, Lamins, Nuclear pore complex, Nucleus, Super-resolution imaging, TPR, Translocated promoter region,
• MeSH
• HeLa buňky MeSH
• jaderná lamina metabolismus   ultrastruktura   MeSH
• jaderný obal metabolismus   ultrastruktura   MeSH
• komplex proteinů jaderného póru antagonisté a inhibitory   genetika   metabolismus   MeSH
• lamin typ A antagonisté a inhibitory   genetika   metabolismus   MeSH
• lamin typ B genetika   metabolismus   MeSH
• lidé MeSH
• malá interferující RNA genetika   metabolismus   MeSH
• molekulární zobrazování MeSH
• protoonkogenní proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• komplex proteinů jaderného póru MeSH
• lamin typ A MeSH
• lamin typ B MeSH
• LMNA protein, human MeSH Prohlížeč
• malá interferující RNA MeSH
• protoonkogenní proteiny MeSH
• TPR protein, human MeSH Prohlížeč

PURPOSE: The quality and precision of post-mortem MRI microscopy may vary depending on the embedding medium used. To investigate this, our study evaluated the impact of 5 widely used media on: (1) image quality, (2) contrast of high spatial resolution gradient-echo (T1 and T2* -weighted) MR images, (3) effective transverse relaxation rate (R2* ), and (4) quantitative susceptibility measurements (QSM) of post-mortem brain specimens. METHODS: Five formaldehyde-fixed brain slices were scanned using 7.0T MRI in: (1) formaldehyde solution (formalin), (2) phosphate-buffered saline (PBS), (3) deuterium oxide (D2 O), (4) perfluoropolyether (Galden), and (5) agarose gel. SNR and contrast-to-noise ratii (SNR/CNR) were calculated for cortex/white matter (WM) and basal ganglia/WM regions. In addition, median R2* and QSM values were extracted from caudate nucleus, putamen, globus pallidus, WM, and cortical regions. RESULTS: PBS, Galden, and agarose returned higher SNR/CNR compared to formalin and D2 O. Formalin fixation, and its use as embedding medium for scanning, increased tissue R2* . Imaging with agarose, D2 O, and Galden returned lower R2* values than PBS (and formalin). No major QSM offsets were observed, although spatial variance was increased (with respect to R2* behaviors) for formalin and agarose. CONCLUSIONS: Embedding media affect gradient-echo image quality, R2* , and QSM in differing ways. In this study, PBS embedding was identified as the most stable experimental setup, although by a small margin. Agarose and Galden were preferred to formalin or D2 O embedding. Formalin significantly increased R2* causing noisier data and increased QSM variance.

• Klíčová slova
• ex vivo imaging, formaldehyde fixation, post-mortem imaging, quantitative susceptibility mapping, ultra-high field MRI,
• MeSH
• ethery MeSH
• fluorokarbony MeSH
• formaldehyd MeSH
• fosfáty MeSH
• kontrastní látky MeSH
• lidé středního věku MeSH
• lidé MeSH
• magnetická rezonanční tomografie přístrojové vybavení   metody   MeSH
• mapování mozku metody   MeSH
• mozek diagnostické zobrazování   patologie   MeSH
• odběr biologického vzorku MeSH
• oxid deuteria MeSH
• pitva přístrojové vybavení   metody   MeSH
• počítačové zpracování obrazu MeSH
• poměr signál - šum MeSH
• sefarosa chemie   MeSH
• senioři MeSH
• zalévání tkání přístrojové vybavení   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ethery MeSH
• fluorokarbony MeSH
• formaldehyd MeSH
• fosfáty MeSH
• kontrastní látky MeSH
• oxid deuteria MeSH
• perfluoropolyether MeSH Prohlížeč
• sefarosa MeSH

Integrins are transmembrane cell receptors involved in two crucial mechanisms for successful fertilization, namely, mammalian intracellular signaling and cell adhesion. Integrins α6β4, α3β1 and α6β1 are three major laminin receptors expressed on the surface of mammalian cells including gametes, and the presence of individual integrin subunits α3, α6, β1 and β4 has been previously detected in mammalian sperm. However, to date, proof of the existence of individual heterodimer pairs in sperm and their detailed localization is missing. The major conclusion of this study is evidence that the β4 integrin subunit is expressed in mouse sperm and that it pairs with subunit α6; additionally, there is a detailed identification of integrin heterodimer pairs across individual membranes in an intact mouse sperm head. We also demonstrate the existence of β4 integrin mRNAs in round spermatids and spermatogonia by q-RT-PCR, which was further supported by sequencing the PCR products. Using super-resolution microscopy accompanied by colocalization analysis, we located integrin subunits as follows: α6/β4-inner apical acrosomal membrane and equatorial segment; α3, α6/β1, β4-plasma membrane overlaying the apical acrosome; and α3/β1-outer acrosomal membrane. The existence of α6β4, α3β1 and α6β1 heterodimers was further confirmed by proximity ligation assay (PLA). In conclusion, we delivered detailed characterization of α3, α6, β1 and β4 integrin subunits, showing their presence in distinct compartments of the intact mouse sperm head. Moreover, we identified sperm-specific localization for heterodimers α6β4, α3β1 and α6β1, and their membrane compartmentalization and the presented data show a complexity of membranes overlaying specialized microdomain structures in the sperm head. Their different protein compositions of these individual membrane rafts may play a specialized role, based on their involvement in sperm-epithelium and sperm-egg interaction.

• Klíčová slova
• integrin heterodimers, integrins, sperm head, α3β1, α6β1, α6β4,
• MeSH
• biologické modely MeSH
• integriny chemie   metabolismus   MeSH
• kompartmentace buňky * MeSH
• multimerizace proteinu * MeSH
• myši inbrední C57BL MeSH
• podjednotky proteinů metabolismus   MeSH
• proteinové domény MeSH
• spermie metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• integriny MeSH
• podjednotky proteinů MeSH

In the recent past, we demonstrated that a great deal is going on in the salivary glands of Drosophila in the interval after they release their glycoprotein-rich secretory glue during pupariation. The early-to-mid prepupal salivary glands undergo extensive endocytosis with widespread vacuolation of the cytoplasm followed by massive apocrine secretion. Here, we describe additional novel properties of these endosomes. The use of vital pH-sensitive probes provided confirmatory evidence that these endosomes have acidic contents and that there are two types of endocytosis seen in the prepupal glands. The salivary glands simultaneously generate mildly acidic, small, basally-derived endosomes and strongly acidic, large and apical endosomes. Staining of the large vacuoles with vital acidic probes is possible only after there is ambipolar fusion of both basal and apical endosomes, since only basally-derived endosomes can bring fluorescent probes into the vesicular system. We obtained multiple lines of evidence that the small basally-derived endosomes are chiefly involved in the uptake of dietary Fe3+ iron. The fusion of basal endosomes with the larger and strongly acidic apical endosomes appears to facilitate optimal conditions for ferrireductase activity inside the vacuoles to release metabolic Fe2+ iron. While iron was not detectable directly due to limited staining sensitivity, we found increasing fluorescence of the glutathione-sensitive probe CellTracker Blue CMAC in large vacuoles, which appeared to depend on the amount of iron released by ferrireductase. Moreover, heterologous fluorescently-labeled mammalian iron-bound transferrin is actively taken up, providing direct evidence for active iron uptake by basal endocytosis. In addition, we serendipitously found that small (basal) endosomes were uniquely recognized by PNA lectin, whereas large (apical) vacuoles bound DBA lectin.

• Klíčová slova
• basal and apical endosomes, iron reallocation, metamorphosis, prepupal period, salivary glands, transferrin uptake,
• MeSH
• Drosophila melanogaster anatomie a histologie   cytologie   MeSH
• endozomy metabolismus   MeSH
• fluorescenční barviva chemie   MeSH
• kukla cytologie   MeSH
• slinné žlázy cytologie   metabolismus   MeSH
• sloučeniny železa metabolismus   MeSH
• vakuoly metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fluorescenční barviva MeSH
• sloučeniny železa MeSH

The novel synthesis of a polymeric interface grown from the surface of bright fluorescent nanodiamonds is reported. The polymer enables bioorthogonal attachment of various molecules by click chemistry; the particles are resistant to nonspecific protein adsorption and show outstanding colloidal stability in buffers and biological media. The coating fully preserves the unique optical properties of the nitrogen-vacancy centers that are crucial for bioimaging and sensoric applications.

• Klíčová slova
• click chemistry, fluorescence, nanoparticles, polymerization, solubilization,
• Publikační typ
• časopisecké články MeSH