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Cell cycle studies in plants and algae are highly dependent on reliable methods for detecting cellular DNA replication. With its short growth cycle and ease of genetic transformation, Phaeodactylum tricornutum is an important model organism for the study of pennate diatoms. Here we explored two different methods to detect the cell cycle of P. tricornutum, one using SYBR‐green I to via flow cytometry, and the other using EdU labeling to observe cell cycle changes under fluorescence microscopy. Both EdU labeling fluorescence microscopy and SYBR‐green I staining flow cytometry accurately indicated that the cells of P. tricornutum enter the G2/M phase after 12 h of light exposure. The results indicate that SYBR Green I was an adequate detection method for nuclear DNA quantitation in cells of P. tricornutum using a flow cytometer and without RNase A treatment. In addition, EdU can be applied to P. tricornutum to reliably detect cell proliferation. Besides, Mg‐ProtoIX was able to reverse the cell cycle division inhibition of P. tricornutum and allow the nuclear DNA replication to proceed normally. Taken together, the photoperiodic division time point was clearly identified, which sheds light on the regulation of cell division mechanism in P. tricornutum. [ABSTRACT FROM AUTHOR]

Summary: N‐(3‐Dimethylaminopropyl)‐N′‐ethylcarbodiimide (EDC) is a carbodiimide coupling reagent commonly used for the preparation of amides from carboxylic acids and amines. Because of initial concerns regarding the genotoxicity of EDC and its use in GMP syntheses at Bristol Myers Squibb, the quantitation of residual EDC and its by‐product N‐(3‐dimethylaminopropyl)‐N′‐ethylurea (EDU) by liquid chromatography–mass spectrometry (LCMS) impurity analysis was required. These analyses required the use of stable‐isotope‐labeled EDC and EDU to serve as internal standards. To meet this need, stable‐isotope‐labeled EDC 9 and EDU 10 were prepared from [1,2‐13C2] ethylene glycol and [13C,15N] potassium cyanide in overall yields of 6% and 8%, respectively. [ABSTRACT FROM AUTHOR]

Two decades after the discovery of adult‐born neurons in the brains of decapod crustaceans, the deutocerebral proliferative system (DPS) producing these neural lineages has become a model of adult neurogenesis in invertebrates. Studies on crayfish have provided substantial insights into the anatomy, cellular dynamics, and regulation of the DPS. Contrary to traditional thinking, recent evidence suggests that the neurogenic niche in the crayfish DPS lacks self‐renewing stem cells, its cell pool being instead sustained via integration of hemocytes generated by the innate immune system. Here, we investigated the origin, division and migration patterns of the adult‐born neural progenitor (NP) lineages in detail. We show that the niche cell pool is not only replenished by hemocyte integration but also by limited numbers of symmetric cell divisions with some characteristics reminiscent of interkinetic nuclear migration. Once specified in the niche, first generation NPs act as transit‐amplifying intermediate NPs that eventually exit and produce multicellular clones as they move along migratory streams toward target brain areas. Different clones may migrate simultaneously in the streams but occupy separate tracks and show spatio‐temporally flexible division patterns. Based on this, we propose an extended DPS model that emphasizes structural similarities to pseudostratified neuroepithelia in other arthropods and vertebrates. This model includes hemocyte integration and intrinsic cell proliferation to synergistically counteract niche cell pool depletion during the animal's lifespan. Further, we discuss parallels to recent findings on mammalian adult neurogenesis, as both systems seem to exhibit a similar decoupling of proliferative replenishment divisions and consuming neurogenic divisions. [ABSTRACT FROM AUTHOR]

To develop treatments for salivary gland dysfunction, it is important to understand how human salivary glands are maintained under normal homeostasis. Previous data from our lab demonstrated that murine salivary acinar cells maintain the acinar cell population through self‐duplication under conditions of homeostasis, as well as after injury. Early studies suggested that human acinar cells are mitotically active, but the identity of the resultant daughter cells was not clear. Using markers of cell cycle activity and mitosis, as well as an ex vivo 5‐Ethynyl‐2´‐deoxyuridine assay, we show that human salivary gland acinar cells divide to generate daughter acinar cells. As in mouse, our data indicate that human salivary gland homeostasis is supported by the intrinsic mitotic capacity of acinar cells. [ABSTRACT FROM AUTHOR]

In our previous study, a Met-to-Ile substitution at amino acid 256 in the catalytic domain of Uba1 was determined in temperature-sensitive CHO-K1 mutant tsTM3 cells, which exhibited chromosomal instability and cell-cycle arrest in the S to G 2 phases with decreased DNA synthesis at the nonpermissive temperature, 39 °C. Mutant cells were also characterized by a significant decrease of Uba1 in the nucleus with decreased ubiquitination activity at 39 °C. Defects of ubiquitination activity in the nucleus resulted in an inappropriate balance between Cdt1 and geminin, a licensing factor of DNA replication and its inhibitor. In the present study, we found that the Cu(I)-catalyzed [3 + 2] cycloaddition (click) reaction inhibits the subsequent indirect immunolabeling of Cdt1 but allows for the detection of PCNA with nascent DNA. Using a procedure without the click reaction, we also demonstrated that Cdt1 remained close to active replication sites in tsTM3 cells at the nonpermissive temperature. Analysis of genome replication by DNA fiber spreading revealed that DNA synthesis continues for at least 10 h after incubation at 39 °C, suggesting that impaired ubiquitination in the nucleus, caused by the defect of Uba1, affected DNA replication only after a long delay. [ABSTRACT FROM AUTHOR]

Telocytes ( TCs) are a novel type of interstitial cells which are potentially involved in tissue regeneration and repair (). Previously, we documented the presence of TCs in liver. However, the possible roles of TCs in liver regeneration remain unknown. In this study, a murine model of partial hepatectomy ( PH) was used to induce liver regeneration. The number of TCs detected by double labelling immunofluorescence methods ( CD34/ PDGFR-α, CD34/ PDGFR-ß and CD34/Vimentin) was significantly increased when a high level of hepatic cell proliferation rate (almost doubled) as shown by 5-ethynyl-2′-deoxyuridine (EdU) immunostaining and Western Blot of Proliferating cell nuclear antigen ( PCNA) was found at 48 and 72 hrs post- PH. Meanwhile, the number of CK-19 positive-hepatic stem cells peaked at 72 hrs post- PH, co-ordinating with the same time-point, when the number of TCs was most significantly increased. Taken together, the results indicate a close relationship between TCs and the cells essentially involved in liver regeneration: hepatocytes and stem cells. It remains to be determined how TCs affect hepatocytes proliferation and/or hepatic stem cell differentiation in liver regeneration. Besides intercellular junctions, we may speculate a paracrine effect via ectovesicles. [ABSTRACT FROM AUTHOR]

The pituitary gland is a highly mitotically active tissue after birth. Various cell types are known to undergo proliferation in the anterior pituitary. However, little is known about the mechanisms regulating mitotic activity in this tissue. When searching for genes specifically expressed in the pituitary gland among those that we previously screened in Drosophila, we found epithelial cell-transforming gene 2 ( Ect2). Ect2 is a guanine nucleotide exchange factor for Rho GTPases, which is known to play an essential role in cytokinesis. Although there have been many cellular studies regarding the function of Ect2, the temporal and spatial expression patterns of Ect2 in vivo have not been determined. In the present study, we examined the postnatal developmental expression of Ect2 in the mouse pituitary. Enhanced Ect2 expression was detected in the mouse pituitary gland during the first 3 weeks after birth, which coincided well with the period of rapid pituitary expansion associated with increased growth rate. Immunostaining analysis showed that Ect2-expressing cells were distributed in the anterior and intermediate lobes, but not the posterior lobe, of the pituitary. These Ect2-expressing cells frequently incorporated the thymidine analogue, EdU (5-ethynyl-2′-deoxyuridine), indicating that these cells were mitotically active. Taken together, the results demonstrate the functional role of Ect2 in postnatal proliferating cells in the two lobes of the pituitary, thereby suggesting roles in developmental growth of the mammalian pituitary. [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis: The avian cochlea regenerates hair cells following aminoglycoside treatment through supporting cell proliferation. Immunocytochemical labeling of 5-bromo-2′-deoxyuridine (BrdU), a thymidine analog, is a popular nonradioactive marker for identifying cells in the DNA synthesis (S phase) of the cell cycle. However, it requires harsh treatments to denature double-stranded DNA for the antibody to bind BrdU. We explored a new method using 5-ethynyl-2′-deoxyuridine (EdU) as a thymidine analog and a nonantibody azide/alkyne reaction between EdU and the fluorescent probe. We propose that EdU is as effective as BrdU, but without the requirement for harsh denaturation or the use of antibodies for detection. Study Design: Two-week-old chicks received a single gentamicin injection followed by a single EdU injection 72 hours later. Cochleae were extracted 4-8 hours later, fixed, and processed for fluorescent detection of EdU. Methods: Cochleae were processed for detection of incorporated EdU using the Click-iT Imaging Kit (Invitrogen/Molecular Probes, Carlsbad, CA) and colabeled with Sox2, myosin VI, or myosin VIIa antibodies. Whole-mount cochlear preparations were examined with confocal microscopy. Results: Supporting cells incorporated EdU into their newly synthesized DNA during the 4-8 hours following the EdU injection and were readily detected with little background signal. The intensity and quantity of cells labeled were similar to or better than that seen for BrdU. Conclusions: The EdU method is as effective as BrdU, without requiring harsh denaturation or secondary antibodies to identify proliferating cells. Thus, the nonantibody EdU system allows more flexibility by enabling colabeling with multiple antibodies to other cellular proteins involved in regeneration. Laryngoscope, 2009 [ABSTRACT FROM AUTHOR]

The cytokinin-like activity of the growth regulating chemical EDU, N-[2-(2-oxo-limidazolidinyl)ethyl]-N'-phenylurea, was determined and compared with the acitivity of kinetin using the tobacco callus bioassay. EDU has a pronounced stimulatory effect on callus growth at concentrations of 5 × 10-4 and 1 × 10-3 M but was 5000 times less potent than the synthetic cytokinin, kinetin. Senescence regulation and oxidant resistance induced by EDU and kinetin were also studied. EDU retarded the breakdown of chlorophyll, protein and RNA in O3-sensitive tobacco leaf discs during senescence. EDU was much more effective in arresting senescence and in protecting against O3 injury than kinetin. Results indicate the EDU-induced plant tolerance to O3 phytotoxicity may be indirect through enzyme induction regulation. [ABSTRACT FROM AUTHOR]