Constancy and variability in the content of DNA in cerebellar Purkinje cell nuclei

Summary A cytophotometric study of DNA content in Purkinje cells of the cerebellum of rats, cats, chicken and humans (Feulgen staining) revealed that in a certain number of cells the amount of DNA ranged between the diploid and tetraploid level (H2C cells). The incidence of H2C Purkinje cells varied among the species studied. In rats, which were studied most thoroughly, these cells amounted on average to 3%. In some rats, as well as in some cats and chickens H2C Purkinje cells were entirely absent. In the group of animals possesing H2C Purkinje cells, great interindividual differences were observed. In rats for instance, the incidence of these cells varied from 1 to 23 per cent. Topographic analyses carried out in rat and human cerebellum revealed that H2C Purkinje cells occurred more frequently in the hemispheres than in the vermis. No significant differences were found in the number of H2C Purkinje cells in healthy and Kilham-DNA-virus infected rats.Densitometric analysis of the distribution of nuclear chromatin showed that H2C Purkinje cells were richer in condensed chromatin, especially in the region of the nucleolus, which apparently contains the hyperploid surplus of DNA. It is proposed that the phenomenon of DNA hyperdiploidy arises as a result of either incomplete S-phase in some immature Purkinje cell precursors or the amplification of some DNA sequences particularly those localized in the nucleolar region.     

Bacterial periphyton of the Vltava River

Bacterial periphyton formed during 48 hours was studied by glass slide method and direct counting in Vltava River in Praha, Czechoslovakia. At water temperatures 8–11°C the numbers of rods ranged between 24,000 and 336,000 per 1 cm² and those of cocci between 30,000 and 228,000. The relation rods: cocci ranged between 0,9 and 2,4 with an average value of 1,7, whereas in a fishpond this average was 0,5. Among the periphyton 81,3% bacterial cells were active. The rods: cocci relation seems to be a good indicator of water pollution by organic matter, but numbers distinguishing the individual saprobic levels cannot be given yet.

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Zusammenfassung Es wurde der bakterielle Bewuchs von Glassplatten im Vltava-Fluss in Praha untersucht. Zweitägige Exposition erwies sich als am meisten geeignet, um den Bewuchs mikroskopisch durch direkte Zählung quantitativ zu erfassen. In Abhängigkeit von der Tiefe, Wassertemperatur, Verunreinigungsgrad, Sonnenstrahlung u.a. entwickelte sich der Bewuchs quantitativ unterschiedlich und zeigte auch eine unterschiedliche Relation von Stäbchen zu Kokken. Im beta-mesosaproben Bereich wurden im Herbst (Wassertemperaturen 8–11 °C) 24.000 bis 336.000 Stäbchen und 30.000 bis 228.000 Kokken per 1 cm² gefunden, mit den Mittelwerten 163.100 Stäbchen und 104.100 Kokken. Die Vergleichswerte von einem Fischteich in Motol lagen für Stäbchen im gleichen Bereich, während die Kokken, bis 254.000 erreichten.Die Relation Stäbchen: Kokken variierte im Vltava-Fluss zwischen 0,9 (nur einmal) und 2,4 mit dem Mittelwert 1,7, im Fischteich zwischen 0,4 ufnd 0,7 mit dem Mittelwert 0,5. Es hat sich erwiesen, dass diese Relation brauchbare Angaben über die organische Verunreinigung gibt. Leider liegen bisher zu wenige Ergebnisse vor, um eine Skale gegenüber den Saprobitätsstufen zu errichten.Unter den Bewuchsbakterien gab es in der Vltava durchschnittlich 81,3% aktive Zellen, wie durch Fluoreszenzanalyse festgestellt wurde.Fast alle Daten indizieren eine schwächere Verunreinigung des Wassers am linken Ufer als am rechten.

     

Circular RNA AFF4 modulates osteogenic differentiation in BM-MSCs by activating SMAD1/5 pathway through miR-135a-5p/FNDC5/Irisin axis

Bone marrow-derived mesenchymal stem cells (BM-MSCs), the common progenitor cells of adipocytes and osteoblasts, have been recognized as the key mediator during bone formation. Herein, our study aim to investigate molecular mechanisms underlying circular RNA (circRNA) AFF4 (circ_AFF4)-regulated BM-MSCs osteogenesis. BM-MSCs were characterized by FACS, ARS, and ALP staining. Expression patterns of circ_AFF4, miR-135a-5p, FNDC5/Irisin, SMAD1/5, and osteogenesis markers, including ALP, BMP4, RUNX2, Spp1, and Colla1 were detected by qRT-PCR, western blot, or immunofluorescence staining, respectively. Interactions between circ_AFF4 and miR-135a-5p, FNDC5, and miR-135a-5p were analyzed using web tools including TargetScan, miRanda, and miRDB, and further confirmed by luciferase reporter assay and RNA pull-down. Complex formation between Irisin and Integrin αV was verified by Co-immunoprecipitation. To further verify the functional role of circ_AFF4 in vivo during bone formation, we conducted animal experiments harboring circ_AFF4 knockdown, and born samples were evaluated by immunohistochemistry, hematoxylin and eosin, and Masson staining. Circ_AFF4 was upregulated upon osteogenic differentiation induction in BM-MSCs, and miR-135a-5p expression declined as differentiation proceeds. Circ_AFF4 knockdown significantly inhibited osteogenesis potential in BM-MSCs. Circ_AFF4 stimulated FNDC5/Irisin expression through complementary binding to its downstream target molecule miR-135a-5p. Irisin formed an intermolecular complex with Integrin αV and activated the SMAD1/5 pathway during osteogenic differentiation. Our work revealed that circ_AFF4, acting as a sponge of miR-135a-5p, triggers the promotion of FNDC5/Irisin via activating the SMAD1/5 pathway to induce osteogenic differentiation in BM-MSCs. These findings gained a deeper insight into the circRNA-miRNA regulatory system in the bone marrow microenvironment and may improve our understanding of bone formation-related diseases at physiological and pathological levels.Subject terms: Stem cells, Diseases     

Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression

Proteins associated with the centrosome play key roles in mitotic progression in mammalian cells. The activity of Cdk1-opposing phosphatases at the centrosome must be inhibited during early mitosis to prevent premature dephosphorylation of Cdh1—an activator of the ubiquitin ligase anaphase-promoting complex/cyclosome—and the consequent premature degradation of mitotic activators. In this paper, we show that reversible oxidative inactivation of centrosome-bound protein phosphatases such as Cdc14B by H₂O₂ is likely responsible for this inhibition. The intracellular concentration of H₂O₂ increases as the cell cycle progresses. Whereas the centrosome is shielded from H₂O₂ through its association with the H₂O₂-eliminating enzyme peroxiredoxin I (PrxI) during interphase, the centrosome-associated PrxI is selectively inactivated through phosphorylation by Cdk1 during early mitosis, thereby exposing the centrosome to H₂O₂ and facilitating inactivation of centrosome-bound phosphatases. Dephosphorylation of PrxI by okadaic acid–sensitive phosphatases during late mitosis again shields the centrosome from H₂O₂ and thereby allows the reactivation of Cdk1-opposing phosphatases at the organelle.     

Allyl Isothiocyanate Ameliorates Angiogenesis and Inflammation in Dextran Sulfate Sodium-Induced Acute Colitis

Allyl isothiocyanate (AITC) is a phytochemical found in cruciferous vegetables that has known chemopreventive and chemotherapeutic activities. Thus far, the antiangiogenic activity of AITC has not been reported in in vivo studies. Herein, we investigated the effect of AITC on angiogenesis and inflammation in a mouse model of colitis. Experimental colitis was induced in mice by administering 3% dextran sulfate sodium via drinking water. To monitor the activity of AITC in this model, we measured body weight, disease activity indices, histopathological scores, microvascular density, myeloperoxidase activity, F4/80 staining, inducible nitric oxide synthase (iNOS) expression, cyclooxygenase-2 (COX-2) expression, and vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (VEGFR2) expression in the mice. We found that AITC-treated mice showed less weight loss, fewer clinical signs of colitis, and longer colons than vehicle-treated mice. AITC treatment also significantly lessened the disruption of colonic architecture that is normally associated with colitis and repressed the microvascularization response. Further, AITC treatment reduced both leukocyte recruitment and macrophage infiltration into the inflamed colon, and the mechanism these activities involved repressing iNOS and COX-2 expression. Finally, AITC attenuated the expression of VEGF-A and VEGFR2. Thus, AITC may have potential application in treating conditions marked by inflammatory-driven angiogenesis and mucosal inflammation.     

THDP17 Decreases Ammonia Production through Glutaminase Inhibition. A New Drug for Hepatic Encephalopathy Therapy

Ammonia production is implicated in the pathogenesis of hepatic encephalopathy (HE), being intestinal glutaminase activity the main source for ammonia. Management of ammonia formation can be effective in HE treatment by lowering intestinal ammonia production. The use of glutaminase inhibitors represents one way to achieve this goal. In this work, we have performed a search for specific inhibitors that could decrease glutaminase activity by screening two different groups of compounds: i) a group integrated by a diverse, highly pure small molecule compounds derived from thiourea ranging from 200 to 800 Daltons; and ii) a group integrated by commonly use compounds in the treatment of HE. Results shown that THDP-17 (10 µM), a thiourea derivate product, could inhibit the intestinal glutaminase activity (57.4±6.7%). Inhibitory effect was tissue dependent, ranging from 40±5.5% to 80±7.8% in an uncompetitive manner, showing V_max and K_m values of 384.62 µmol min⁻¹, 13.62 mM with THDP-17 10 µM, respectively. This compound also decreased the glutaminase activity in Caco-2 cell cultures, showing a reduction of ammonia and glutamate production, compared to control cultures. Therefore, the THDP-17 compound could be a good candidate for HE management, by lowering ammonia production.     

Microtubule-assisted mechanism for toxisome assembly in Fusarium graminearum

In Fusarium graminearum, a trichothecene biosynthetic complex known as the toxisome forms ovoid and spherical structures in the remodelled endoplasmic reticulum (ER) under mycotoxin-inducing conditions. Previous studies also demonstrated that disruption of actin and tubulin results in a significant decrease in deoxynivalenol (DON) biosynthesis in F. graminearum. However, the functional association between the toxisome and microtubule components has not been clearly defined. In this study we tested the hypothesis that the microtubule network provides key support for toxisome assembly and thus facilitates DON biosynthesis. Through fluorescent live cell imaging, knockout mutant generation, and protein–protein interaction assays, we determined that two of the four F. graminearum tubulins, α₁ and β₂ tubulins, are indispensable for DON production. We also showed that these two tubulins are directly associated. When the α₁–β₂ tubulin heterodimer is disrupted, the metabolic activity of the toxisome is significantly suppressed, which leads to significant DON biosynthesis impairment. Similar phenotypic outcomes were shown when F. graminearum wild type was treated with carbendazim, a fungicide that binds to microtubules and disrupts spindle formation. Based on our results, we propose a model where α₁–β₂ tubulin heterodimer serves as the scaffold for functional toxisome assembly in F. graminearum.     

The adhesion of thin carbon films to metallic substrates.

As part of the development of carbon-coated prosthetic devices, the adhesion of thin carbon films to metallic substrates has been studied. The bond strength of carbon films about 5000 A thick on Ti-6A1-4V and stainless steel was measured in a pull test and found to be greater than 4700 psi. Auger electron spectroscopy showed a reactive film/substrate interface. The ultimate bond strength was found to be dependent on the substrate and the deposition parameters.