cells failed cycles: Topics by Science.gov

Sample records for cells failed cycles

Cycle life test and failure model of nickel-hydrogen cells

NASA Technical Reports Server (NTRS)

Smithrick, J. J.

1983-01-01

Six ampere hour individual pressure vessel nickel hydrogen cells were charge/discharge cycled to failure. Failure as used here is defined to occur when the end of discharge voltage degraded to 0.9 volts. They were cycled under a low earth orbit cycle regime to a deep depth of discharge (80 percent of rated ampere hour capacity). Both cell designs were fabricated by the same manufacturer and represent current state of the art. A failure model was advanced which suggests both cell designs have inadequate volume tolerance characteristics. The limited existing data base at a deep depth of discharge (DOD) was expanded. Two cells of each design were cycled. One COMSAT cell failed at cycle 1712 and the other failed at cycle 1875. For the Air Force/Hughes cells, one cell failed at cycle 2250 and the other failed at cycle 2638. All cells, of both designs, failed due to low end of discharge voltage (0.9 volts). No cell failed due to electrical shorts. After cell failure, three different reconditioning tests (deep discharge, physical reorientation, and open circuit voltage stand) were conducted on all cells of each design. A fourth reconditioning test (electrolyte addition) was conducted on one cell of each design. In addition post cycle cell teardown and failure analysis were performed on the one cell of each design which did not have electrolyte added after failure.
Space Station Freedom NiH2 cell testing program

NASA Technical Reports Server (NTRS)

Moore, Bruce; Frate, Dave

1994-01-01

Testing for the Space Station Freedom Nickel Hydrogen Cell Test Program began in 1990 at Crave Division, Naval Surface Warfare Center. The program has included receipt inspection, random vibration, acceptance, characterization, and life cycle testing of Ni-H2 cells in accordance with the NASA LeRC Interagency Order C-31001-J. A total of 400 Ni-H2 cells have been received at NAVSURFWARCENDIV Crane from three separate manufacturers; Yardney Technical Products (Yardney), Eagle Picher Industries (Eagle Picher), and Gates Energy Products (Gates). Of those, 308 cells distributed among 39 packs have undergone life cycle testing under a test regime simulating low earth orbit conditions. As of 30 September 1993, there are 252 cells assembled into 32 packs still on life cycle test. Since the beginning of the program, failed cells have been detected in all phases of testing. The failures include the following; seven 65 AmpHr and 81 AmpHr Yardney cells were found to be leaking KOH on receipt, one 65 AmpHr Eagle Picher cell failed the acceptance test, one 65 AmpHr Gates cell failed during the characterization test, and six 65 AmpHr Gates cells failed the random vibration test. Of the 39 life cycle packs, testing on seven packs, 56 cells, has been suspended because of low end of discharge voltages. All of the failed life cycle packs were cycled at 60% depth of discharge.
Glioblastoma Stem Cells Respond to Differentiation Cues but Fail to Undergo Commitment and Terminal Cell-Cycle Arrest

PubMed Central

Carén, Helena; Stricker, Stefan H.; Bulstrode, Harry; Gagrica, Sladjana; Johnstone, Ewan; Bartlett, Thomas E.; Feber, Andrew; Wilson, Gareth; Teschendorff, Andrew E.; Bertone, Paul; Beck, Stephan; Pollard, Steven M.

2015-01-01

Summary Glioblastoma (GBM) is an aggressive brain tumor whose growth is driven by stem cell-like cells. BMP signaling triggers cell-cycle exit and differentiation of GBM stem cells (GSCs) and, therefore, might have therapeutic value. However, the epigenetic mechanisms that accompany differentiation remain poorly defined. It is also unclear whether cell-cycle arrest is terminal. Here we find only a subset of GSC cultures exhibit astrocyte differentiation in response to BMP. Although overtly differentiated non-cycling astrocytes are generated, they remain vulnerable to cell-cycle re-entry and fail to appropriately reconfigure DNA methylation patterns. Chromatin accessibility mapping identified loci that failed to alter in response to BMP and these were enriched in SOX transcription factor-binding motifs. SOX transcription factors, therefore, may limit differentiation commitment. A similar propensity for cell-cycle re-entry and de-differentiation was observed in GSC-derived oligodendrocyte-like cells. These findings highlight significant obstacles to BMP-induced differentiation as therapy for GBM. PMID:26607953
Destructive physical analysis results of Ni/H2 cells cycled in LEO regime

NASA Technical Reports Server (NTRS)

Lim, Hong S.; Zelter, Gabriela R.; Smithrick, John J.; Hall, Stephen W.

1991-01-01

Six 48-Ah individual pressure vessel (IPV) Ni/H2 cells containing 26 and 31 percent KOH electrolyte were life cycle tested in low Earth orbit. All three cells containing 31 percent KOH failed (3729, 4165, and 11,355 cycles), while those with 26 percent KOH were cycled over 14,000 times in the continuing test. Destructive physical analysis (DPA) of the failed cells included visual inspections, measurements of electrode thickness, scanning electron microscopy, chemical analysis, and measurements of nickel electrode capacity in an electrolyte flooded cell. The cycling failure was due to a decrease of nickel electrode capacity. As possible causes of the capacity decrease, researchers observed electrode expansion, rupture, and corrosion of the nickel electrode substrate, active material redistribution, and accumulation of electrochemically undischargeable active material with cycling.
NASA Lewis advanced IPV nickel-hydrogen technology

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Britton, Doris L.

1993-01-01

Individual pressure vessel (IPV) nickel-hydrogen technology was advanced at NASA Lewis and under Lewis contracts. Some of the advancements are as follows: to use 26 percent potassium hydroxide electrolyte to improve cycle life and performance, to modify the state of the art cell design to eliminate identified failure modes and further improve cycle life, and to develop a lightweight nickel electrode to reduce battery mass, hence reduce launch and/or increase satellite payload. A breakthrough in the LEO cycle life of individual pressure vessel nickel-hydrogen battery cells was reported. The cycle life of boiler plate cells containing 26 percent KOH electrolyte was about 40,000 accelerated LEO cycles at 80 percent DOD compared to 3,500 cycles for cells containing 31 percent KOH. Results of the boiler plate cell tests have been validated at NWSC, Crane, Indiana. Forty-eight ampere-hour flight cells containing 26 and 31 percent KOH have undergone real time LEO cycle life testing at an 80 percent DOD, 10 C. The three cells containing 26 percent KOH failed on the average at cycle 19,500. The three cells containing 31 percent KOH failed on the average at cycle 6,400. Validation testing of NASA Lewis 125 Ah advanced design IPV nickel-hydrogen flight cells is also being conducted at NWSC, Crane, Indiana under a NASA Lewis contract. This consists of characterization, storage, and cycle life testing. There was no capacity degradation after 52 days of storage with the cells in the discharged state, on open circuit, 0 C, and a hydrogen pressure of 14.5 psia. The catalyzed wall wick cells have been cycled for over 22,694 cycles with no cell failures in the continuing test. All three of the non-catalyzed wall wick cells failed (cycles 9,588; 13,900; and 20,575). Cycle life test results of the Fibrex nickel electrode has demonstrated the feasibility of an improved nickel electrode giving a higher specific energy nickel-hydrogen cell. A nickel-hydrogen boiler plate cell using an 80 mil thick, 90 percent porous Fibrex nickel electrode has been cycled for 10,000 cycles at 40 percent DOD.
Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight cell - Update II

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1992-01-01

An update of validation test results confirming the breakthrough in LEO cycle life of nickel-hydrogen cells containing 26 percent KOH electrolyte is presented. A breakthrough in the LEO cycle life of individual pressure vessel (IPV) nickel-hydrogen cells has been previously reported. The cycle life of boiler plate cells containing 26 percent potassium hydroxide (KOH) electrolyte was about 40,000 LEO cycles, compared to 3500 cycles for cells containing 31 percent KOH. The cycle regime was a stressful accelerated LEO, which consisted of a 27.5 min charge followed by a 17.5 min discharge (2X normal rate). The depth-of-discharge was 80 percent. Six 48-Ah Hughes recirculation design IPV nickel-hydrogen flight battery cells are being evaluated. Three of the cells contain 26 percent KOH (test cells), and three contain 31 percent KOH (control cells). They are undergoing real time LEO cycle life testing. The cycle regime is a 90-min LEO orbit consisting of a 54-min charge followed by a 36-min discharge. The depth-of-discharge is 80 percent. The cell temperature is maintained at 10 C. The three 31 percent KOH cells failed (cycles 3729, 4165, and 11355). One of the 26 percent KOH cells failed at cycle 15314. The other two 26 percent KOH cells were cycled for over 16,000 cycles during the continuing test.
Effect of LEO cycling on 125 Ah advanced design IPV nickel-hydrogen flight cells. An update

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1991-01-01

Validation testing of the NASA Lewis 125 Ah advanced design individual pressure vessel (IPV) nickel-hydrogen flight cells was conducted. Work consisted of characterization, storage, and cycle life testing. There was no capacity degradation after 52 days of storage with the cells in the discharged state, an open circuit, 0 C, and a hydrogen pressure of 14.5 psia. The catalyzed wall wick cells were cycled for over 11,000 cycles with no cell failures in the continuing test. One of the noncatalyzed wall wick cells failed.
Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight cells-update 2

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1991-01-01

An update of validation test results confirming the breakthrough in low earth orbit (LEO) cycle life of nickel-hydrogen cells containing 26 percent KOH electrolyte is presented. A breakthrough in the LEO cycle life of individual pressure vessel (IPV nickel-hydrogen cells has been previously reported. The cycle life of boiler plate cells containing 26 percent potassium hydroxide (KOH) electrolyte was about 40 000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH. This test was conducted at Hughes Aircraft Company under a NASA Lewis contract. The purpose was to investigate the effect of KOH concentration on cycle life. The cycle regime was a stressful accelerated LEO, which consisted of a 27.5 min charge followed by a 17.5 min discharge (2x normal rate). The depth of discharge (DOD) was 80 percent. The cell temperature was maintained at 23 C. The boiler plate test results are in the process of being validated using flight hardware and real time LEO test at the Naval Weapons Support Center (NWSC), Crane, Indiana under a NASA Lewis Contract. Six 48 Ah Hughes recirculation design IPV nickel-hydrogen flight battery cells are being evaluated. Three of the cells contain 26 percent KOH (test cells), and three contain 31 percent KOH (control cells). They are undergoing real time LEO cycle life testing. The cycle regime is a 90-min LEO orbit consisting of a 54-min charge followed by a 36-min discharge. The depth-of-discharge is 80 percent. The cell temperature is maintained at 10 C. The three 31 percent KOH cells failed (cycles 3729, 4165, and 11355). One of the 26 percent KOH cells failed at cycle 15314. The other two 26 percent KOH cells were cycled for over 16600 cycles during the continuing test.
Impedance measurements on a spiral-wound nickel/metal hydride cell cycled in a simulated Leo orbit

NASA Technical Reports Server (NTRS)

Reid, Margaret A.

1993-01-01

A spiral-wound size C cell was cycled at 25 C in a low earth orbit (LEO) regime at 50 percent depth of discharge (DOD) with approximately five percent over-charge. The nominal capacity was 3.5 AH. The cell was cycled for 2000 cycles. Capacity checks and impedance measurements over the complete range of state of charge were made upon receipt and after 500, 1000, and 2000 cycles. The capacity of the cell was essentially unchanged until after the impedance measurements at 2000 cycles. Only small changes in the impedance parameters were observed, but there was somewhat more scatter in the data after 2000 cycles. When the cell was returned to LEO cycling after 2000 cycles, only 38 percent of the capacity could be obtained. It is believed that the cell failed because of an equipment failure at the end of the final impedance measurements which allowed an over-discharge.
Nickel hydrogen cell tests. [recharging

NASA Technical Reports Server (NTRS)

Mueller, V. C.

1981-01-01

Some parametric tests followed by cycling tests are described for the characterization of the service life of nickel hydrogen cells. Three cells were automatically cycled in simulated low Earth orbit in 35 minute discharge, 55 minute charge, with charging voltage limited, temperature compensated. The cells were mounted in a fixture that conducts heat to an aluminum baseplate. The baseplate in turn, is bounded in a temperature controlled bath to remove the heat from the mounted fixture. One cell was tested with a zircar separator, which failed after 2473 cyles. Two other cells were tested one with a zircar separator; the other with asbestos. More than 400 cycles were achieved.
Development of single-cell protectors for sealed silver-zinc cells

NASA Technical Reports Server (NTRS)

Lear, J. W.; Donovan, R. L.; Imamura, M. S.

1978-01-01

Three design approaches to cell-level protection were developed, fabricated, and tested. These systems are referred to as the single-cell protector (SCP), multiplexed-cell protector(MCP). To evaluate the systems 18-cell battery packs without cell level control were subjected to cycle life test. A total of five batteries were subjected to simulate synchronous orbit cycling at 40% depth of discharge at 22C. Batteries without cell-level protection failed between 345 and 255 cycles. Cell failure in the cell level protected batteries occurred between 412 and 540. It was determined that the cell-level monitoring and protection is necessary to attain the long cycle life of a AgZn battery. The best method of providing control and protection of the AgZn cells depends on the specific application and capability of the user.
Effects of 12-crown-4 ether on the electrochemical performance of CoO2 and TiS2 cathodes in Li polymer electrolyte cells

NASA Technical Reports Server (NTRS)

Nagasubramanian, G.; Attia, Alan I.; Halpert, G.

1992-01-01

The effect of adding 12-crown-4 ether (12Cr4) to the polyethylene oxide (PEO) electrolyte on the electrochemical properties of cells with Li(x)CoO2 or TiS2 as the cathode was investigated. The polymer electrolyte films were: (1) PEO, LiBF4; (2) PEO, LiBF4 with 12Cr4; (3) Li(x)CoO2, PEO, and LiBF4; and (4) Li(x)CoO2, PEO, LiBF4, and 12Cr4. It was found that, although 12Cr4 improved the cell performance over cells without 12Cr4 in the shallow c/d cycles (cyclic voltammetric behavior), it did not seem to prolong the active life of the cell. The cells with CoO2 as the cathode failed after a few c/d cycles, while similar cells with TiS2 did not fail even after 12 c/d cycles. The probable cause of failure in the case of CoO2 is ascribed to the instability of the CoO2 cathode.
Cycles till failure of silver-zinc cells with competing failure modes - Preliminary data analysis

NASA Technical Reports Server (NTRS)

Sidik, S. M.; Leibecki, H. F.; Bozek, J. M.

1980-01-01

The data analysis of cycles to failure of silver-zinc electrochemical cells with competing failure modes is presented. The test ran 129 cells through charge-discharge cycles until failure; preliminary data analysis consisted of response surface estimate of life. Batteries fail through low voltage condition and an internal shorting condition; a competing failure modes analysis was made using maximum likelihood estimation for the extreme value life distribution. Extensive residual plotting and probability plotting were used to verify data quality and selection of model.
KOH concentration effect on cycle life of nickel-hydrogen cells

NASA Technical Reports Server (NTRS)

Lim, Hong S.; Verzwyvelt, S. A.

1987-01-01

A cycle life test of Ni/H2 cells containing electrolytes of various KOH concentrations and a sintered type nickel electrode was carried out at 23 C using a 45 min accelerated low Earth orbit (LEO) cycle regime at 80 percent depth of discharge. One of three cells containing 26 percent KOH has achieved over 28,000 cycles, and the other two 19,000 cycles, without a sign of failure. Two other cells containing 31 percent KOH electrolyte, which is the concentration presently used in aerospace cells, failed after 2,979 and 3,620 cycles. This result indicates that the cycle life of the present type of Ni/H2 cells may be extended by a factor of 5 to 10 simply by lowering the KOH concentration. Long cycle life of a Ni/H2 battery at high depth-of-discharge operation is desired, particularly for an LEO spacecraft application. Typically, battery life of about 30,000 cycles is required for a five year mission in an LEO. Such a cycle life with presently available cells can be assured only at a very low depth-of-discharge operation. Results of testing already show that the cycle life of an Ni/H2 cell is tremendously improved by simply using an electrolyte of low KOH concentration.
Evaluation program for secondary spacecraft cells

NASA Technical Reports Server (NTRS)

Harkness, J. D.

1975-01-01

The cycle life tests to determine the performance capabilities of packs of cells under different loads and temperature conditions are reported. Results are summarized, and the failure of 14 failed cells is analyzed. It was found that the main cause of failure was separator deterioration and migration of the negative plate material.
Cyclin A and the retinoblastoma gene product complex with a common transcription factor.

PubMed

Bandara, L R; Adamczewski, J P; Hunt, T; La Thangue, N B

1991-07-18

The retinoblastoma gene (Rb) product is a negative regulator of cellular proliferation, an effect that could be mediated in part at the transcriptional level through its ability to complex with the sequence-specific transcription factor DRTF1. This interaction is modulated by adenovirus E1a, which sequesters the Rb protein and several other cellular proteins, including cyclin A, a molecule that undergoes cyclical accumulation and destruction during each cell cycle and which is required for cell cycle progression. Cyclin A, which also complexes with DRTF1, facilitates the efficient assembly of the Rb protein into the complex. This suggests a role for cyclin A in regulating transcription and defines a transcription factor through which molecules that regulate the cell cycle in a negative fashion, such as Rb, and in a positive fashion, such as cyclin A, interact. Mutant loss-of-function Rb alleles, which occur in a variety of tumour cells, also fail to complex with E1a and large T antigen. Here we report on a naturally occurring loss-of-function Rb allele encoding a protein that fails to complex with DRTF1. This might explain how mutation in the Rb gene prevents negative growth control.
Mutations in new cell cycle genes that fail to complement a multiply mutant third chromosome of Drosophila.

PubMed

White-Cooper, H; Carmena, M; Gonzalez, C; Glover, D M

1996-11-01

We have simultaneously screened for new alleles and second site mutations that fail to complement five cell cycle mutations of Drosphila carried on a single third chromosome (gnu, polo, mgr, asp, stg). Females that are either transheterozygous for scott of the antartic (scant) and polo, or homozygous for scant produce embryos that show mitotic defects. A maternal effect upon embryonic mitoses is also seen in embryos derived from females transheterozygous with helter skelter (hsk) and either mgr or asp. cleopatra (cleo), fails to complement asp but is not uncovered by a deficiency for asp. The mitotic phenotype of larvae heterozygous for cleo and the multiple mutant chromosome is similar to weak alleles of asp, but there are no defects in male meiosis. Mutations that failed to complement stg fell into two complementation groups corresponding to stg and a new gene noose. Three of the new stg alleles are early zygotic lethals, whereas the fourth is a pharate adult lethal allele that affects both mitosis and meiosis. Mutations in noose fully complement a small deficiency that removes stg, but when placed in trans to certain stg alleles, result in late lethality and mitotic abnormalities in larval brains.
The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1→S transition.

PubMed

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay; Sarvepalli, Kavitha; Sadhale, Parag P; Nath, Utpal

2011-07-01

The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, their exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1→S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1→S arrest is discussed. Copyright © 2011 Elsevier Inc. All rights reserved.
Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells

PubMed Central

Wang, Jennifer T; Kong, Dong; Hoerner, Christian R; Loncarek, Jadranka

2017-01-01

Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next. PMID:28906251
Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells.

PubMed

Wang, Jennifer T; Kong, Dong; Hoerner, Christian R; Loncarek, Jadranka; Stearns, Tim

2017-09-14

Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.

Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells.

PubMed

Xiao, Dong; Johnson, Candace S; Trump, Donald L; Singh, Shivendra V

2004-05-01

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, offers significant protection against cancer in animals induced by a variety of carcinogens. The present study demonstrates that PEITC suppresses proliferation of PC-3 cells in a dose-dependent manner by causing G(2)-M-phase cell cycle arrest and apoptosis. Interestingly, phenyl isothiocyanate (PITC), which is a structural analogue of PEITC but lacks the -CH(2) spacers that link the aromatic ring to the -N=C=S group, neither inhibited PC-3 cell viability nor caused cell cycle arrest or apoptosis. These results indicated that even a subtle change in isothiocyanate (ITC) structure could have a significant impact on its biological activity. The PEITC-induced cell cycle arrest was associated with a >80% reduction in the protein levels of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C (Cdc25C; 24 h after treatment with 10 micro M PEITC), which led to an accumulation of Tyr(15) phosphorylated (inactive) Cdk1. On the other hand, PITC treatment neither reduced protein levels of Cdk1 or Cdc25C nor affected Cdk1 phosphorylation. The PEITC-induced decline in Cdk1 and Cdc25C protein levels and cell cycle arrest were significantly blocked on pretreatment of PC-3 cells with proteasome inhibitor lactacystin. A 24 h exposure of PC-3 cells to 10 micro M PEITC, but not PITC, resulted in about 56% and 44% decrease in the levels of antiapoptotic proteins Bcl-2 and Bcl-X(L), respectively. However, ectopic expression of Bcl-2 failed to alter sensitivity of PC-3 cells to growth inhibition or apoptosis induction by PEITC. Treatment of cells with PEITC, but not PITC, also resulted in cleavage of procaspase-3, procaspase-9, and procaspase-8. Moreover, the PEITC-induced apoptosis was significantly attenuated in the presence of general caspase inhibitor and specific inhibitors of caspase-8 and caspase-9. In conclusion, our data indicate that PEITC-induced cell cycle arrest in PC-3 cells is likely due to proteasome-mediated degradation of Cdc25C and Cdk1, and ectopic expression of Bcl-2 fails to confer resistance to PEITC-induced apoptosis. Furthermore, the results of the present study point toward involvement of both caspase-8- and caspase-9-mediated pathways in apoptosis induction by PEITC.
cdc-25.4, a Caenorhabditis elegans Ortholog of cdc25, Is Required for Male Mating Behavior

PubMed Central

Oh, Sangmi; Kawasaki, Ichiro; Park, Jae-Hyung; Shim, Yhong-Hee

2016-01-01

Cell division cycle 25 (cdc25) is an evolutionarily conserved phosphatase that promotes cell cycle progression. Among the four cdc25 orthologs in Caenorhabditis elegans, we found that cdc-25.4 mutant males failed to produce outcrossed progeny. This was not caused by defects in sperm development, but by defects in male mating behavior. The cdc-25.4 mutant males showed various defects during male mating, including contact response, backing, turning, and vulva location. Aberrant turning behavior was the most prominent defect in the cdc-25.4 mutant males. We also found that cdc-25.4 is expressed in many neuronal cells throughout development. The turning defect in cdc-25.4 mutant males was recovered by cdc-25.4 transgenic expression in neuronal cells, suggesting that cdc-25.4 functions in neurons for male mating. However, the neuronal morphology of cdc-25.4 mutant males appeared to be normal, as examined with several neuronal markers. Also, RNAi depletion of wee-1.3, a C. elegans ortholog of Wee1/Myt1 kinase, failed to suppress the mating defects of cdc-25.4 mutant males. These findings suggest that, for successful male mating, cdc-25.4 does not target cell cycles that are required for neuronal differentiation and development. Rather, cdc-25.4 likely regulates noncanonical substrates in neuronal cells. PMID:27770028
cdc-25.4, a Caenorhabditis elegans Ortholog of cdc25, Is Required for Male Mating Behavior.

PubMed

Oh, Sangmi; Kawasaki, Ichiro; Park, Jae-Hyung; Shim, Yhong-Hee

2016-12-07

Cell division cycle 25 (cdc25) is an evolutionarily conserved phosphatase that promotes cell cycle progression. Among the four cdc25 orthologs in Caenorhabditis elegans, we found that cdc-25.4 mutant males failed to produce outcrossed progeny. This was not caused by defects in sperm development, but by defects in male mating behavior. The cdc-25.4 mutant males showed various defects during male mating, including contact response, backing, turning, and vulva location. Aberrant turning behavior was the most prominent defect in the cdc-25.4 mutant males. We also found that cdc-25.4 is expressed in many neuronal cells throughout development. The turning defect in cdc-25.4 mutant males was recovered by cdc-25.4 transgenic expression in neuronal cells, suggesting that cdc-25.4 functions in neurons for male mating. However, the neuronal morphology of cdc-25.4 mutant males appeared to be normal, as examined with several neuronal markers. Also, RNAi depletion of wee-1.3, a C. elegans ortholog of Wee1/Myt1 kinase, failed to suppress the mating defects of cdc-25.4 mutant males. These findings suggest that, for successful male mating, cdc-25.4 does not target cell cycles that are required for neuronal differentiation and development. Rather, cdc-25.4 likely regulates noncanonical substrates in neuronal cells. Copyright © 2016 Oh et al.
A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression.

PubMed

Mir, Riyaz A; Bele, Aditya; Mirza, Sameer; Srivastava, Shashank; Olou, Appolinaire A; Ammons, Shalis A; Kim, Jun Hyun; Gurumurthy, Channabasavaiah B; Qiu, Fang; Band, Hamid; Band, Vimla

2015-12-28

Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression

PubMed Central

Mir, Riyaz A.; Bele, Aditya; Mirza, Sameer; Srivastava, Shashank; Olou, Appolinaire A.; Ammons, Shalis A.; Kim, Jun Hyun; Gurumurthy, Channabasavaiah B.; Qiu, Fang; Band, Hamid

2015-01-01

Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function. PMID:26711270
Validation test of 125 Ah advanced design IPV nickel-hydrogen flight cells

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1993-01-01

An update of validation test results confirming the advanced design nickel-hydrogen cell is presented. An advanced 125 Ah individual pressure vessel Ni-H cell was designed. The primary function of the advanced cell is to store and deliver energy for long-term LEO spacecraft missions. The new features of this design are: (1) use of 26 percent rather than 31 percent KOH electrolyte; (2) use of a patented catalyzed wall wick; (3) use of serrated-edge separators to facilitate gaseous O and H flow within the cell, while maintaining physical contact with the wall wick for electrolyte management; and (4) use of a floating rather than a fixed stack to accommodate Ni electrode expansion due to charge/discharge cycling. The significant improvements resulting from these innovations are extended cycle life; enhanced thermal, electrolyte, and oxygen management; and accommodation of Ni electrode expansion. Six 125 Ah flight cells based on this design were fabricated; the catalyzed wall wick cells have been cycled for over 19,000 cycles with no cell failures in the continuing test. Two of the noncatalyzed wall wick cells failed (cycles 9588 and 13,900).
Improving the Safety of Lithium-Ion Battery via a Redox Shuttle Additive 2,5-Di- tert-butyl-1,4-bis(2-methoxyethoxy)benzene (DBBB).

PubMed

Leonet, Olatz; Colmenares, Luis C; Kvasha, Andriy; Oyarbide, Mikel; Mainar, Aroa R; Glossmann, Tobias; Blázquez, J Alberto; Zhang, Zhengcheng

2018-03-21

2,5-Di- tert-butyl-1,4-bis(2-methoxyethoxy)benzene (DBBB) is studied as a redox shuttle additive for overcharge protection for a 1.5 Ah graphite/C-LFP lithium-ion pouch cell for the first time. The electrochemical performance demonstrated that the protecting additive remains inert during the extended standard cycling for 4000 cycles. When a 100% overcharge is introduced in the charging protocol, the baseline cell fails rapidly during the first abusive event, whereas the cell containing DBBB additive withstands 700 overcharge cycles with 87% capacity retention and no gas evolution or cell swelling was observed. It is the first time the effectiveness of the DBBB as overcharge protection additive in a large pouch cell format is demonstrated.
Progranulin regulates neurogenesis in the developing vertebrate retina.

PubMed

Walsh, Caroline E; Hitchcock, Peter F

2017-09-01

We evaluated the expression and function of the microglia-specific growth factor, Progranulin-a (Pgrn-a) during developmental neurogenesis in the embryonic retina of zebrafish. At 24 hpf pgrn-a is expressed throughout the forebrain, but by 48 hpf pgrn-a is exclusively expressed by microglia and/or microglial precursors within the brain and retina. Knockdown of Pgrn-a does not alter the onset of neurogenic programs or increase cell death, however, in its absence, neurogenesis is significantly delayed-retinal progenitors fail to exit the cell cycle at the appropriate developmental time and postmitotic cells do not acquire markers of terminal differentiation, and microglial precursors do not colonize the retina. Given the link between Progranulin and cell cycle regulation in peripheral tissues and transformed cells, we analyzed cell cycle kinetics among retinal progenitors following Pgrn-a knockdown. Depleting Pgrn-a results in a significant lengthening of the cell cycle. These data suggest that Pgrn-a plays a dual role during nervous system development by governing the rate at which progenitors progress through the cell cycle and attracting microglial progenitors into the embryonic brain and retina. Collectively, these data show that Pgrn-a governs neurogenesis by regulating cell cycle kinetics and the transition from proliferation to cell cycle exit and differentiation. © 2017 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 77: 1114-1129, 2017. © 2017 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc.
Cell cycle re-entry sensitizes podocytes to injury induced death.

PubMed

Hagen, Manuel; Pfister, Eva; Kosel, Andrea; Shankland, Stuart; Pippin, Jeffrey; Amann, Kerstin; Daniel, Christoph

2016-07-17

Podocytes are terminally differentiated renal cells, lacking the ability to regenerate by proliferation. However, during renal injury, podocytes re-enter into the cell cycle but fail to divide. Earlier studies suggested that re-entry into cell cycle results in loss of podocytes, but a direct evidence for this is lacking. Therefore, we established an in vitro model to test the consequences of re-entry into the cell cycle on podocyte survival. A mouse immortalized podocyte cell line was differentiated to non-permissive podocytes and stimulated with e.g. growth factors. Stimulated cells were analyzed for mRNA-expression or stained for cell cycle analysis using flow cytometry and immunocytofluorescence microscopy. After stimulation to re-entry into cell cycle, podocytes were stressed with puromycin aminonucleoside (PAN) and analyzed for survival. During permissive stage more than 40% of immortalized podocytes were in the S-phase. In contrast, S-phase in non-permissive differentiated podocytes was reduced to 5%. Treatment with b-FGF dose dependently induced re-entry into cell cycle increasing the number of podocytes in the S-phase to 10.7% at an optimal bFGF dosage of 10 ng/ml. Forty eight hours after stimulation with bFGF the number of bi-nucleated podocytes significantly increased. A secondary injury stimulus significantly reduced podocyte survival preferentially in bi-nucleated podocytes In conclusion, stimulation of podocytes using bFGF was able to induce re-entry of podocytes into the cell cycle and to sensitize the cells for cell death by secondary injuries. Therefore, this model is appropriate for testing new podocyte protective substances that can be used for therapy.
Cycles till failure of silver-zinc cells with completing failures modes: Preliminary data analysis

NASA Technical Reports Server (NTRS)

Sidik, S. M.; Leibecki, H. F.; Bozek, J. M.

1980-01-01

One hundred and twenty nine cells were run through charge-discharge cycles until failure. The experiment design was a variant of a central composite factorial in five factors. Preliminary data analysis consisted of response surface estimation of life. Batteries fail under two basic modes; a low voltage condition and an internal shorting condition. A competing failure modes analysis using maximum likelihood estimation for the extreme value life distribution was performed. Extensive diagnostics such as residual plotting and probability plotting were employed to verify data quality and choice of model.
Cyclebase 3.0: a multi-organism database on cell-cycle regulation and phenotypes.

PubMed

Santos, Alberto; Wernersson, Rasmus; Jensen, Lars Juhl

2015-01-01

The eukaryotic cell division cycle is a highly regulated process that consists of a complex series of events and involves thousands of proteins. Researchers have studied the regulation of the cell cycle in several organisms, employing a wide range of high-throughput technologies, such as microarray-based mRNA expression profiling and quantitative proteomics. Due to its complexity, the cell cycle can also fail or otherwise change in many different ways if important genes are knocked out, which has been studied in several microscopy-based knockdown screens. The data from these many large-scale efforts are not easily accessed, analyzed and combined due to their inherent heterogeneity. To address this, we have created Cyclebase--available at http://www.cyclebase.org--an online database that allows users to easily visualize and download results from genome-wide cell-cycle-related experiments. In Cyclebase version 3.0, we have updated the content of the database to reflect changes to genome annotation, added new mRNA and protein expression data, and integrated cell-cycle phenotype information from high-content screens and model-organism databases. The new version of Cyclebase also features a new web interface, designed around an overview figure that summarizes all the cell-cycle-related data for a gene. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
Effect of storage and LEO cycling on manufacturing technology IPV nickel-hydrogen cells

NASA Technical Reports Server (NTRS)

Smithrick, John J.

1987-01-01

Yardney Manufacturing Technology (MANTECH) 50 A-hr space weight individual pressure vessel nickel-hydrogen cells were evaluated. This consisted of investigating: the effect of storage and charge/discharge cycling on cell performance. For the storage test the cells were precharged with hydrogen, by the manufacturer, to a pressure of 14.5 psia. After undergoing activation and acceptance tests, the cells were discharged at C/10 rate (5A) to 0.1 V or less. The terminals were then shorted. The cells were shipped to NASA Lewis Research Center where they were stored at room temperature in the shorted condition for 1 year. After storage, the acceptance tests were repeated at NASA Lewis. A comparison of test results indicate no significant degradation in electrical performance due to 1 year storage. For the cycle life test the regime was a 90 minute low earth orbit at deep depths of discharge (80 and 60 percent). At the 80 percent DOD the three cells failed on the average at cycle 741. Failure for this test was defined to occur when the cell voltage degraded to 1 V prior to completion of the 35 min discharge. The DOD was reduced to 60 percent. The cycle life test was continued.
Cell cycle re-entry sensitizes podocytes to injury induced death

PubMed Central

Hagen, Manuel; Pfister, Eva; Kosel, Andrea; Shankland, Stuart; Pippin, Jeffrey; Amann, Kerstin; Daniel, Christoph

2016-01-01

ABSTRACT Podocytes are terminally differentiated renal cells, lacking the ability to regenerate by proliferation. However, during renal injury, podocytes re-enter into the cell cycle but fail to divide. Earlier studies suggested that re-entry into cell cycle results in loss of podocytes, but a direct evidence for this is lacking. Therefore, we established an in vitro model to test the consequences of re-entry into the cell cycle on podocyte survival. A mouse immortalized podocyte cell line was differentiated to non-permissive podocytes and stimulated with e.g. growth factors. Stimulated cells were analyzed for mRNA-expression or stained for cell cycle analysis using flow cytometry and immunocytofluorescence microscopy. After stimulation to re-entry into cell cycle, podocytes were stressed with puromycin aminonucleoside (PAN) and analyzed for survival. During permissive stage more than 40% of immortalized podocytes were in the S-phase. In contrast, S-phase in non-permissive differentiated podocytes was reduced to 5%. Treatment with b-FGF dose dependently induced re-entry into cell cycle increasing the number of podocytes in the S-phase to 10.7% at an optimal bFGF dosage of 10 ng/ml. Forty eight hours after stimulation with bFGF the number of bi-nucleated podocytes significantly increased. A secondary injury stimulus significantly reduced podocyte survival preferentially in bi-nucleated podocytes In conclusion, stimulation of podocytes using bFGF was able to induce re-entry of podocytes into the cell cycle and to sensitize the cells for cell death by secondary injuries. Therefore, this model is appropriate for testing new podocyte protective substances that can be used for therapy. PMID:27232327
The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 {yields} S transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay

2011-07-01

Highlights: {yields} TCP4 is a class II TCP transcription factor, that represses cell division in Arabidopsis. {yields} TCP4 expression in yeast retards cell division by blocking G1 {yields} S transition. {yields} Genome-wide expression studies and Western analysis reveals stabilization of cell cycle inhibitor Sic1, as possible mechanism. -- Abstract: The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, theirmore » exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 {yields} S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 {yields} S arrest is discussed.« less
Isotretinoin revisited: pluripotent effects on human sebaceous gland cells.

PubMed

Zouboulis, Christos C

2006-10-01

Nelson et al. confirmed the previously described antiproliferative effect of isotretinoin on human sebocytes. They attributed a portion of this decrease to cell cycle arrest and detected sebocyte apoptosis, which was not recapitulated by alitretinoin or tretinoin. These events were specific to sebocytes, as isotretinoin failed to induce apoptosis in keratinocytes. Isotretinoin-induced apoptosis was shown to be an RAR-independent mechanism.
Recovery from the DNA Replication Checkpoint

PubMed Central

Chaudhury, Indrajit; Koepp, Deanna M.

2016-01-01

Checkpoint recovery is integral to a successful checkpoint response. Checkpoint pathways monitor progress during cell division so that in the event of an error, the checkpoint is activated to block the cell cycle and activate repair pathways. Intrinsic to this process is that once repair has been achieved, the checkpoint signaling pathway is inactivated and cell cycle progression resumes. We use the term “checkpoint recovery” to describe the pathways responsible for the inactivation of checkpoint signaling and cell cycle re-entry after the initial stress has been alleviated. The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. When replication stress is encountered, replication forks are stalled, and the checkpoint signaling pathway is activated. Central to recovery from the S-phase checkpoint is the restart of stalled replication forks. If checkpoint recovery fails, stalled forks may become unstable and lead to DNA breaks or unusual DNA structures that are difficult to resolve, causing genomic instability. Alternatively, if cell cycle resumption mechanisms become uncoupled from checkpoint inactivation, cells with under-replicated DNA might proceed through the cell cycle, also diminishing genomic stability. In this review, we discuss the molecular mechanisms that contribute to inactivation of the S-phase checkpoint signaling pathway and the restart of replication forks during recovery from replication stress. PMID:27801838
Tumor suppressor Lzap regulates cell cycle progression, doming and zebrafish epiboly

PubMed Central

Liu, Dan; Wang, Wen-Der; Melville, David B.; Cha, Yong I.; Yin, Zhirong; Issaeva, Natalia; Knapik, Ela W.; Yarbrough, Wendell G.

2012-01-01

Initial stages of embryonic development rely on rapid, synchronized cell divisions of the fertilized egg followed by a set of morphogenetic movements collectively called epiboly and gastrulation. Lzap is a putative tumor suppressor whose expression is lost in 30% of head and neck squamous cell carcinomas. Lzap activities include regulation of cell cycle progression and response to therapeutic agents. Here we explore developmental roles of the lzap gene during zebrafish morphogenesis. Lzap is highly conserved among vertebrates and is maternally deposited. Expression is initially ubiquitous during gastrulation, and later becomes more prominent in the pharyngeal arches, digestive tract and brain. Antisense morpholino-mediated depletion of Lzap resulted in delayed cell divisions and apoptosis during blastomere formation, resulting in fewer, larger cells. Cell cycle analysis suggested that Lzap loss in early embryonic cells resulted in a G2/M arrest. Furthermore, the Lzap-deficient embryos failed to initiate epiboly – the earliest morphogenetic movement in animal development – which has been shown to be dependent on cell adhesion and migration of epithelial sheets. Our results strongly implicate Lzap in regulation of cell cycle progression, adhesion and migratory activity of epithelial cell sheets during early development. These functions provide further insight into Lzap activity that may contribute not only to development, but also to tumor formation. PMID:21523853
Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels.

PubMed

Yang, Ke; Guo, Yang; Stacey, William C; Harwalkar, Jyoti; Fretthold, Jonathan; Hitomi, Masahiro; Stacey, Dennis W

2006-08-30

The expression level of cyclin D1 plays a vital role in the control of proliferation. This protein is reported to be degraded following phosphorylation by glycogen synthase kinase 3 (GSK3) on Thr-286. We recently showed that phosphorylation of Thr-286 is responsible for a decline in cyclin D1 levels during S phase, an event required for efficient DNA synthesis. These studies were undertaken to test the possibility that phosphorylation by GSK3 is responsible for the S phase specific decline in cyclin D1 levels, and that this event is regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway which controls GSK3. We found, however, that neither PI3K, AKT, GSK3, nor proliferative signaling activity in general is responsible for the S phase decline in cyclin D1 levels. In fact, the activity of these signaling kinases does not vary through the cell cycle of proliferating cells. Moreover, we found that GSK3 activity has little influence over cyclin D1 expression levels during any cell cycle phase. Inhibition of GSK3 activity by siRNA, LiCl, or other chemical inhibitors failed to influence cyclin D1 phosphorylation on Thr-286, even though LiCl efficiently blocked phosphorylation of beta-catenin, a known substrate of GSK3. Likewise, the expression of a constitutively active GSK3 mutant protein failed to influence cyclin D1 phosphorylation or total protein expression level. Because we were unable to identify any proliferative signaling molecule or pathway which is regulated through the cell cycle, or which is able to influence cyclin D1 levels, we conclude that the suppression of cyclin D1 levels during S phase is regulated by cell cycle position rather than signaling activity. We propose that this mechanism guarantees the decline in cyclin D1 levels during each S phase; and that in so doing it reduces the likelihood that simple over expression of cyclin D1 can lead to uncontrolled cell growth.
C/EBPα regulates CRL4Cdt2-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint

PubMed Central

Hall, Jonathan R; Bereman, Michael S; Nepomuceno, Angelito I; Thompson, Elizabeth A; Muddiman, David C; Smart, Robert C

2014-01-01

The bZIP transcription factor, C/EBPα is highly inducible by UVB and other DNA damaging agents in keratinocytes. C/EBPα-deficient keratinocytes fail to undergo cell cycle arrest in G1 in response to UVB-induced DNA damage and mice lacking epidermal C/EBPα are highly susceptible to UVB-induced skin cancer. The mechanism through which C/EBPα regulates the cell cycle checkpoint in response to DNA damage is unknown. Here we report untreated C/EBPα-deficient keratinocytes have normal levels of the cyclin-dependent kinase inhibitor, p21, however, UVB-treated C/EBPα-deficient keratinocytes fail to up-regulate nuclear p21 protein levels despite normal up-regulation of Cdkn1a mRNA levels. UVB-treated C/EBPα-deficient keratinocytes displayed a 4-fold decrease in nuclear p21 protein half-life due to the increased proteasomal degradation of p21 via the E3 ubiquitin ligase CRL4Cdt2. Cdt2 is the substrate recognition subunit of CRL4Cdt2 and Cdt2 mRNA and protein levels were up-regulated in UVB-treated C/EBPα-deficient keratinocytes. Knockdown of Cdt2 restored p21 protein levels in UVB-treated C/EBPα-deficient keratinocytes. Lastly, the failure to accumulate p21 in response to UVB in C/EBPα-deficient keratinocytes resulted in decreased p21 interactions with critical cell cycle regulatory proteins, increased CDK2 activity, and inappropriate entry into S-phase. These findings reveal C/EBPα regulates G1/S cell cycle arrest in response to DNA damage via the control of CRL4Cdt2 mediated degradation of p21. PMID:25483090
Regulation of male germ cell cycle arrest and differentiation by DND1 is modulated by genetic background

PubMed Central

Cook, Matthew S.; Munger, Steven C.; Nadeau, Joseph H.; Capel, Blanche

2011-01-01

Human germ cell tumors show a strong sensitivity to genetic background similar to Dnd1Ter/Ter mutant mice, where testicular teratomas arise only on the 129/SvJ genetic background. The introduction of the Bax mutation onto mixed background Dnd1Ter/Ter mutants, where teratomas do not typically develop, resulted in a high incidence of teratomas. However, when Dnd1Ter/Ter; Bax–/– double mutants were backcrossed to C57BL/6J, no tumors arose. Dnd1Ter/Ter germ cells show a strong downregulation of male differentiation genes including Nanos2. In susceptible strains, where teratomas initiate around E15.5-E17.5, many mutant germ cells fail to enter mitotic arrest in G0 and do not downregulate the pluripotency markers NANOG, SOX2 and OCT4. We show that DND1 directly binds a group of transcripts that encode negative regulators of the cell cycle, including p27Kip1 and p21Cip1. P27Kip1 and P21Cip1 protein are both significantly decreased in Dnd1Ter/Ter germ cells on all strain backgrounds tested, strongly suggesting that DND1 regulates mitotic arrest in male germ cells through translational regulation of cell cycle genes. Nonetheless, in C57BL/6J mutants, germ cells arrest prior to M-phase of the cell cycle and downregulate NANOG, SOX2 and OCT4. Consistent with their ability to rescue cell cycle arrest, C57BL/6J germ cells overexpress negative regulators of the cell cycle relative to 129/SvJ. This work suggests that reprogramming of pluripotency in germ cells and prevention of tumor formation requires cell cycle arrest, and that differences in the balance of cell cycle regulators between 129/SvJ and C57BL/6 might underlie differences in tumor susceptibility. PMID:21115610

Mcl1 regulates the terminal mitosis of neural precursor cells in the mammalian brain through p27Kip1.

PubMed

Hasan, S M Mahmudul; Sheen, Ashley D; Power, Angela M; Langevin, Lisa Marie; Xiong, Jieying; Furlong, Michael; Day, Kristine; Schuurmans, Carol; Opferman, Joseph T; Vanderluit, Jacqueline L

2013-08-01

Cortical development requires the precise timing of neural precursor cell (NPC) terminal mitosis. Although cell cycle proteins regulate terminal mitosis, the factors that influence the cell cycle machinery are incompletely understood. Here we show in mice that myeloid cell leukemia 1 (Mcl1), an anti-apoptotic Bcl-2 protein required for the survival of NPCs, also regulates their terminal differentiation through the cell cycle regulator p27(Kip1). A BrdU-Ki67 cell profiling assay revealed that in utero electroporation of Mcl1 into NPCs in the embryonic neocortex increased NPC cell cycle exit (the leaving fraction). This was further supported by a decrease in proliferating NPCs (Pax6(+) radial glial cells and Tbr2(+) neural progenitors) and an increase in differentiating cells (Dcx(+) neuroblasts and Tbr1(+) neurons). Similarly, BrdU birth dating demonstrated that Mcl1 promotes premature NPC terminal mitosis giving rise to neurons of the deeper cortical layers, confirming their earlier birthdate. Changes in Mcl1 expression within NPCs caused concomitant changes in the levels of p27(Kip1) protein, a key regulator of NPC differentiation. Furthermore, in the absence of p27(Kip1), Mcl1 failed to induce NPC cell cycle exit, demonstrating that p27(Kip1) is required for Mcl1-mediated NPC terminal mitosis. In summary, we have identified a novel physiological role for anti-apoptotic Mcl1 in regulating NPC terminal differentiation.
Validation test of 125 Ah advanced design IPV nickel-hydrogen flight cells

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1993-01-01

An update of validation test results confirming the advanced design nickel-hydrogen cell is presented. An advanced 125 Ah individual pressure vessel (IPV) nickel-hydrogen cell was designed. The primary function of the advanced cell is to store and deliver energy for long-term, Low-Earth-Orbit (LEO) spacecraft missions. The new features of this design, which are not incorporated in state-of-the-art design cells, are: (1) use of 26 percent rather than 31 percent potassium hydroxide (KOH) electrolyte; (2) use of a patented catalyzed wall wick; (3) use of serrated-edge separators to facilitate gaseous oxygen and hydrogen flow within the cell, while still maintaining physical contact with the wall wick for electrolyte management; and (4) use of a floating rather than a fixed stack (state-of-the-art) to accommodate nickel electrode expansion due to charge/discharge cycling. The significant improvements resulting from these innovations are extended cycle life; enhanced thermal, electrolyte, and oxygen management; and accommodation of nickel electrode expansion. Six 125 Ah flight cells based on this design were fabricated by Eagle-Picher. Three of the cells contain all of the advanced features (test cells) and three are the same as the test cells except they do not have catalyst on the wall wick (control cells). All six cells are in the process of being evaluated in a LEO cycle life test at the Naval Weapons Support Center, Crane, IN, under a NASA Lewis Research Center contract. The catalyzed wall wick cells have been cycled for over 19000 cycles with no cell failures in the continuing test. Two of the noncatalyzed wall wick cells failed (cycles 9588 and 13,900).
Concerted control of Escherichia coli cell division

PubMed Central

Osella, Matteo; Nugent, Eileen; Cosentino Lagomarsino, Marco

2014-01-01

The coordination of cell growth and division is a long-standing problem in biology. Focusing on Escherichia coli in steady growth, we quantify cell division control using a stochastic model, by inferring the division rate as a function of the observable parameters from large empirical datasets of dividing cells. We find that (i) cells have mechanisms to control their size, (ii) size control is effected by changes in the doubling time, rather than in the single-cell elongation rate, (iii) the division rate increases steeply with cell size for small cells, and saturates for larger cells. Importantly, (iv) the current size is not the only variable controlling cell division, but the time spent in the cell cycle appears to play a role, and (v) common tests of cell size control may fail when such concerted control is in place. Our analysis illustrates the mechanisms of cell division control in E. coli. The phenomenological framework presented is sufficiently general to be widely applicable and opens the way for rigorous tests of molecular cell-cycle models. PMID:24550446
Conserved cell cycle regulatory properties within the amino terminal domain of the Epstein-Barr virus nuclear antigen 3C

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Nikhil; Knight, Jason S.; Robertson, Erle S.

The gammaherpesviruses Rhesus lymphocryptovirus (LCV) and Epstein-Barr virus (EBV) are closely related phylogenetically. Rhesus LCV efficiently immortalizes Rhesus B cells in vitro. However, despite a high degree of conservation between the Rhesus LCV and EBV genomes, Rhesus LCV fails to immortalize human B cells in vitro. This species restriction may, at least in part, be linked to the EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), known to be essential for B cell transformation. We compared specific properties of EBNA3C, a well-characterized and essential EBV protein, with its Rhesus counterpart to determine whether EBNA3C phenotypes which contribute to cellmore » cycle regulation are conserved in the Rhesus LCV. We show that both EBNA3C and Rhesus EBNA3C bind to a conserved region of mammalian cyclins, regulate pRb stability, and modulate SCF{sup Skp2}-dependent ubiquitination. These results suggest that Rhesus LCV restriction from human B cell immortalization is independent of the conserved cell cycle regulatory functions of the EBNA3C protein.« less
Cytokinesis-Based Constraints on Polarized Cell Growth in Fission Yeast

PubMed Central

Bohnert, K. Adam; Gould, Kathleen L.

2012-01-01

The rod-shaped fission yeast Schizosaccharomyces pombe, which undergoes cycles of monopolar-to-bipolar tip growth, is an attractive organism for studying cell-cycle regulation of polarity establishment. While previous research has described factors mediating this process from interphase cell tips, we found that division site signaling also impacts the re-establishment of bipolar cell growth in the ensuing cell cycle. Complete loss or targeted disruption of the non-essential cytokinesis protein Fic1 at the division site, but not at interphase cell tips, resulted in many cells failing to grow at new ends created by cell division. This appeared due to faulty disassembly and abnormal persistence of the cell division machinery at new ends of fic1Δ cells. Moreover, additional mutants defective in the final stages of cytokinesis exhibited analogous growth polarity defects, supporting that robust completion of cell division contributes to new end-growth competency. To test this model, we genetically manipulated S. pombe cells to undergo new end take-off immediately after cell division. Intriguingly, such cells elongated constitutively at new ends unless cytokinesis was perturbed. Thus, cell division imposes constraints that partially override positive controls on growth. We posit that such constraints facilitate invasive fungal growth, as cytokinesis mutants displaying bipolar growth defects formed numerous pseudohyphae. Collectively, these data highlight a role for previous cell cycles in defining a cell's capacity to polarize at specific sites, and they additionally provide insight into how a unicellular yeast can transition into a quasi-multicellular state. PMID:23093943
PD1 blockade with pembrolizumab is highly effective in relapsed or refractory NK/T-cell lymphoma failing l-asparaginase.

PubMed

Kwong, Yok-Lam; Chan, Thomas S Y; Tan, Daryl; Kim, Seok Jin; Poon, Li-Mei; Mow, Benjamin; Khong, Pek-Lan; Loong, Florence; Au-Yeung, Rex; Iqbal, Jabed; Phipps, Colin; Tse, Eric

2017-04-27

Natural killer (NK)/T-cell lymphomas failing L-asparaginse regimens have no known salvage and are almost invariably fatal. Seven male patients with NK/T-cell lymphoma (median age, 49 years; range, 31-68 years) for whom a median of 2 (range, 1-5) regimens (including l-asparaginase regimens and allogeneic hematopoietic stem-cell transplantation [HSCT] in 2 cases) failed were treated with the anti-programmed death 1 (PD1) antibody pembrolizumab. All patients responded, according to various clinical, radiologic (positron emission tomography), morphologic, and molecular (circulating Epstein-Barr virus [EBV] DNA) criteria. Two patients achieved complete response (CR) in all parameters. Three patients achieved clinical and radiologic CRs, with two having molecular remission (undetectable EBV DNA) but minimal EBV-encoded RNA-positive cells in lesions comprising predominantly CD3 + CD4 + and CD3 + CD8 + T cells (which ultimately disappeared, suggesting they represented pseudoprogression) and one having detectable EBV DNA despite morphologic CR. Two patients achieved partial response (PR). After a median of 7 (range, 2-13) cycles of pembrolizumab and a follow-up of a median of 6 (range, 2-10) months, all five CR patients were still in remission. The only adverse event was grade 2 skin graft-versus-host disease in one patient with previous allogeneic HSCT. Expression of the PD1 ligand was strong in 4 patients (3 achieving CR) and weak in 1 (achieving PR). PD1 blockade with pembrolizumab was a potent strategy for NK/T-cell lymphomas failing l-asparaginase regimens. © 2017 by The American Society of Hematology.
The MAPK Signaling Cascade is a Central Hub in the Regulation of Cell Cycle, Apoptosis and Cytoskeleton Remodeling by Tripeptidyl-Peptidase II

PubMed Central

Sompallae, Ramakrishna; Stavropoulou, Vaia; Houde, Mathieu; Masucci, Maria G.

2008-01-01

Tripeptidyl-peptidase II (TPPII) is a serine peptidase highly expressed in malignant Burkitt’s lymphoma cells (BL). We have previously shown that overexpression of TPPII correlates with chromosomal instability, centrosomal and mitotic spindle abnormalities and resistance to apoptosis induced by spindle poisons. Furthermore, TPPII knockdown by RNAi was associated with endoreplication and the accumulation of polynucleated cells that failed to complete cell division, indicating a role of TPPII in the cell cycle. Here we have applied a global approach of gene expression analysis to gain insights on the mechanism by which TPPII regulates this phenotype. mRNA profiling of control and TPPII knockdown BL cells identified one hundred and eighty five differentially expressed genes. Functional categorization of these genes highlighted major physiological functions such as apoptosis, cell cycle progression, cytoskeleton remodeling, proteolysis, and signal transduction. Pathways and protein interactome analysis revealed a significant enrichment in components of MAP kinases signaling. These findings suggest that TPPII influences a wide network of signaling pathways that are regulated by MAPKs and exerts thereby a pleiotropic effect on biological processes associated with cell survival, proliferation and genomic instability. PMID:19787088
Life testing of secondary silver-zinc cells

NASA Technical Reports Server (NTRS)

Brewer, Jeffrey C.; Doreswamy, Rajiv

1991-01-01

Testing on a variety of secondary silver-zinc (Ag-Zn) cells has been in progress at the Marshall Space Flight Center (MSFC) for over six years. The latest test involves a 350-Ah cell design that has been cycled at 10 C for 16 months. This design has achieved over 7200 low-earth-orbit (LEO) cycles as well as 17 deep discharges at an 85 percent depth of discharge. This test not only is a life test on these cells but also addresses different methods of storing these cells between the deep discharges. As the test is approaching completion, some interesting results are being seen. In particular, two of the four packs currently on test have failed to meet the 35-h (295-Ah) deep discharge requirement that was arbitrarily set at the beginning of the test. This capacity loss failure is likely a result of the storage method used on these two packs between deep discharges. The two packs are LEO cycled in such a way as to minimize overcharge in an attempt to prolong life.
Evaluation program for secondary spacecraft cells: Acceptance tests of Eagle-Picher 12.0 ampere-hour nickel-cadmium cells with auxiliary electrodes

NASA Technical Reports Server (NTRS)

Christy, D. E.

1971-01-01

An acceptance test program was conducted on 24 cells to insure that all cells put into the life cycle program were of high quality by the removal of cells found to have electrolyte leakage, internal shorts, low capacity, or inability of any cell to recover its open circuit voltage above 1.150 volts after the cell short test. The cells were rated at 12.0 ampere-hours and equipped with auxiliary electrodes. Test results were: (1) The capacity of the 24 cells ranged from 14.6 to 16.8 ah. All the cells exceeded the rated capacity on all three capacity checks. (2) One cell failed to recover to 1.150 volts after the cell short test. (3) During the overcharge tests, all cells but one failed the test at the c/10 rate after the first minute. (4) A special resistance test was conducted on the auxiliary electrodes of these cells to establish the resistance value necessary which would provide maximum signal power across the auxiliary electrode. The resistance value established was 10 ohms. (5) No electrolyte leakage was observed.
Virtex-5 CN Package Daisy Chain Evaluation Test Report

NASA Technical Reports Server (NTRS)

Suh, Jong-ook

2016-01-01

The board-level temperature cycling reliability of Xilinx Virtex-5 (V5) CN package was investigated. V5s were temperature cycled under two conditions, 0 to +100 C (0/100) and -55 to +100 C (-55/100). During the 0/100 test, no part out of 8 parts failed up to 6586 cycles. During the -55/100 test, one part out of 8 parts failed at 1236 cycle, and there were no additional failures up to 1705 cycles. The failure mode of the part that failed at 1236 cycles indicated that most likely the failure was not a solder fatigue failure, and therefore no obvious solder fatigue failure was observed throughout the tests.
Comparison of Accelerated Testing with Modeling to Predict Lifetime of CPV Solder Layers (Presentation)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Silverman, T. J.; Bosco, N.; Kurtz, S.

2012-03-01

Concentrating photovoltaic (CPV) cell assemblies can fail due to thermomechanical fatigue in the die-attach layer. In this presentation, we show the latest results from our computational model of thermomechanical fatigue. The model is used to estimate the relative lifetime of cell assemblies exposed to various temperature histories consistent with service and with accelerated testing. We also present early results from thermal cycling experiments designed to help validate the computational model.
The SUMO pathway is essential for nuclear integrity and chromosome segregation in mice.

PubMed

Nacerddine, Karim; Lehembre, François; Bhaumik, Mantu; Artus, Jérôme; Cohen-Tannoudji, Michel; Babinet, Charles; Pandolfi, Pier Paolo; Dejean, Anne

2005-12-01

Covalent modification by SUMO regulates a wide range of cellular processes, including transcription, cell cycle, and chromatin dynamics. To address the biological function of the SUMO pathway in mammals, we generated mice deficient for the SUMO E2-conjugating enzyme Ubc9. Ubc9-deficient embryos die at the early postimplantation stage. In culture, Ubc9 mutant blastocysts are viable, but fail to expand after 2 days and show apoptosis of the inner cell mass. Loss of Ubc9 leads to major chromosome condensation and segregation defects. Ubc9-deficient cells also show severe defects in nuclear organization, including nuclear envelope dysmorphy and disruption of nucleoli and PML nuclear bodies. Moreover, RanGAP1 fails to accumulate at the nuclear pore complex in mutant cells that show a collapse in Ran distribution. Together, these findings reveal a major role for Ubc9, and, by implication, for the SUMO pathway, in nuclear architecture and function, chromosome segregation, and embryonic viability in mammals.
Heat-shock protein 60 is required for blastema formation and maintenance during regeneration

PubMed Central

Makino, Shinji; Whitehead, Geoffrey G.; Lien, Ching-Ling; Kim, Soo; Jhawar, Payal; Kono, Akane; Kawata, Yasushi; Keating, Mark T.

2005-01-01

Zebrafish fin regeneration requires the formation and maintenance of blastema cells. Blastema cells are not derived from stem cells but behave as such, because they are slow-cycling and are thought to provide rapidly proliferating daughter cells that drive regenerative outgrowth. The molecular basis of blastema formation is not understood. Here, we show that heat-shock protein 60 (hsp60) is required for blastema formation and maintenance. We used a chemical mutagenesis screen to identify no blastema (nbl), a zebrafish mutant with an early fin regeneration defect. Fin regeneration failed in nbl due to defective blastema formation. nbl also failed to regenerate hearts. Positional cloning and mutational analyses revealed that nbl results from a V324E missense mutation in hsp60. This mutation reduced hsp60 function in binding and refolding denatured proteins. hsp60 expression is increased during formation of blastema cells, and dysfunction leads to mitochondrial defects and apoptosis in these cells. These data indicate that hsp60 is required for the formation and maintenance of regenerating tissue. PMID:16204379
X-33 Tank Failure During Autoclave Fabrication

NASA Technical Reports Server (NTRS)

Nettles, Alan T.; Munafo, Paul (Technical Monitor)

2001-01-01

During a repair cure cycle on tank #1 of the X-33 liquid hydrogen tanks, a skin to core disbond occurred. Both the inner skin and outer skin of the lobe #1 sandwich panel was noted to have been disbonded and cracked- An investigation was undertaken to determine the cause of this failure. The investigation consisted of reviewing all of the processing data and performing testing on the failed lobe #1, as well as the other lobes, which did not fail during the cure cycle. The tests consisted of residual stress measurements in one of the intact lobes and "plug-pulls" to assess skin to core strength on all of the remaining lobes. Results showed an extremely low bondline strength due to lack of proper filleting of the adhesive, in addition, tests showed a very rapid decrease in strength with increasing temperature, as well as a further decrease in strength with a larger number of cycles. Also, the honeycomb used was not vented so pressure could build up within the cells. All of these factors appeared to be contributors to the failure.
CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

PubMed

Převorovský, Martin; Oravcová, Martina; Zach, Róbert; Jordáková, Anna; Bähler, Jürg; Půta, František; Folk, Petr

2016-11-16

For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.
A specifically designed nanoconstruct associates, internalizes, traffics in cardiovascular cells, and accumulates in failing myocardium: a new strategy for heart failure diagnostics and therapeutics.

PubMed

Ruiz-Esparza, Guillermo U; Segura-Ibarra, Victor; Cordero-Reyes, Andrea M; Youker, Keith A; Serda, Rita E; Cruz-Solbes, Ana S; Amione-Guerra, Javier; Yokoi, Kenji; Kirui, Dickson K; Cara, Francisca E; Paez-Mayorga, Jesus; Flores-Arredondo, Jose H; Guerrero-Beltrán, Carlos E; Garcia-Rivas, Gerardo; Ferrari, Mauro; Blanco, Elvin; Torre-Amione, Guillermo

2016-02-01

Ongoing inflammation and endothelial dysfunction occurs within the local microenvironment of heart failure, creating an appropriate scenario for successful use and delivery of nanovectors. This study sought to investigate whether cardiovascular cells associate, internalize, and traffic a nanoplatform called mesoporous silicon vector (MSV), and determine its intravenous accumulation in cardiac tissue in a murine model of heart failure. In vitro cellular uptake and intracellular trafficking of MSVs was examined by scanning electron microscopy, confocal microscopy, time-lapse microscopy, and flow cytometry in cardiac myocytes, fibroblasts, smooth muscle cells, and endothelial cells. The MSVs were internalized within the first hours, and trafficked to perinuclear regions in all the cell lines. Cytotoxicity was investigated by annexin V and cell cycle assays. No significant evidence of toxicity was found. In vivo intravenous cardiac accumulation of MSVs was examined by high content fluorescence and confocal microscopy, with results showing increased accumulation of particles in failing hearts compared with normal hearts. Similar to observations in vitro, MSVs were able to associate, internalize, and traffic to the perinuclear region of cardiomyocytes in vivo. Results show that MSVs associate, internalize, and traffic in cardiovascular cells without any significant toxicity. Furthermore, MSVs accumulate in failing myocardium after intravenous administration, reaching intracellular regions of the cardiomyocytes. These findings represent a novel avenue to develop nanotechnology-based therapeutics and diagnostics in heart failure. © 2016 The Authors European Journal of Heart Failure © 2016 European Society of Cardiology.
Enforced expression of the c-myc oncogene inhibits cell differentiation by precluding entry into a distinct predifferentiation state in G/sub 0//G/sub 1/

DOE Office of Scientific and Technical Information (OSTI.GOV)

Freytag, S.O.

1988-04-01

A broad base of data has implicated a role for the c-myc proto-oncogene in the control of the cell cycle and cell differentiation. To further define the role of myc in these processes, the authors examined the effect of enforced myc expression on several events that are thought to be important steps leading to the terminally differentiated state: (i) the ability to arrest growth in G/sub 0//G/sub 1/, (ii) the ability to replicate the genome upon initiation of the differentiation program, and (iii) the ability to loose responsiveness to mitogens and withdraw from the cell cycle. 3T3-L1 preadipocyte cell linesmore » expressing various levels of myc mRNA were established by transfection with a recombinant myc gene under the transcriptional control of the Rous sarcoma virus (RSV) promoter. Cells that expressed high constitutive levels of pRSV myc mRNA arrested in G/sub 0//G/sub 1/ at densities similar to those of normal cells at confluence. Upon initiation of the differentiation program, such cells traversed the cell cycle with kinetics similar to those of normal cells and subsequently arrested in G/sub 0//G/sub 1/. Thus, enforced expression of myc had no effect on the ability of cells to arrest growth in G/sub 0//G/sub 1/ or to replicate the genome upon initiation of the differentiation program. Cells were then tested for their ability to reenter the cell cycle upon exposure to high concentrations of serum and for their capacity to differentiate. In contrast to normal cells, cells expressing high constitutive levels of myc RNA reentered the cell cycle when challenged with 30% serum and failed to terminally differentiate.« less
FGFR and PTEN signaling interact during lens development to regulate cell survival

PubMed Central

Chaffee, Blake R.; Hoang, Thanh V.; Leonard, Melissa R.; Bruney, Devin G.; Wagner, Brad D.; Dowd, Joseph Richard; Leone, Gustavo; Ostrowski, Michael C.; Robinson, Michael L.

2016-01-01

Lens epithelial cells express many receptor tyrosine kinases (RTKs) that stimulate PI3K-AKT and RAS-RAF-MEK-ERK intracellular signaling pathways. These pathways ultimately activate the phosphorylation of key cellular transcription factors and other proteins that control proliferation, survival, metabolism, and differentiation in virtually all cells. Among RTKs in the lens, only stimulation of fibroblast growth factor receptors (FGFRs) elicits a lens epithelial cell to fiber cell differentiation response in mammals. Moreover, although the lens expresses three different Fgfr genes, the isolated removal of Fgfr2 at the lens placode stage inhibits both lens cell survival and fiber cell differentiation. Phosphatase and tensin homolog (PTEN), commonly known as a tumor suppressor, inhibits ERK and AKT activation and initiates both apoptotic pathways, and cell cycle arrest. Here, we show that the combined deletion of Fgfr2 and Pten rescues the cell death phenotype associated with Fgfr2 loss alone. Additionally, Pten removal increased AKT and ERK activation, above the levels of controls, in the presence or absence of Fgfr2. However, isolated deletion of Pten failed to stimulate ectopic fiber cell differentiation, and the combined deletion of Pten and Fgfr2 failed to restore differentiation-specific Aquaporin0 and DnaseIIβ expression in the lens fiber cells. PMID:26764128
A role for the ESCRT system in cell division in archaea.

PubMed

Samson, Rachel Y; Obita, Takayuki; Freund, Stefan M; Williams, Roger L; Bell, Stephen D

2008-12-12

Archaea are prokaryotic organisms that lack endomembrane structures. However, a number of hyperthermophilic members of the Kingdom Crenarchaea, including members of the Sulfolobus genus, encode homologs of the eukaryotic endosomal sorting system components Vps4 and ESCRT-III (endosomal sorting complex required for transport-III). We found that Sulfolobus ESCRT-III and Vps4 homologs underwent regulation of their expression during the cell cycle. The proteins interacted and we established the structural basis of this interaction. Furthermore, these proteins specifically localized to the mid-cell during cell division. Overexpression of a catalytically inactive mutant Vps4 in Sulfolobus resulted in the accumulation of enlarged cells, indicative of failed cell division. Thus, the archaeal ESCRT system plays a key role in cell division.
P53-dependent antiproliferative and pro-apoptotic effects of trichostatin A (TSA) in glioblastoma cells.

PubMed

Bajbouj, K; Mawrin, C; Hartig, R; Schulze-Luehrmann, J; Wilisch-Neumann, A; Roessner, A; Schneider-Stock, R

2012-05-01

Glioblastomas are known to be highly chemoresistant, but HDAC inhibitors (HDACi) have been shown to be of therapeutic relevance for this aggressive tumor type. We treated U87 glioblastoma cells with trichostatin A (TSA) to define potential epigenetic targets for HDACi-mediated antitumor effects. Using a cDNA array analysis covering 96 cell cycle genes, cyclin-dependent kinase inhibitor p21(WAF1) was identified as the major player in TSA-induced cell cycle arrest. TSA slightly inhibited proliferation and viability of U87 cells, cumulating in a G1/S cell cycle arrest. This effect was accompanied by a significant up-regulation of p53 and its transcriptional target p21(WAF1) and by down-regulation of key G1/S regulators, such as cdk4, cdk6, and cyclin D1. Nevertheless, TSA did not induce apoptosis in U87 cells. As expected, TSA promoted the accumulation of total acetylated histones H3 and H4 and a decrease in endogenous HDAC activity. Characterizing the chromatin modulation around the p21(WAF1) promoter after TSA treatment using chromatin immunoprecipitation, we found (1) a release of HDAC1, (2) an increase of acetylated H4 binding, and (3) enhanced recruitment of p53. p53-depleted U87 cells showed an abrogation of the G1/S arrest and re-entered the cell cycle. Immunofluorescence staining revealed that TSA induced the nuclear translocation of p21(WAF1) verifying a cell cycle arrest. On the other hand, a significant portion of p21(WAF1) was present in the cytoplasmic compartment causing apoptosis resistance. Furthermore, TSA-treated p53-mutant cell line U138 failed to show an induction in p21(WAF1), showed a deficient G2/M checkpoint, and underwent mitotic catastrophe. We suggest that HDAC inhibition in combination with other clinically used drugs may be considered an effective strategy to overcome chemoresistance in glioblastoma cells.

Proliferation kinetics and cyclic AMP as prognostic factors in adult acute leukemia.

PubMed

Paietta, E; Mittermayer, K; Schwarzmeier, J

1980-07-01

In 41 adult patients with acute leukemia (myeloblastic, lymphoblastic, and undifferentiated), proliferation kinetics (as determined by double-label autoradiography) and cyclic adenosine 3',5'-monophosphate (cAMP) concentration were studied for their significance in the prediction of responsiveness to cytostatic therapy. Patients with good clinical response had significantly shorter turnover times and higher labeling indices in the bone marrow than did those who failed to respond to treatment. Cases for which cell kinetics did not correlate with clinical response were explained by variance in the distribution of leukemic blasts between the proliferative cell cycle and the resting pool. Good clinical response was also found to be associated with low levels of cAMP in leukemic cells prior to therapy, whereas high cAMP contents predicted failure. Low cAMP concentrations, however, did not necessarily correlate with short turnover times and vice versa. This might be due to fluctuations of the cAMP concentrations during the cell cycle.
Sporophytic and gametophytic functions of the cell cycle-associated Mob1 gene in Arabidopsis thaliana L.

PubMed

Galla, Giulio; Zenoni, Sara; Marconi, Gianpiero; Marino, Giada; Botton, Alessandro; Pinosa, Francesco; Citterio, Sandra; Ruperti, Benedetto; Palme, Klaus; Albertini, Emidio; Pezzotti, Mario; Mau, Martin; Sharbel, Timothy F; De Storme, Nico; Geelen, Danny; Barcaccia, Gianni

2011-09-15

Mob1 genes are primarily involved in the cell cycle progression and mitosis exit in yeasts and animals. The function of a Mob1-like gene (At5g45550) from Arabidopsis thaliana was investigated using RNAi and immunological staining. AtMob1-like RNAi silenced lines showed a reduced radial expansion of the inflorescence stem and a reduced elongation zone of the primary root. Morphological features of plant organs were accompanied by a reduction in cell size. The fertility of AtMob1-like RNAi silenced lines was very low as seed production was strongly reduced. About 2% of the progeny of AtMob1-like RNAi silenced plants were tetraploid. The female and male sporogenesis was affected differentially. The ovules developed irregularly and one third of the megaspores and embryo sacs degenerated prematurely. Up to 20% of the ovules produced binucleated megaspores that failed to develop further, being their degeneration likely accompanied with a delayed programmed cell death. The anthers produced about 30% of aborted pollen grains, showing also a strong variation in their size. Together, the results show that Arabidopsis MOB1-like is required to regulate cell expansion and cell division, presumably by affecting the mitotic as well as the meiotic cell cycle. Copyright © 2011 Elsevier B.V. All rights reserved.
DNA hypomethylation induces a DNA replication-associated cell cycle arrest to block hepatic outgrowth in uhrf1 mutant zebrafish embryos

PubMed Central

Jacob, Vinitha; Chernyavskaya, Yelena; Chen, Xintong; Tan, Poh Seng; Kent, Brandon; Hoshida, Yujin; Sadler, Kirsten C.

2015-01-01

UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 to hemimethylated DNA during replication and is essential for maintaining DNA methylation. uhrf1 mutant zebrafish have global DNA hypomethylation and display embryonic defects, including a small liver, and they die as larvae. We make the surprising finding that, despite their reduced organ size, uhrf1 mutants express high levels of genes controlling S-phase and have many more cells undergoing DNA replication, as measured by BrdU incorporation. In contrast to wild-type hepatocytes, which are continually dividing during hepatic outgrowth and thus dilute the BrdU label, uhrf1 mutant hepatocytes retain BrdU throughout outgrowth, reflecting cell cycle arrest. Pulse-chase-pulse experiments with BrdU and EdU, and DNA content analysis indicate that uhrf1 mutant cells undergo DNA re-replication and that apoptosis is the fate of many of the re-replicating and arrested hepatocytes. Importantly, the DNA re-replication phenotype and hepatic outgrowth failure are preceded by global loss of DNA methylation. Moreover, uhrf1 mutants are phenocopied by mutation of dnmt1, and Dnmt1 knockdown in uhrf1 mutants enhances their small liver phenotype. Together, these data indicate that unscheduled DNA replication and failed cell cycle progression leading to apoptosis are the mechanisms by which DNA hypomethylation prevents organ expansion in uhrf1 mutants. We propose that cell cycle arrest leading to apoptosis is a strategy that restricts propagation of epigenetically damaged cells during embryogenesis. PMID:25564650
DNA hypomethylation induces a DNA replication-associated cell cycle arrest to block hepatic outgrowth in uhrf1 mutant zebrafish embryos.

PubMed

Jacob, Vinitha; Chernyavskaya, Yelena; Chen, Xintong; Tan, Poh Seng; Kent, Brandon; Hoshida, Yujin; Sadler, Kirsten C

2015-02-01

UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 to hemimethylated DNA during replication and is essential for maintaining DNA methylation. uhrf1 mutant zebrafish have global DNA hypomethylation and display embryonic defects, including a small liver, and they die as larvae. We make the surprising finding that, despite their reduced organ size, uhrf1 mutants express high levels of genes controlling S-phase and have many more cells undergoing DNA replication, as measured by BrdU incorporation. In contrast to wild-type hepatocytes, which are continually dividing during hepatic outgrowth and thus dilute the BrdU label, uhrf1 mutant hepatocytes retain BrdU throughout outgrowth, reflecting cell cycle arrest. Pulse-chase-pulse experiments with BrdU and EdU, and DNA content analysis indicate that uhrf1 mutant cells undergo DNA re-replication and that apoptosis is the fate of many of the re-replicating and arrested hepatocytes. Importantly, the DNA re-replication phenotype and hepatic outgrowth failure are preceded by global loss of DNA methylation. Moreover, uhrf1 mutants are phenocopied by mutation of dnmt1, and Dnmt1 knockdown in uhrf1 mutants enhances their small liver phenotype. Together, these data indicate that unscheduled DNA replication and failed cell cycle progression leading to apoptosis are the mechanisms by which DNA hypomethylation prevents organ expansion in uhrf1 mutants. We propose that cell cycle arrest leading to apoptosis is a strategy that restricts propagation of epigenetically damaged cells during embryogenesis. © 2015. Published by The Company of Biologists Ltd.
Loss of PodJ in Agrobacterium tumefaciens Leads to Ectopic Polar Growth, Branching, and Reduced Cell Division

PubMed Central

Anderson-Furgeson, James C.; Zupan, John R.; Grangeon, Romain

2016-01-01

ABSTRACT Agrobacterium tumefaciens is a rod-shaped Gram-negative bacterium that elongates by unipolar addition of new cell envelope material. Approaching cell division, the growth pole transitions to a nongrowing old pole, and the division site creates new growth poles in sibling cells. The A. tumefaciens homolog of the Caulobacter crescentus polar organizing protein PopZ localizes specifically to growth poles. In contrast, the A. tumefaciens homolog of the C. crescentus polar organelle development protein PodJ localizes to the old pole early in the cell cycle and accumulates at the growth pole as the cell cycle proceeds. FtsA and FtsZ also localize to the growth pole for most of the cell cycle prior to Z-ring formation. To further characterize the function of polar localizing proteins, we created a deletion of A. tumefaciens podJ (podJAt). ΔpodJAt cells display ectopic growth poles (branching), growth poles that fail to transition to an old pole, and elongated cells that fail to divide. In ΔpodJAt cells, A. tumefaciens PopZ-green fluorescent protein (PopZAt-GFP) persists at nontransitioning growth poles postdivision and also localizes to ectopic growth poles, as expected for a growth-pole-specific factor. Even though GFP-PodJAt does not localize to the midcell in the wild type, deletion of podJAt impacts localization, stability, and function of Z-rings as assayed by localization of FtsA-GFP and FtsZ-GFP. Z-ring defects are further evidenced by minicell production. Together, these data indicate that PodJAt is a critical factor for polar growth and that ΔpodJAt cells display a cell division phenotype, likely because the growth pole cannot transition to an old pole. IMPORTANCE How rod-shaped prokaryotes develop and maintain shape is complicated by the fact that at least two distinct species-specific growth modes exist: uniform sidewall insertion of cell envelope material, characterized in model organisms such as Escherichia coli, and unipolar growth, which occurs in several alphaproteobacteria, including Agrobacterium tumefaciens. Essential components for unipolar growth are largely uncharacterized, and the mechanism constraining growth to one pole of a wild-type cell is unknown. Here, we report that the deletion of a polar development gene, podJAt, results in cells exhibiting ectopic polar growth, including multiple growth poles and aberrant localization of cell division and polar growth-associated proteins. These data suggest that PodJAt is a critical factor in normal polar growth and impacts cell division in A. tumefaciens. PMID:27137498
In vitro recapitulation of the urea cycle using murine embryonic stem cell-derived in vitro liver model.

PubMed

Tamai, Miho; Aoki, Mami; Nishimura, Akihito; Morishita, Koji; Tagawa, Yoh-ichi

2013-12-01

Ammonia, a toxic metabolite, is converted to urea in hepatocytes via the urea cycle, a process necessary for cell/organismal survival. In liver, hepatocytes, polygonal and multipolar structures, have a few sides which face hepatic sinusoids and adjacent hepatocytes to form intercellular bile canaliculi connecting to the ductules. The critical nature of this three-dimensional environment should be related to the maintenance of hepatocyte function such as urea synthesis. Recently, we established an in vitro liver model derived from murine embryonic stem cells, IVL(mES), which included the hepatocyte layer and a surrounding sinusoid vascular-like network. The IVL(mES) culture, where the hepatocyte is polarized in a similar fashion to its in vivo counterpart, could successfully recapitulate in vivo results. L-Ornithine is an intermediate of the urea cycle, but supplemental L-ornithine does not activate the urea cycle in the apolar primary hepatocyte of monolayer culture. In the IVL(mES), supplemental L-ornithine could activate the urea cycle, and also protect against ammonium/alcohol-induced hepatocyte death. While the IVL(mES) displays architectural and functional properties similar to the liver, primary hepatocyte of monolayer culture fail to model critical functional aspects of liver physiology. We propose that the IVL(mES) will represent a useful, humane alternative to animal studies for drug toxicity and mechanistic studies of liver injury.
DEC1/STRA13 is a key negative regulator of activation-induced proliferation of human B cells highly expressed in anergic cells.

PubMed

Camponeschi, Alessandro; Todi, Laura; Cristofoletti, Cristina; Lazzeri, Cristina; Carbonari, Maurizio; Mitrevski, Milica; Marrapodi, Ramona; Del Padre, Martina; Fiorilli, Massimo; Casato, Milvia; Visentini, Marcella

2018-06-01

The transcription factor DEC1/STRA13 (also known as BHLHE40 and SHARP2) is involved in a number of processes including inhibition of cell proliferation and delay of cell cycle, and is a negative regulator of B cell activation and development in mice. We show here that, unlike in mice, DEC1/STRA13 expression is induced in human naïve and memory resting B cells by activation through the B-cell receptor (BCR) or Toll-like receptor 9 (TLR9). siRNA silencing of DEC1/STRA13 increases the capacity of activated B cells to perform a high number of divisions after TLR9 ligation. This identifies DEC1/STRA13 as a critical negative regulator of clonal expansion of activated human B cells. We also show that DEC1/STRA13 is upregulated in human anergic CD21 low B cells clonally expanded in patients with HCV-associated mixed cryoglobulinemia, which fail to proliferate in response to BCR or TLR9 ligation. siRNA knockdown of DEC1/STRA13, however, fails to restore responsiveness to stimuli in these cells, although it might improve the proliferative capacity in a subset of anergic cells with less pronounced proliferative defect. Copyright © 2018 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
Alteration of the carbohydrate for deoxyguanosine analogs markedly changes DNA replication fidelity, cell cycle progression and cytotoxicity

PubMed Central

O’Konek, Jessica J.; Ladd, Brendon; Flanagan, Sheryl A.; Im, Mike M.; Boucher, Paul D.; Thepsourinthone, Tico S.; Secrist, John A.; Shewach, Donna S.

2011-01-01

Nucleoside analogs are efficacious cancer chemotherapeutics due to their incorporation into tumor cell DNA. However, they exhibit vastly different antitumor efficacies, suggesting that incorporation produces divergent effects on DNA replication. Here we have evaluated the consequences of incorporation on DNA replication and its fidelity for three structurally related deoxyguanosine analogs: ganciclovir (GCV), currently in clinical trials in a suicide gene therapy approach for cancer, D-carbocyclic 2′-deoxyguanosine (CdG) and penciclovir (PCV). GCV and CdG elicited similar cytotoxicity at low concentrations, whereas PCV was 10–100-fold less cytotoxic in human tumor cells. DNA replication fidelity was evaluated using a supF plasmid-based mutation assay. Only GCV induced a dose-dependent increase in mutation frequency, predominantly GC→TA transversions, which contributed to cytotoxicity and implicated the ether oxygen in mutagenicity. Activation of mismatch repair with hydroxyurea decreased mutations but failed to repair the GC→TA transversions. GCV slowed S-phase progression and CdG also induced a G2/M block, but both drugs allowed completion of one cell cycle after drug treatment followed by cell death in the second cell cycle. In contrast, PCV induced a lengthy early S-phase block due to profound suppression of DNA synthesis, with cell death in the first cell cycle after drug treatment. These data suggest that GCV and CdG elicit superior cytotoxicity due to their effects in template DNA, whereas strong inhibition of nascent strand synthesis by PCV may protect against cytotoxicity. Nucleoside analogs based on the carbohydrate structures of GCV and CdG is a promising area for antitumor drug development. PMID:20004674
N-acetyl-L-cysteine increases MnSOD activity and enhances the recruitment of quiescent human fibroblasts to the proliferation cycle during wound healing.

PubMed

Mao, Gaowei; Goswami, Monali; Kalen, Amanda L; Goswami, Prabhat C; Sarsour, Ehab H

2016-01-01

The rebuilding of the connective tissue during wound healing requires the recruitment of fibroblasts to the wound area as well as reentry of quiescent fibroblasts to the proliferative cycle. Whether this process can be modulated by a small molecular weight thiol antioxidant N-acetyl-L-cysteine (NAC) was tested in normal human skin fibroblasts (NHFs) using a uni-directional wound healing assay. NAC treated cells demonstrated a decreased migration rate but increased number of proliferating cells recruited into the wound area post wounding. Fifteen day quiescent control and NAC treated NHFs were re-plated at a lower density and cell numbers counted at different days post-plating. Interestingly, NAC treated cells exhibited increased cellular proliferation indicated by both decreased cell population doubling time and increased S phase cells. NAC treated cells demonstrated decreased steady state levels of reactive oxygen species as well as increased protein and activity levels of manganese superoxide dismutase (MnSOD). NAC treatment failed to induce proliferation in quiescent cells lacking MnSOD expression. These results demonstrate that NAC enhanced the recruitment of quiescent NHFs into proliferation cycle during wound healing. Our results also suggest that the wound healing properties of NAC might be due to its ability to induce and enhance MnSOD expression and activity. Altogether, these findings suggest NAC might be potentially developed as a dietary intervention to improve tissue injury in animals and humans.
TRMM Solar Array

NASA Technical Reports Server (NTRS)

1996-01-01

Basic requirement of 978.59 watts per Panel output @ 58.9 volts B.O.L. was met on an average basis per agreement with NASA. Lower grade Cells were used on the shadowed Panel (Boom shadow) to maximize available power to the Spacecraft. The average output @ 58.9 volts was 991 watts. The outputs of the four t4) Panels ranged from 960 to 1,022 watts. The Panels successfully passed environmental testing at TRW to the contract specification and subsequent testing at NASA which involved output measurements at elevated temperatures. As this type of Array had never previously been built by TRW (aluminum Substrate with 4 cm x 4.4 cm GaAs Cells), the TRMM Program was a development effort combined with a Qual and Flight production effort. The most significant technical problem was Cell cracking during Qual thermal cycling. The cracking problem was determined to be generic within our Solar Array factory in the application of GaAs Cells to our designs. As a result, a TRW funded manufacturing process verification panel (known as the Manufacturing Verification Panel) was built to demonstrate our ability to properly apply GaAs Cells. The original Qual Panel comprised three (3) design variations with respect to Coverglass-to-Cell and Cell-to-Substrate adhesives. The intent was to qualify multiple designs in case one or more failed. When two of the three combinations failed due to excessive Cell breakage during thermal cycling, NASA was reluctant to allow Flight production based on the one remaining good Qual Panel Quadrant. This issue was pivotal for continuing the contract. Facts and recommendations are as follows: (1) The cause of the excessive cracking was never determined. and (2) The areas where the excessive cracking occurred utilized DC93-500 glassing adhesive which was NASA approved, and had been widely used by TRW on a multitude of projects.
Cryopreserved embryo transfer: adjacent or non-adjacent to failed fresh long GnRH-agonist protocol IVF cycle.

PubMed

Volodarsky-Perel, Alexander; Eldar-Geva, Talia; Holzer, Hananel E G; Schonberger, Oshrat; Reichman, Orna; Gal, Michael

2017-03-01

The optimal time to perform cryopreserved embryo transfer (CET) after a failed oocyte retrieval-embryo transfer (OR-ET) cycle is unknown. Similar clinical pregnancy rates were recently reported in immediate and delayed CET, performed after failed fresh OR-ET, in cycles with the gonadotrophin-releasing hormone (GnRH) antagonist protocol. This study compared outcomes of CET performed adjacently (<50 days, n = 67) and non-adjacently (≥50 to 120 days, n = 62) to the last OR-day of cycles with the GnRH agonist down-regulation protocol. Additional inclusion criteria were patients' age 20-38 years, the transfer of only 1-2 cryopreserved embryos, one treatment cycle per patient and artificial preparation for CET. Significantly higher implantation, clinical pregnancy and live birth rates were found in the non-adjacent group than in the adjacent group: 30.5% versus 11.3% (P = 0.001), 41.9% versus 17.9% (P = 0.003) and 32.3% versus 13.4% (P = 0.01), respectively. These results support the postponement of CET after a failed OR-ET for at least one menstrual cycle, when a preceding long GnRH-agonist protocol is used. Copyright Â© 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.

PubMed

Frémin, Christophe; Bessard, Anne; Ezan, Frédéric; Gailhouste, Luc; Régeard, Morgane; Le Seyec, Jacques; Gilot, David; Pagès, Gilles; Pouysségur, Jacques; Langouët, Sophie; Baffet, Georges

2009-03-01

We investigated the specific role of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long-term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)-dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF-dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2-dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2. Our results emphasize that transient MAPK inhibition allows multiple cell cycles in primary cultures of hepatocytes and that ERK2 has a key role in the regulation of S phase entry. Moreover, we revealed a major and distinct role of ERK1 in the regulation of hepatocyte survival. Taken together, our results represent an important advance in understanding long-term survival and cell cycle regulation of hepatocytes.
Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses

PubMed Central

Rubbi, Carlos P.; Milner, Jo

2003-01-01

p53 protects against cancer through its capacity to induce cell cycle arrest or apoptosis under a large variety of cellular stresses. It is not known how such diversity of signals can be integrated by a single molecule. However, the literature reveals that a common denominator in all p53-inducing stresses is nucleolar disruption. We thus postulated that the impairment of nucleolar function might stabilize p53 by preventing its degradation. Using micropore irradiation, we demonstrate that large amounts of nuclear DNA damage fail to stabilize p53 unless the nucleolus is also disrupted. Forcing nucleolar disruption by anti-upstream binding factor (UBF) microinjection (in the absence of DNA damage) also causes p53 stabilization. We propose that the nucleolus is a stress sensor responsible for maintenance of low levels of p53, which are automatically elevated as soon as nucleolar function is impaired in response to stress. Our model integrates all known p53-inducing agents and also explains cell cycle-related variations in p53 levels which correlate with established phases of nucleolar assembly/disassembly through the cell cycle. PMID:14609953
Dual Functions of α-Ketoglutarate Dehydrogenase E2 in the Krebs Cycle and Mitochondrial DNA Inheritance in Trypanosoma brucei

PubMed Central

Sykes, Steven E.

2013-01-01

The dihydrolipoyl succinyltransferase (E2) of the multisubunit α-ketoglutarate dehydrogenase complex (α-KD) is an essential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally regulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. We found that despite the absence of a functional α-KD, bloodstream form (BF) trypanosomes express α-KDE2, which localized to the mitochondrial matrix and inner membrane. Furthermore, α-KDE2 fractionated with the mitochondrial genome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for α-KDE2 in kDNA maintenance was revealed in α-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showed pronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation of cells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochondrial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that α-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribution of kDNA networks to daughter cells at cytokinesis. PMID:23125353
Dual functions of α-ketoglutarate dehydrogenase E2 in the Krebs cycle and mitochondrial DNA inheritance in Trypanosoma brucei.

PubMed

Sykes, Steven E; Hajduk, Stephen L

2013-01-01

The dihydrolipoyl succinyltransferase (E2) of the multisubunit α-ketoglutarate dehydrogenase complex (α-KD) is an essential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally regulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. We found that despite the absence of a functional α-KD, bloodstream form (BF) trypanosomes express α-KDE2, which localized to the mitochondrial matrix and inner membrane. Furthermore, α-KDE2 fractionated with the mitochondrial genome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for α-KDE2 in kDNA maintenance was revealed in α-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showed pronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation of cells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochondrial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that α-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribution of kDNA networks to daughter cells at cytokinesis.
Cardiac gene transfer of short hairpin RNA directed against phospholamban effectively knocks down gene expression but causes cellular toxicity in canines.

PubMed

Bish, Lawrence T; Sleeper, Meg M; Reynolds, Caryn; Gazzara, Jeffrey; Withnall, Elanor; Singletary, Gretchen E; Buchlis, George; Hui, Daniel; High, Katherine A; Gao, Guangping; Wilson, James M; Sweeney, H Lee

2011-08-01

Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways.
Cardiac Gene Transfer of Short Hairpin RNA Directed Against Phospholamban Effectively Knocks Down Gene Expression but Causes Cellular Toxicity in Canines

PubMed Central

Sleeper, Meg M.; Reynolds, Caryn; Gazzara, Jeffrey; Withnall, Elanor; Singletary, Gretchen E.; Buchlis, George; Hui, Daniel; High, Katherine A.; Gao, Guangping; Wilson, James M.; Sweeney, H. Lee

2011-01-01

Abstract Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways. PMID:21542669
The DivJ, CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti

PubMed Central

Pini, Francesco; Frage, Benjamin; Ferri, Lorenzo; De Nisco, Nicole J.; Mohapatra, Saswat S.; Taddei, Lucilla; Fioravanti, Antonella; Dewitte, Frederique; Galardini, Marco; Brilli, Matteo; Villeret, Vincent; Bazzicalupo, Marco; Mengoni, Alessio; Walker, Graham C.; Becker, Anke; Biondi, Emanuele G.

2013-01-01

SUMMARY Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis. PMID:23909720
Fumarate hydratase is a critical metabolic regulator of hematopoietic stem cell functions.

PubMed

Guitart, Amelie V; Panagopoulou, Theano I; Villacreces, Arnaud; Vukovic, Milica; Sepulveda, Catarina; Allen, Lewis; Carter, Roderick N; van de Lagemaat, Louie N; Morgan, Marcos; Giles, Peter; Sas, Zuzanna; Gonzalez, Marta Vila; Lawson, Hannah; Paris, Jasmin; Edwards-Hicks, Joy; Schaak, Katrin; Subramani, Chithra; Gezer, Deniz; Armesilla-Diaz, Alejandro; Wills, Jimi; Easterbrook, Aaron; Coman, David; So, Chi Wai Eric; O'Carroll, Donal; Vernimmen, Douglas; Rodrigues, Neil P; Pollard, Patrick J; Morton, Nicholas M; Finch, Andrew; Kranc, Kamil R

2017-03-06

Strict regulation of stem cell metabolism is essential for tissue functions and tumor suppression. In this study, we investigated the role of fumarate hydratase (Fh1), a key component of the mitochondrial tricarboxylic acid (TCA) cycle and cytosolic fumarate metabolism, in normal and leukemic hematopoiesis. Hematopoiesis-specific Fh1 deletion (resulting in endogenous fumarate accumulation and a genetic TCA cycle block reflected by decreased maximal mitochondrial respiration) caused lethal fetal liver hematopoietic defects and hematopoietic stem cell (HSC) failure. Reexpression of extramitochondrial Fh1 (which normalized fumarate levels but not maximal mitochondrial respiration) rescued these phenotypes, indicating the causal role of cellular fumarate accumulation. However, HSCs lacking mitochondrial Fh1 (which had normal fumarate levels but defective maximal mitochondrial respiration) failed to self-renew and displayed lymphoid differentiation defects. In contrast, leukemia-initiating cells lacking mitochondrial Fh1 efficiently propagated Meis1 / Hoxa9 -driven leukemia. Thus, we identify novel roles for fumarate metabolism in HSC maintenance and hematopoietic differentiation and reveal a differential requirement for mitochondrial Fh1 in normal hematopoiesis and leukemia propagation. © 2017 Guitart et al.
ADP-ribosylation factor arf6p may function as a molecular switch of new end take off in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujita, Atsushi

2008-02-01

Small GTPases act as molecular switches in a wide variety of cellular processes. In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from a monopolar manner to a bipolar manner, which is known as 'New End Take Off' (NETO). Here I report the identification of a gene, arf6{sup +}, encoding an ADP-ribosylation factor small GTPase, that may be essential for NETO. arf6{delta} cells completely fail to undergo NETO. arf6p localizes at both cell ends and presumptive septa in a cell-cycle dependent manner. And its polarized localization is not dependent on microtubules, actin cytoskeletons and some NETO factors (bud6p,more » for3p, tea1p, tea3p, and tea4p). Notably, overexpression of a fast GDP/GTP-cycling mutant of arf6p can advance the timing of NETO. These findings suggest that arf6p functions as a molecular switch for the activation of NETO in fission yeast.« less

K48-linked KLF4 ubiquitination by E3 ligase Mule controls T-cell proliferation and cell cycle progression.

PubMed

Hao, Zhenyue; Sheng, Yi; Duncan, Gordon S; Li, Wanda Y; Dominguez, Carmen; Sylvester, Jennifer; Su, Yu-Wen; Lin, Gloria H Y; Snow, Bryan E; Brenner, Dirk; You-Ten, Annick; Haight, Jillian; Inoue, Satoshi; Wakeham, Andrew; Elford, Alisha; Hamilton, Sara; Liang, Yi; Zúñiga-Pflücker, Juan C; He, Housheng Hansen; Ohashi, Pamela S; Mak, Tak W

2017-01-13

T-cell proliferation is regulated by ubiquitination but the underlying molecular mechanism remains obscure. Here we report that Lys-48-linked ubiquitination of the transcription factor KLF4 mediated by the E3 ligase Mule promotes T-cell entry into S phase. Mule is elevated in T cells upon TCR engagement, and Mule deficiency in T cells blocks proliferation because KLF4 accumulates and drives upregulation of its transcriptional targets E2F2 and the cyclin-dependent kinase inhibitors p21 and p27. T-cell-specific Mule knockout (TMKO) mice develop exacerbated experimental autoimmune encephalomyelitis (EAE), show impaired generation of antigen-specific CD8 + T cells with reduced cytokine production, and fail to clear LCMV infections. Thus, Mule-mediated ubiquitination of the novel substrate KLF4 regulates T-cell proliferation, autoimmunity and antiviral immune responses in vivo.
Interaction of the Tumor Suppressor p53 with Replication Protein A.

DTIC Science & Technology

1996-08-01

The DNA replication factor RPA physically associates with the tumor suppressor protein p53, an interaction that could be important for the function...binding single-stranded DNA, this mutant of RPA fails to support DNA replication . Therefore the region of RPA which interacts with p53 is essential for...of p53, p21/WAFl/CIPl, inhibits the cell-cycle by associating with cyclin-cdk kinases. It also inhibits DNA replication by interacting with a
Extensive disease small cell carcinoma of the lung; trial of non-cross resistant chemotherapy and consolidation radiotherapy. [X ray

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dillman, R.O., Taelle, R., Segren, S.; Royston, I.; Koziol, J.

1982-05-15

Twenty-nine patients with extensive disease, small-cell carcinoma of the lung, were treated with two cycles of intensive combination chemotherapy: HexaVAC (hexamethylmelamine, vincristine, Adriamycin, cyclophosphamide). Responders received prophylactic cranial radiation (2000 rad/10 fractions) and non cross resistant chemotherapy via a schedule of alternating cycles of CMV (cyclophosphamide, methotrexate, VP-16-213) and AMV (Adriamycin, methotrexate, VP-16-213). Whenever a complete response was achieved, consolidation radiotherapy was given to the lung primary (4000 rad/20 fractions, split dose) and abdominal metastases (2000 rad/10 fractions) synchronous with CMV therapy. The complete response rate was 14% with HexaVAC, but increased to 38% during CMV/AMV. Total response rate (completemore » and partial) was 59% and median survival was 42 weeks. Prophylactic brain radiation prevented clinical relapse in the brain in all 14 patients who received it. However, consolidation radiotherapy failed to prevent clinical relapse in the lung and/or liver, and therapeutic brain radiation (3000 rad) failed to prevent relapse in that site. The simultaneous administration of radiotherapy and chemotherapy was well-tolerated although two patients with poor performance status died of infectious complications while leukopenic. In spite of the high response rate, durable remissions with prolonged disease free survival were rare. Further evaluation of induction, consolidation, and maintenance modes of therapy are indicated.« less
Fibroblasts Lead the Way: A Unified View of 3D Cell Motility.

PubMed

Petrie, Ryan J; Yamada, Kenneth M

2015-11-01

Primary human fibroblasts are remarkably adaptable, able to migrate in differing types of physiological 3D tissue and on rigid 2D tissue culture surfaces. The crawling behavior of these and other vertebrate cells has been studied intensively, which has helped generate the concept of the cell motility cycle as a comprehensive model of 2D cell migration. However, this model fails to explain how cells force their large nuclei through the confines of a 3D matrix environment and why primary fibroblasts can use more than one mechanism to move in 3D. Recent work shows that the intracellular localization of myosin II activity is governed by cell-matrix interactions to both force the nucleus through the extracellular matrix (ECM) and dictate the type of protrusions used to migrate in 3D. Published by Elsevier Ltd.
N-acetyl-L-cysteine increases MnSOD activity and enhances the recruitment of quiescent human fibroblasts to the proliferation cycle during wound healing

PubMed Central

Mao, Gaowei; Goswami, Monali; Kalen, Amanda L.; Goswami, Prabhat C.; Sarsour, Ehab H.

2016-01-01

Background The rebuilding of the connective tissue during wound healing requires the recruitment of fibroblasts to the wound area as well as reentry of quiescent fibroblasts to the proliferative cycle. Whether this process can be modulated by a small molecular weight thiol antioxidant N-acetyl-L-cysteine (NAC) was tested in normal human skin fibroblasts (NHFs) in this study. Methods and Results By using a uni-directional wound healing assay, NAC treated cells demonstrated a decreased migration rate but increased number of proliferating cells recruited into the wound area post wounding. Fifteen day quiescent control and NAC treated NHFs were re-plated at a lower density and cell numbers counted at different days post-plating. Interestingly, NAC treated cells exhibited increased cellular proliferation indicated by both decreased cell population doubling time and increased S phase cells. NAC treated cells demonstrated decreased steady state levels of reactive oxygen species as well as increased protein and activity levels of manganese superoxide dismutase (MnSOD). NAC treatment failed to induce proliferation in quiescent cells lacking MnSOD expression. Conclusions These results demonstrate that NAC enhanced the recruitment of quiescent NHFs into proliferation cycle during wound healing. Our results also suggest that the wound healing properties of NAC might be due to its ability to induce and enhance MnSOD expression and activity. Altogether, these findings suggest NAC might be potentially developed as a dietary intervention to improve tissue injury in animals and humans. PMID:26671656
Mitochondrial DNA polymerase editing mutation, PolgD257A, disturbs stem-progenitor cell cycling in the small intestine and restricts excess fat absorption.

PubMed

Fox, Raymond G; Magness, Scott; Kujoth, Gregory C; Prolla, Tomas A; Maeda, Nobuyo

2012-05-01

Changes in intestinal absorption of nutrients are important aspects of the aging process. To address this issue, we investigated the impact of accelerated mitochondrial DNA mutations on the stem/progenitor cells in the crypts of Lieberkühn in mice homozygous for a mitochondrial DNA polymerase gamma mutation, Polg(D257A), that exhibit accelerated aging phenotype. As early as 3-7 mo of age, the small intestine was significantly enlarged in the PolgD257A mice. The crypts of the PolgD257A mice contained 20% more cells than those of their wild-type littermates and exhibited a 10-fold increase in cellular apoptosis primarily in the stem/progenitor cell zones. Actively dividing cells were proportionally increased, yet a significantly smaller proportion of cells was in the S phase of the cell cycle. Stem cell-derived organoids from PolgD257A mice failed to develop fully in culture and exhibited fewer crypt units, indicating an impact of the mutation on the intestinal epithelial stem/progenitor cell maintenance. In addition, epithelial cell migration along the crypt-villus axis was slowed and less organized, and the ATP content in the villi was significantly reduced. On a high-fat, high-carbohydrate diet, PolgD257A mice showed significantly restricted absorption of excess lipids accompanied by an increase in fecal steatocrits. We conclude that the PolgD257A mutation causes cell cycle dysregulation in the crypts leading to the age-associated changes in the morphology of the small intestine and contributes to the restricted absorption of dietary lipids.
17{alpha}-Estradiol arrests cell cycle progression at G{sub 2}/M and induces apoptotic cell death in human acute leukemia Jurkat T cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jun, Do Youn; Park, Hae Sun; Kim, Jun Seok

2008-09-15

A pharmacological dose (2.5-10 {mu}M) of 17{alpha}-estradiol (17{alpha}-E{sub 2}) exerted a cytotoxic effect on human leukemias Jurkat T and U937 cells, which was not suppressed by the estrogen receptor (ER) antagonist ICI 182,780. Along with cytotoxicity in Jurkat T cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, PARP degradation, and DNA fragmentation were induced. The cytotoxicity of 17{alpha}-E{sub 2} was not blocked by the anti-Fas neutralizing antibody ZB-4. While undergoing apoptosis, there was a remarkable accumulation of G{sub 2}/M cells with the upregulatoin of cdc2 kinase activity, which was reflected in the Thr56more » phosphorylation of Bcl-2. Dephosphorylation at Tyr15 and phosphorylation at Thr161 of cdc2, and significant increase in the cyclin B1 level were underlying factors for the cdc2 kinase activation. Whereas the 17{alpha}-E{sub 2}-induced apoptosis was completely abrogated by overexpression of Bcl-2 or by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G{sub 2}/M cells significantly increased. The caspase-8 inhibitor z-IETD-fmk failed to influence 17{alpha}-E{sub 2}-mediated caspase-9 activation, but it markedly reduced caspase-3 activation and PARP degradation with the suppression of apoptosis, indicating the contribution of caspase-8; not as an upstream event of the mitochondrial cytochrome c release, but to caspase-3 activation. In the presence of hydroxyurea, which blocked the cell cycle progression at the G{sub 1}/S boundary, 17{alpha}-E{sub 2} failed to induce the G{sub 2}/M arrest as well as apoptosis. These results demonstrate that the cytotoxicity of 17{alpha}-E{sub 2} toward Jurkat T cells is attributable to apoptosis mainly induced in G{sub 2}/M-arrested cells, in an ER-independent manner, via a mitochondria-dependent caspase pathway regulated by Bcl-2.« less
Extended Pulse-Powered Humidity-Freeze Cycling for Testing Module-Level Power Electronics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hacke, Peter L; Rodriguez, Miguel; Kempe, Michael D

An EMI suppression capacitor (polypropylene film type) failed by 'popcorning' due to vapor outgassing in pulse powered humidity-freeze cycles. No shorts or shunts could be detected despite mildly corroded metallization visible in the failed capacitor. Humidity-freeze cycling is optimized to break into moisture barriers. However, further studies will be required on additional module level power electronic (MLPE) devices to optimize the stress testing for condensation to precipitate any weakness to short circuiting and other humidity/bias failure modes.
TNF-{alpha} promotes cell survival through stimulation of K{sup +} channel and NF{kappa}B activity in corneal epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Ling; Reinach, Peter; Lu, Luo

2005-11-15

Tumor necrosis factor (TNF-{alpha}) in various cell types induces either cell death or mitogenesis through different signaling pathways. In the present study, we determined in human corneal epithelial cells how TNF-{alpha} also promotes cell survival. Human corneal epithelial (HCE) cells were cultured in DMEM/F-12 medium containing 10% FBS. TNF-{alpha} stimulation induced activation of a voltage-gated K{sup +} channel detected by measuring single channel activity using patch clamp techniques. The effect of TNF-{alpha} on downstream events included NF{kappa}B nuclear translocation and increases in DNA binding activities, but did not elicit ERK, JNK, or p38 limb signaling activation. TNF-{alpha} induced increases inmore » p21 expression resulting in partial cell cycle attenuation in the G{sub 1} phase. Cell cycle progression was also mapped by flow cytometer analysis. Blockade of TNF-{alpha}-induced K{sup +} channel activity effectively prevented NF{kappa}B nuclear translocation and binding to DNA, diminishing the cell-survival protective effect of TNF-{alpha}. In conclusion, TNF-{alpha} promotes survival of HCE cells through sequential stimulation of K{sup +} channel and NF{kappa}B activities. This response to TNF-{alpha} is dependent on stimulating K{sup +} channel activity because following suppression of K{sup +} channel activity TNF-{alpha} failed to activate NF{kappa}B nuclear translocation and binding to nuclear DNA.« less
CacyBP/SIP nuclear translocation regulates p27Kip1 stability in gastric cancer cells

PubMed Central

Niu, Ying-Lin; Li, Ya-Jun; Wang, Jing-Bo; Lu, Yuan-Yuan; Liu, Zhen-Xiong; Feng, Shan-Shan; Hu, Jian-Guo; Zhai, Hui-Hong

2016-01-01

AIM: To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP) nuclear translocation in promoting the proliferation of gastric cancer (GC) cells. METHODS: The effect of CacyBP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Co-immunoprecipitation (co-IP) analysis was performed to examine the binding of CacyBP/SIP with Skp1. A CacyBP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1 protein expression assessed by Western blot analysis. RESULTS: CacyBP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while CacyBP/SIP nuclear translocation was inhibited using siRNA to suppress CacyBP/SIP expression, cell cycle was clearly inhibited. CacyBP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of CacyBP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, CacyBP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant CacyBP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus. CONCLUSION: CacyBP/SIP nuclear translocation contributes to the proliferation of GC cells, and CacyBP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27Kip1. PMID:27099442
The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells.

PubMed

Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe; Sosa-Pineda, Beatriz; Dussaud, Sébastien; Billestrup, Nils; Madsen, Ole D; Serup, Palle; Heimberg, Harry; Mansouri, Ahmed

2009-08-07

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.
Induction of tumor cell death through targeting tubulin and evoking dysregulation of cell cycle regulatory proteins by multifunctional cinnamaldehydes.

PubMed

Nagle, Amrita A; Gan, Fei-Fei; Jones, Gavin; So, Choon-Leng; Wells, Geoffrey; Chew, Eng-Hui

2012-01-01

Multifunctional trans-cinnamaldehyde (CA) and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA) and 5-fluoro-2-hydroxycinnamaldehyde (FHCA) being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA), were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G(2)/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G(2) phase. G(2) arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G(2) to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM) labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G(2) phase, resulting in apoptotic cell death characterized by emergence of cleaved forms of caspase 3 and poly (ADP-ribose) polymerase (PARP). Results presented in this study have thus provided further insights into the intricate network of cellular events by which cinnamaldehydes induce tumor cell death.
Adenovirally mediated p53 overexpression diversely influence the cell cycle of HEp-2 and CAL 27 cell lines upon cisplatin and methotrexate treatment.

PubMed

Kraljević Pavelić, Sandra; Marjanović, Marko; Poznić, Miroslav; Kralj, Marijeta

2009-12-01

p53 gene plays a crucial role in the response to therapy. Since it is inactivated in the majority of human cancers, it is strongly believed that the p53 mutations confer resistance to therapeutics. In this paper we analyzed the influence of two mechanistically diverse antitumor agents--cisplatin and methotrexate on the proliferation and cell cycle of two head and neck squamous cancer cell lines HEp-2 (wild type p53 gene, but HPV 18/E6-inactivated protein) and CAL 27 (mutated p53 gene), along with the influence of adenovirally mediated p53 overexpression in modulation of cisplatin and methoterexate effects, whereby subtoxic vector/compound concentrations were employed. p53 gene was introduced into tumor cells using adenoviral vector (AdCMV-p53). The cell cycle perturbations were measured by two parameter flow cytometry. The expression of p53, p21(WAF1/CIP1) and cyclin B1 proteins was examined using immunocytochemistry and western blot methods. In CAL 27 cells overexpression of p53 completely abrogated high S phase content observed in methotrexate-treated cells into a G1 and slight G2 arrest, while it sustained G2 arrest of the cells treated with cisplatin, along with the reduction of DNA synthesis and cyclin B1 expression. On the other hand, in HEp-2 cell line p53 overexpression slightly slowed down the progression through S phase in cells treated with methotrexate, decreased the cyclin B1 expression only after 24 h, and failed to sustain the G2 arrest after treatment with cisplatin alone. Instead, it increased the population of S phase cells that were not actively synthesizing DNA, sustained cyclin B1 expression and allowed the G2 cells to progress through mitosis. This study demonstrates that adenovirally mediated p53 overexpression at sub-cytotoxic levels enhanced the activity of low doses of cisplatin and methotrexate in HEp-2 and CAL 27 cells through changes in the cell cycle. However, the mechanisms of these effects differ depending on the genetic context and on the chemotherapeutics' modality of action.
A comparison of lateral ankle ligament suture anchor strength.

PubMed

Barber, F Alan; Herbert, Morley A; Crates, John M

2013-06-01

Lateral ankle ligament repairs increasingly use suture anchors instead of bone tunnels. Our purpose was to compare the biomechanical properties of a knotted and knotless suture anchor appropriate for a lateral ankle ligament reconstruction. In porcine distal fibulae, 10 samples of 2 different PEEK anchors were inserted. The attached sutures were cyclically loaded between 10N and 60N for 200 cycles. A destructive pull was performed and failure loads, cyclic displacement, stiffness, and failure mode recorded. PushLock 2.5 anchors failed before 200 cycles. PushLock 100 cycle displacement was less than Morphix 2.5 displacement (p<0.001). Ultimate failure load for anchors completing 200 cycles was 86.5N (PushLock) and 252.1N (Morphix) (p<0.05). The failure mode was suture breaking for all PushLocks while the Morphix failed equally by anchor breaking and suture breakage. The knotted Morphix demonstrated more displacement and greater failure strength than the knotless PushLock. The PushLock failed consistently with suture breaking. The Morphix anchor failed both by anchor breaking and by suture breaking. Copyright © 2012 European Foot and Ankle Society. Published by Elsevier Ltd. All rights reserved.
Flat-plate photovoltaic array design optimization

NASA Technical Reports Server (NTRS)

Ross, R. G., Jr.

1980-01-01

An analysis is presented which integrates the results of specific studies in the areas of photovoltaic structural design optimization, optimization of array series/parallel circuit design, thermal design optimization, and optimization of environmental protection features. The analysis is based on minimizing the total photovoltaic system life-cycle energy cost including repair and replacement of failed cells and modules. This approach is shown to be a useful technique for array optimization, particularly when time-dependent parameters such as array degradation and maintenance are involved.
Comparison of ``AA`` nickel metal hydride cells with ``AA`` Ni-Cd cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alminauskas, V.; Johnson, W.

1996-12-31

This paper compares ``AA`` size nickel metal hydride (Ni-HM) cells with comparable ``AA;; nickel cadmium (Ni-Cd) cells both of which were obtained in 1993. The Ni-MH cells were found to be a suitable substitute for conventional Ni-Cd cells. Both these cell types have similar voltages and discharge characteristics. The Ni-MH cells, though had nearly twice the capacity as comparable Ni-Cd cells. There was no significant difference in self discharge between the two types of cells. The Ni-MH cells also performed as well as Ni-Cd cells at rates lower than 5 amperes and at temperatures higher than 0 C (32 F).more » The most interesting finding is that the Ni-MH cells showed an irreversible decay of the discharge voltage with each cycle which was more noticeable during pulses. Eventually the Ni-MH packs fail, not because of loss of capacity, but because of low voltage during the pulse.« less
TGFβ restores hematopoietic homeostasis after myelosuppressive chemotherapy

PubMed Central

Brenet, Fabienne; Kermani, Pouneh; Spektor, Roman; Rafii, Shahin

2013-01-01

Myelosuppression is a life-threatening complication of antineoplastic therapy, but treatment is restricted to a few cytokines with unilineage hematopoietic activity. Although hematopoietic stem cells (HSCs) are predominantly quiescent during homeostasis, they are rapidly recruited into cell cycle by stresses, including myelosuppressive chemotherapy. Factors that induce HSCs to proliferate during stress have been characterized, but it is not known how HSC quiescence is then reestablished. In this study, we show that TGFβ signaling is transiently activated in hematopoietic stem and progenitor cells (HSPCs) during hematopoietic regeneration. Blockade of TGFβ signaling after chemotherapy accelerates hematopoietic reconstitution and delays the return of cycling HSCs to quiescence. In contrast, TGFβ blockade during homeostasis fails to induce cycling of HSPCs. We identified the cyclin-dependent kinase inhibitor Cdkn1c (p57) as a key downstream mediator of TGFβ during regeneration because the recovery of chimeric mice, incapable of expressing p57 in HSPCs, phenocopies blockade of TGFβ signaling after chemotherapy. This study demonstrates that context-dependent activation of TGFβ signaling is central to an unrecognized counterregulatory mechanism that promotes homeostasis once hematopoiesis has sufficiently recovered from myelosuppressive chemotherapy. These results open the door to new, potentially superior, approaches to promote multilineage hematopoietic recovery by blocking the TGFβ signaling that dampens regeneration. PMID:23440043
The Activity of Differentiation Factors Induces Apoptosis in Polyomavirus Large T-Expressing Myoblasts

PubMed Central

Fimia, Gian Maria; Gottifredi, Vanesa; Bellei, Barbara; Ricciardi, Maria Rosaria; Tafuri, Agostino; Amati, Paolo; Maione, Rossella

1998-01-01

It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G00 arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis. PMID:9614186
EBNA3C-Mediated Regulation of Aurora Kinase B Contributes to Epstein-Barr Virus-Induced B-Cell Proliferation through Modulation of the Activities of the Retinoblastoma Protein and Apoptotic Caspases

PubMed Central

Jha, Hem Chandra; Lu, Jie; Saha, Abhik; Cai, Qiliang; Banerjee, Shuvomoy; Prasad, Mahadesh A. J.

2013-01-01

Epstein-Barr virus (EBV) is an oncogenic gammaherpesvirus that is implicated in several human malignancies, including Burkitt's lymphoma (BL), posttransplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma (NPC), and AIDS-associated lymphomas. Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential EBV latent antigens, can induce mammalian cell cycle progression through its interaction with cell cycle regulators. Aurora kinase B (AK-B) is important for cell division, and deregulation of AK-B is associated with aneuploidy, incomplete mitotic exit, and cell death. Our present study shows that EBNA3C contributes to upregulation of AK-B transcript levels by enhancing the activity of its promoter. Further, EBNA3C also increased the stability of the AK-B protein, and the presence of EBNA3C leads to reduced ubiquitination of AK-B. Importantly, EBNA3C in association with wild-type AK-B but not with its kinase-dead mutant led to enhanced cell proliferation, and AK-B knockdown can induce nuclear blebbing and cell death. This phenomenon was rescued in the presence of EBNA3C. Knockdown of AK-B resulted in activation of caspase 3 and caspase 9, along with poly(ADP-ribose) polymerase 1 (PARP1) cleavage, which is known to be an important contributor to apoptotic signaling. Importantly, EBNA3C failed to stabilize the kinase-dead mutant of AK-B compared to wild-type AK-B, which suggests a role for the kinase domain in AK-B stabilization and downstream phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). This study demonstrates the functional relevance of AK-B kinase activity in EBNA3C-regulated B-cell proliferation and apoptosis. PMID:23986604
Enhanced charging capability of lithium metal batteries based on lithium bis(trifluoromethanesulfonyl)imide-lithium bis(oxalato)borate dual-salt electrolytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiang, Hongfa; Shi, Pengcheng; Bhattacharya, Priyanka

2016-06-01

Rechargeable lithium (Li) metal batteries with conventional LiPF6-carbonate electrolytes have been reported to fail quickly at charging current densities of about 1.0 mA cm-2 and above. In this work, we demonstrate the rapid charging capability of the Li||LiNi0.8Co0.15Al0.05O2 (NCA) cells enabled by a dual-salt electrolyte of LiTFSI-LiBOB in a carbonate solvent mixture. It is found that the thickness of solid electrolyte interphase (SEI) layer on Li metal anode largely increases with increasing charging current density. However, the cells using the LiTFSI-LiBOB dual-salt electrolyte significantly outperforms those using the LiPF6 electrolyte at high charging current densities. At the charging current densitymore » of 1.50 mA cm-2, the Li||NCA cells with the dual-salt electrolyte can still deliver a discharge capacity of 131 mAh g-1 and a capacity retention of 80% after 100 cycles, while those with the LiPF6 electrolyte start to show fast capacity fading after the 30th cycle and only exhibit a low capacity of 25 mAh g-1 and a low retention of 15% after 100 cycles. The reasons for the good chargeability and cycling stability of the cells using LiTFSI-LiBOB dual-salt electrolyte can be attributed to the good film-formation ability of the electrolyte on lithium metal anode and the highly conductive nature of the sulfur-rich interphase layer.« less

Hot fire test results of subscale tubular combustion chambers

NASA Technical Reports Server (NTRS)

Kazaroff, John M.; Jankovsky, Robert S.; Pavli, Albert J.

1992-01-01

Advanced, subscale, tubular combustion chambers were built and test fired with hydrogen-oxygen propellants to assess the increase in fatigue life that can be obtained with this type of construction. Two chambers were tested: one ran for 637 cycles without failing, compared to a predicted life of 200 cycles for a comparable smooth-wall milled-channel liner configuration. The other chamber failed at 256 cycles, compared to a predicted life of 118 cycles for a comparable smooth-wall milled-channel liner configuration. Posttest metallographic analysis determined that the strain-relieving design (structural compliance) of the tubular configuration was the cause of this increase in life.
Genetic interactions between the hedgehog co-receptors Gas1 and Boc regulate cell proliferation during murine palatogenesis

PubMed Central

Xavier, Guilherme M.; Seppala, Maisa; Papageorgiou, Spyridon N.; Fan, Chen-Ming; Cobourne, Martyn T.

2016-01-01

Abnormal regulation of Sonic hedgehog (Shh) signaling has been described in a variety of human cancers and developmental anomalies, which highlights the essential role of this signaling molecule in cell cycle regulation and embryonic development. Gas1 and Boc are membrane co-receptors for Shh, which demonstrate overlapping domains of expression in the early face. This study aims to investigate potential interactions between these co-receptors during formation of the secondary palate. Mice with targeted mutation in Gas1 and Boc were used to generate Gas1; Boc compound mutants. The expression of key Hedgehog signaling family members was examined in detail during palatogenesis via radioactive in situ hybridization. Morphometric analysis involved computational quantification of BrdU-labeling and cell packing; whilst TUNEL staining was used to assay cell death. Ablation of Boc in a Gas1 mutant background leads to reduced Shh activity in the palatal shelves and an increase in the penetrance and severity of cleft palate, associated with failed elevation, increased proliferation and reduced cell death. Our findings suggest a dual requirement for Boc and Gas1 during early development of the palate, mediating cell cycle regulation during growth and subsequent fusion of the palatal shelves. PMID:27811357
Low dose decitabine in very high risk relapsed or refractory acute myeloid leukaemia in children and young adults.

PubMed

Phillips, Christine L; Davies, Stella M; McMasters, Richard; Absalon, Michael; O'Brien, Maureen; Mo, Jun; Broun, Randall; Moscow, Jeffrey A; Smolarek, Teresa; Garzon, Ramiro; Blum, William; Schwind, Sebastian; Marcucci, Guido; Perentesis, John P

2013-05-01

Low-dose decitabine has encouraging activity and tolerability in adults with acute myeloid leukaemia (AML), but paediatric experience is lacking. We report our retrospective experience with decitabine in eight children and young adults (median age 4 years) with refractory/relapsed AML, who had failed multiple regimens or were not candidates for standard retrieval regimens due to prior toxicities. Three of eight patients (38%) had complete response (CR; 1 each of CR, CR with incomplete platelet recovery and CR with incomplete count recovery). Best responses were observed after a median of 2.5 cycles (range 1-4 cycles). Four patients received subsequent allogeneic stem cell transplant, and two remain in long-term CR. © 2013 Blackwell Publishing Ltd.
Selective dissolution of halide perovskites as a step towards recycling solar cells

NASA Astrophysics Data System (ADS)

Kim, Byeong Jo; Kim, Dong Hoe; Kwon, Seung Lee; Park, So Yeon; Li, Zhen; Zhu, Kai; Jung, Hyun Suk

2016-05-01

Most research on perovskite solar cells has focused on improving power-conversion efficiency and stability. However, if one could refurbish perovskite solar cells, their stability might not be a critical issue. From the perspective of cost effectiveness, if failed, perovskite solar cells could be collected and recycled; reuse of their gold electrodes and transparent conducting glasses could reduce the price per watt of perovskite photovoltaic modules. Herein, we present a simple and effective method for removing the perovskite layer and reusing the mesoporous TiO2-coated transparent conducting glass substrate via selective dissolution. We find that the perovskite layer can be easily decomposed in polar aprotic solvents because of the reaction between polar aprotic solvents and Pb2+ cations. After 10 cycles of recycling, a mesoporous TiO2-coated transparent conducting glass substrate-based perovskite solar cell still shows a constant power-conversion efficiency, thereby demonstrating the possibility of recycling perovskite solar cells.
Selective dissolution of halide perovskites as a step towards recycling solar cells.

PubMed

Kim, Byeong Jo; Kim, Dong Hoe; Kwon, Seung Lee; Park, So Yeon; Li, Zhen; Zhu, Kai; Jung, Hyun Suk

2016-05-23

Most research on perovskite solar cells has focused on improving power-conversion efficiency and stability. However, if one could refurbish perovskite solar cells, their stability might not be a critical issue. From the perspective of cost effectiveness, if failed, perovskite solar cells could be collected and recycled; reuse of their gold electrodes and transparent conducting glasses could reduce the price per watt of perovskite photovoltaic modules. Herein, we present a simple and effective method for removing the perovskite layer and reusing the mesoporous TiO2-coated transparent conducting glass substrate via selective dissolution. We find that the perovskite layer can be easily decomposed in polar aprotic solvents because of the reaction between polar aprotic solvents and Pb(2+) cations. After 10 cycles of recycling, a mesoporous TiO2-coated transparent conducting glass substrate-based perovskite solar cell still shows a constant power-conversion efficiency, thereby demonstrating the possibility of recycling perovskite solar cells.
Persistence of the cell-cycle checkpoint kinase Wee1 in SadA- and SadB-deficient neurons disrupts neuronal polarity.

PubMed

Müller, Myriam; Lutter, Daniela; Püschel, Andreas W

2010-01-15

Wee1 is well characterized as a cell-cycle checkpoint kinase that regulates the entry into mitosis in dividing cells. Here we identify a novel function of Wee1 in postmitotic neurons during the establishment of distinct axonal and dendritic compartments, which is an essential step during neuronal development. Wee1 is expressed in unpolarized neurons but is downregulated after neurons have extended an axon. Suppression of Wee1 impairs the formation of minor neurites but does not interfere with axon formation. However, neuronal polarity is disrupted when neurons fail to downregulate Wee1. The kinases SadA and SadB (Sad kinases) phosphorylate Wee1 and are required to initiate its downregulation in polarized neurons. Wee1 expression persists in neurons that are deficient in SadA and SadB and disrupts neuronal polarity. Knockdown of Wee1 rescues the Sada(-/-);Sadb(-/-) mutant phenotype and restores normal polarity in these neurons. Our results demonstrate that the regulation of Wee1 by SadA and SadB kinases is essential for the differentiation of polarized neurons.
Conditioning monitoring by microstructural evaluation of cumulative fatigue damage

NASA Astrophysics Data System (ADS)

Fukuoka, C.; Nakagawa, Y. G.; Lance, J. J.; Pangborn, R. N.

1996-12-01

The objective of this work is to evaluate the damage induced below and above the fatigue limit (Δ σ t =360 MPa) in pressure vessel steels, such as SA508. Fatigue damage was induced in samples taken from an SA508 steel plate by various loading histories in order to examine the influence of prior cyclic loading below the fatigue limit. Cell-to-cell misorientation differences were measured by the selected area diffraction (SAD) method. Surface cracking was also studied by the replication method. Small cracks were observed after precycling both below and above the fatigue limit. It was, however, found that fatigue test bars had a longer lifetime after precycling below the fatigue limit, while precycling above the fatigue limit caused other specimens to fail even when subsequently cycled below the fatigue limit. Cell-to-cell misorientation usually increases with accumulation of fatigue damage, but it was found that the misorientations measured after precycling below the fatigue limit decreased again at the beginning of the subsequent cycling above the fatigue limit. It should be noted that the misorientation at failure was always about 4 to 5 deg, regardless of loading histories. Misorientation showed good correlation with the fatigue lifetime of the samples.
NG2 expression in glioblastoma identifies an actively proliferating population with an aggressive molecular signature

PubMed Central

Al-Mayhani, M. Talal F.; Grenfell, Richard; Narita, Masashi; Piccirillo, Sara; Kenney-Herbert, Emma; Fawcett, James W.; Collins, V. Peter; Ichimura, Koichi; Watts, Colin

2011-01-01

Glioblastoma multiforme (GBM) is the most common type of primary brain tumor and a highly malignant and heterogeneous cancer. Current conventional therapies fail to eradicate or curb GBM cell growth. Hence, exploring the cellular and molecular basis of GBM cell growth is vital to develop novel therapeutic approaches. Neuroglia (NG)-2 is a transmembrane proteoglycan expressed by NG2+ progenitors and is strongly linked to cell proliferation in the normal brain. By using NG2 as a biomarker we identify a GBM cell population (GBM NG2+ cells) with robust proliferative, clonogenic, and tumorigenic capacity. We show that a significant proportion (mean 83%) of cells proliferating in the tumor mass express NG2 and that over 50% of GBM NG2+ cells are proliferating. Compared with the GBM NG2− cells from the same tumor, the GBM of NG2+ cells overexpress genes associated with aggressive tumorigenicity, including overexpression of Mitosis and Cell Cycling Module genes (e.g., MELK, CDC, MCM, E2F), which have been previously shown to correlate with poor survival in GBM. We also show that the coexpression pattern of NG2 with other glial progenitor markers in GBM does not recapitulate that described in the normal brain. The expression of NG2 by such an aggressive and actively cycling GBM population combined with its location on the cell surface identifies this cell population as a potential therapeutic target in a subset of patients with GBM. PMID:21798846
Marinobufagin, a molecule from poisonous frogs, causes biochemical, morphological and cell cycle changes in human neoplasms and vegetal cells.

PubMed

Machado, Kátia da Conceição; Sousa, Lívia Queiroz de; Lima, Daisy Jereissati Barbosa; Soares, Bruno Marques; Cavalcanti, Bruno Coêlho; Maranhão, Sarah Sant'Anna; Noronha, Janaina da Costa de; Rodrigues, Domingos de Jesus; Militão, Gardenia Carmen Gadelha; Chaves, Mariana Helena; Vieira-Júnior, Gerardo Magela; Pessoa, Cláudia; Moraes, Manoel Odorico de; Sousa, João Marcelo de Castro E; Melo-Cavalcante, Ana Amélia de Carvalho; Ferreira, Paulo Michel Pinheiro

2018-03-15

Skin toad secretion present physiologically active molecules to protect them against microorganisms, predators and infections. This work detailed the antiproliferative action of marinobufagin on tumor and normal lines, investigate its mechanism on HL-60 leukemia cells and its toxic effects on Allium cepa meristematic cells. Initially, cytotoxic action was assessed by colorimetric assays. Next, HL-60 cells were analyzed by morphological and flow cytometry techniques and growing A. cepa roots were examined after 72 h exposure. Marinobufagin presented high antiproliferative action against all human tumor lines [IC 50 values ranging from 0.15 (leukemia) to 7.35 (larynx) μM] and it failed against human erythrocytes and murine lines. Human normal peripheral blood mononuclear cells (PBMC) were up to 72.5-fold less sensitive [IC 50: 10.88 μM] to marinobufagin than HL-60 line, but DNA strand breaks were no detected. Leukemia treaded cells exhibited cell viability reduction, DNA fragmentation, phosphatidylserine externalization, binucleation, nuclear condensation and cytoplasmic vacuoles. Marinobufagin also reduced the growth of A. cepa roots (EC 50 : 7.5 μM) and mitotic index, caused cell cycle arrest and chromosomal alterations (micronuclei, delays and C-metaphases) in meristematic cells. So, to find out partially targeted natural molecules on human leukemia cells, like marinobufagin, is an amazing and stimulating way to continue the battle against cancer. Copyright © 2017 Elsevier B.V. All rights reserved.
40 CFR 51.357 - Test procedures and standards.

Code of Federal Regulations, 2014 CFR

2014-07-01

... invalid test condition, unsafe conditions, fast pass/fail algorithms, or, in the case of the on-board... using approved fast pass or fast fail algorithms and multiple pass/fail algorithms may be used during the test cycle to eliminate false failures. The transient test procedure, including algorithms and...
40 CFR 51.357 - Test procedures and standards.

Code of Federal Regulations, 2012 CFR

2012-07-01

... invalid test condition, unsafe conditions, fast pass/fail algorithms, or, in the case of the on-board... using approved fast pass or fast fail algorithms and multiple pass/fail algorithms may be used during the test cycle to eliminate false failures. The transient test procedure, including algorithms and...
40 CFR 51.357 - Test procedures and standards.

Code of Federal Regulations, 2011 CFR

2011-07-01

... invalid test condition, unsafe conditions, fast pass/fail algorithms, or, in the case of the on-board... using approved fast pass or fast fail algorithms and multiple pass/fail algorithms may be used during the test cycle to eliminate false failures. The transient test procedure, including algorithms and...
40 CFR 51.357 - Test procedures and standards.

Code of Federal Regulations, 2013 CFR

2013-07-01

... invalid test condition, unsafe conditions, fast pass/fail algorithms, or, in the case of the on-board... using approved fast pass or fast fail algorithms and multiple pass/fail algorithms may be used during the test cycle to eliminate false failures. The transient test procedure, including algorithms and...
Microstructural evaluation of cumulative fatigue damage below the fatigue limit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukuoka, C.; Nakagawa, Y.G.

1996-05-01

The objective of this work is to evaluate the microstructural changes induced near and below the fatigue limit in a pressure vessel steel plate, SA508. Dislocation cell to cell misorientation differences, {theta}, which increase with fatigue damage accumulation, are measured by the Selected Area Diffraction (SAD) method. The misorientation difference, {theta}, of the sample failed just above the fatigue limit is about 4.0 degrees on the average, which is about the same as that for the failure conditions of low cycle fatigue at higher stresses. The {theta} value increases even below the fatigue limit, but it does not increase atmore » stresses which are lower than 50% of the fatigue limit.« less
Long Life Nickel Electrodes for Nickel-Hydrogen Cells: Fiber Substrates Nickel Electrodes

NASA Technical Reports Server (NTRS)

Rogers, Howard H.

2000-01-01

Samples of nickel fiber mat electrodes were investigated over a wide range of fiber diameters, electrode thickness, porosity and active material loading levels. Thickness' were 0.040, 0.060 and 0.080 inches for the plaque: fiber diameters were primarily 2, 4, and 8 micron and porosity was 85, 90, and 95%. Capacities of 3.5 in. diameter electrodes were determined in the flooded condition with both 26 and 31% potassium hydroxide solution. These capacity tests indicated that the highest capacities per unit weight were obtained at the 90% porosity level with a 4 micron diameter fiber plaque. It appeared that the thinner electrodes had somewhat better performance, consistent with sintered electrode history. Limited testing with two-positive-electrode boiler plate cells was also carried out. Considerable difficulty with constructing the cells was encountered with short circuits the major problem. Nevertheless, four cells were tested. The cell with 95% porosity electrodes failed during conditioning cycling due to high voltage during charge. Discharge showed that this cell had lost nearly all of its capacity. The other three cells after 20 conditioning cycles showed capacities consistent with the flooded capacities of the electrodes. Positive electrodes made from fiber substrates may well show a weight advantage of standard sintered electrodes, but need considerably more work to prove this statement. A major problem to be investigated is the lower strength of the substrate compared to standard sintered electrodes. Problems with welding of leads were significant and implications that the electrodes would expand more than sintered electrodes need to be investigated. Loading levels were lower than had been expected based on sintered electrode experiences and the lower loading led to lower capacity values. However, lower loading causes less expansion and contraction during cycling so that stress on the substrate is reduced.
Transition in Survival From Low-Dose Hyper-Radiosensitivity to Increased Radioresistance Is Independent of Activation of ATM SER1981 Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krueger, Sarah A.; Collis, Spencer J.; Joiner, Michael C.

2007-11-15

Purpose: The molecular basis of low-dose hyper-radiosensitivity (HRS) is only partially understood. The aim of this study was to define the roles of ataxia telangiectasia mutated (ATM) activity and the downstream ATM-dependent G{sub 2}-phase cell cycle checkpoint in overcoming HRS and triggering radiation resistance. Methods and Materials: Survival was measured using a high-resolution clonogenic assay. ATM Ser1981 activation was measured by Western blotting. The role of ATM was determined in survival experiments after molecular (siRNA) and chemical (0.4 mM caffeine) inhibition and chemical (20 {mu}g/mL chloroquine, 15 {mu}M genistein) activation 4-6 h before irradiation. Checkpoint responsiveness was assessed in eightmore » cell lines of differing HRS status using flow cytometry to quantify the progression of irradiated (0-2 Gy) G{sub 2}-phase cells entering mitosis, using histone H3 phosphorylation analysis. Results: The dose-response pattern of ATM activation was concordant with the transition from HRS to radioresistance. However, ATM activation did not play a primary role in initiating increased radioresistance. Rather, a relationship was discovered between the function of the downstream ATM-dependent early G{sub 2}-phase checkpoint and the prevalence and overcoming of HRS. Four cell lines that exhibited HRS failed to show low-dose (<0.3-Gy) checkpoint function. In contrast, four HRS-negative cell lines exhibited immediate cell cycle arrest for the entire 0-2-Gy dose range. Conclusion: Overcoming HRS is reliant on the function of the early G{sub 2}-phase checkpoint. These data suggest that clinical exploitation of HRS could be achieved by combining radiotherapy with chemotherapeutic agents that modulate this cell cycle checkpoint.« less
The use of morphokinetics as a predictor of embryo implantation.

PubMed

Meseguer, Marcos; Herrero, Javier; Tejera, Alberto; Hilligsøe, Karen Marie; Ramsing, Niels Birger; Remohí, Jose

2011-10-01

Time-lapse observation presents an opportunity for optimizing embryo selection based on morphological grading as well as providing novel kinetic parameters, which may further improve accurate selection of viable embryos. The objective of this retrospective study was to identify the morphokinetic parameters specific to embryos that were capable of implanting. In order to compare a large number of embryos, with minimal variation in culture conditions, we have used an automatic embryo monitoring system. Using a tri-gas IVF incubator with a built-in camera designed to automatically acquire images at defined time points, we have simultaneously monitored up to 72 individual embryos without removing the embryos from the controlled environment. Images were acquired every 15 min in five different focal planes for at least 64 h for each embryo. We have monitored the development of transferred embryos from 285 couples undergoing their first ICSI cycle. The total number of transferred embryos was 522, of which 247 either failed to implant or fully implanted, with full implantation meaning that all transferred embryos in a treatment implanted. A detailed retrospective analysis of cleavage times, blastomere size and multinucleation was made for the 247 transferred embryos with either failed or full implantation. We found that several parameters were significantly correlated with subsequent implantation (e.g. time of first and subsequent cleavages as well as the time between cleavages). The most predictive parameters were: (i) time of division to 5 cells, t5 (48.8-56.6 h after ICSI); (ii) time between division to 3 cells and subsequent division to 4 cells, s2 (≤ 0.76 h) and (iii) duration of cell cycle two, i.e. time between division to 2 cells and division to 3 cells, cc2 (≤ 11.9 h). We also observed aberrant behavior such as multinucleation at the 4 cell stage, uneven blastomere size at the 2 cell stage and abrupt cell division to three or more cells, which appeared to largely preclude implantation. The image acquisition and time-lapse analysis system makes it possible to determine exact timing of embryo cleavages in a clinical setting. We propose a multivariable model based on our findings to classify embryos according to their probability of implantation. The efficacy of this classification will be evaluated in a prospective randomized study that ultimately will determine if implantation rates can be improved by time-lapse analysis.
CDK2 phosphorylation of Smad2 disrupts TGF-beta transcriptional regulation in resistant primary bone marrow myeloma cells.

PubMed

Baughn, Linda B; Di Liberto, Maurizio; Niesvizky, Ruben; Cho, Hearn J; Jayabalan, David; Lane, Joseph; Liu, Fang; Chen-Kiang, Selina

2009-02-15

Resistance to growth suppression by TGF-beta1 is common in cancer; however, mutations in this pathway are rare in hematopoietic malignancies. In multiple myeloma, a fatal cancer of plasma cells, malignant cells accumulate in the TGF-beta-rich bone marrow due to loss of both cell cycle and apoptotic controls. Herein we show that TGF-beta activates Smad2 but fails to induce cell cycle arrest or apoptosis in primary bone marrow myeloma and human myeloma cell lines due to its inability to activate G(1) cyclin-dependent kinase (CDK) inhibitors (p15(INK4b), p21(CIP1/WAF1), p27(KIP1), p57(KIP2)) or to repress c-myc and Bcl-2 transcription. Correlating with aberrant activation of CDKs, CDK-dependent phosphorylation of Smad2 on Thr(8) (pT8), a modification linked to impaired Smad activity, is elevated in primary bone marrow myeloma cells, even in asymptomatic monoclonal gammopathy of undetermined significance. Moreover, CDK2 is the predominant CDK that phosphorylates Smad2 on T8 in myeloma cells, leading to inhibition of Smad2-Smad4 association that precludes transcriptional regulation by Smad2. Our findings provide the first direct evidence that pT8 Smad2 couples dysregulation of CDK2 to TGF-beta resistance in primary cancer cells, and they suggest that disruption of Smad2 function by CDK2 phosphorylation acts as a mechanism for TGF-beta resistance in multiple myeloma.
Individualized decision-making in IVF: calculating the chances of pregnancy.

PubMed

van Loendersloot, L L; van Wely, M; Repping, S; Bossuyt, P M M; van der Veen, F

2013-11-01

Are we able to develop a model to calculate the chances of pregnancy prior to the start of the first IVF cycle as well as after one or more failed cycles? Our prediction model enables the accurate individualized calculation of the probability of an ongoing pregnancy with IVF. To improve counselling, patient selection and clinical decision-making in IVF, a number of prediction models have been developed. These models are of limited use as they were developed before current clinical and laboratory protocols were established. This was a cohort study. The development set included 2621 cycles in 1326 couples who had been treated with IVF or ICSI between January 2001 and July 2009. The validation set included additional data from 515 cycles in 440 couples treated between August 2009 and April 2011. The outcome of interest was an ongoing pregnancy after transfer of fresh or frozen-thawed embryos from the same stimulated IVF cycle. If a couple became pregnant after an IVF/ICSI cycle, the follow-up was at a gestational age of at least 11 weeks. Women treated with IVF or ICSI between January 2001 and April 2011 in a university hospital. IVF/ICSI cycles were excluded in the case of oocyte or embryo donation, surgically retrieved spermatozoa, patients positive for human immunodeficiency virus, modified natural IVF and cycles cancelled owing to poor ovarian stimulation, ovarian hyperstimulation syndrome or other unexpected medical or non-medical reasons. Thirteen variables were included in the final prediction model. For all cycles, these were female age, duration of subfertility, previous ongoing pregnancy, male subfertility, diminished ovarian reserve, endometriosis, basal FSH and number of failed IVF cycles. After the first cycle: fertilization, number of embryos, mean morphological score per Day 3 embryo, presence of 8-cell embryos on Day 3 and presence of morulae on Day 3 were also included. In validation, the model had moderate discriminative capacity (c-statistic 0.68, 95% confidence interval: 0.63-0.73) but calibrated well, with a range from 0.01 to 0.56 in calculated probabilities. In our study, the outcome of interest was ongoing pregnancy. Live birth may have been a more appropriate outcome, although only 1-2% of all ongoing pregnancies result in late miscarriage or stillbirth. The model was based on data from a single centre. The IVF model presented here is the first to calculate the chances of an ongoing pregnancy with IVF, both for the first cycle and after any number of failed cycles. The generalizability of the model to other clinics has to be evaluated more extensively in future studies (geographical validation). Centres with higher or lower success rates could use the model, after recalibration, by adjusting the intercept to reflect the IVF success rates in their centre. This project was funded by the NutsOhra foundation (Grant 1004-179). The NutsOhra foundation had no role in the development of our study, in the collection, analysis and interpretation of data; in writing of the manuscript, and in the decision to submit the manuscript for publication. There were no competing interests.
miR-181a and miR-630 regulate cisplatin-induced cancer cell death.

PubMed

Galluzzi, Lorenzo; Morselli, Eugenia; Vitale, Ilio; Kepp, Oliver; Senovilla, Laura; Criollo, Alfredo; Servant, Nicolas; Paccard, Caroline; Hupé, Philippe; Robert, Thomas; Ripoche, Hugues; Lazar, Vladimir; Harel-Bellan, Annick; Dessen, Philippe; Barillot, Emmanuel; Kroemer, Guido

2010-03-01

MicroRNAs (miRNA) are noncoding RNAs that regulate multiple cellular processes, including proliferation and apoptosis. We used microarray technology to identify miRNAs that were upregulated by non-small cell lung cancer (NSCLC) A549 cells in response to cisplatin (CDDP). The corresponding synthetic miRNA precursors (pre-miRNAs) per se were not lethal when transfected into A549 cells yet affected cell death induction by CDDP, C2-ceramide, cadmium, etoposide, and mitoxantrone in an inducer-specific fashion. Whereas synthetic miRNA inhibitors (anti-miRNAs) targeting miR-181a and miR-630 failed to modulate the response of A549 to CDDP, pre-miR-181a and pre-miR-630 enhanced and reduced CDDP-triggered cell death, respectively. Pre-miR-181a and pre-miR-630 consistently modulated mitochondrial/postmitochondrial steps of the intrinsic pathway of apoptosis, including Bax oligomerization, mitochondrial transmembrane potential dissipation, and the proteolytic maturation of caspase-9 and caspase-3. In addition, pre-miR-630 blocked early manifestations of the DNA damage response, including the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and of two ATM substrates, histone H2AX and p53. Pharmacologic and genetic inhibition of p53 corroborated the hypothesis that pre-miR-630 (but not pre-miR-181a) blocks the upstream signaling pathways that are ignited by DNA damage and converge on p53 activation. Pre-miR-630 arrested A549 cells in the G0-G1 phase of the cell cycle, correlating with increased levels of the cell cycle inhibitor p27(Kip1) as well as with reduced proliferation rates and resulting in greatly diminished sensitivity of A549 cells to the late S-G2-M cell cycle arrest mediated by CDDP. Altogether, these results identify miR-181a and miR-630 as novel modulators of the CDDP response in NSCLC.

Two LXXLL motifs in the N terminus of Mps1 are required for Mps1 nuclear import during G(2)/M transition and sustained spindle checkpoint responses.

PubMed

Zhang, Xiaojuan; Yin, Qingqing; Ling, Youguo; Zhang, Yanhong; Ma, Runlin; Ma, Qingjun; Cao, Cheng; Zhong, Hui; Liu, Xuedong; Xu, Quanbin

2011-08-15

Spindle assembly checkpoint kinase Mps1 is spatially and temporally regulated during cell cycle progression. Mps1 is predominately localized to the cytosol in interphase cells, whereas it is concentrated on kinetochores in prophase and prometaphase cells. The timing and mechanism of Mps1 redistribution during cell cycle transition is currently poorly understood. Here, we show that Mps1 relocates from the cytosol to the nucleus at the G 2/M boundary prior to nuclear envelope breakdown (NEB). This timely translocation depends on two tandem LXXLL motifs in the N terminus of Mps1, and mutations in either motif abolish Mps1 nuclear accumulation. Furthermore, we found that phosphorylation of Mps1 Ser80 (which is located between the two LXXLL motifs) also plays a role in regulating timely nuclear entry of Mps1. Mps1 that is defective in LXXLL motifs has near wild-type kinase activity. Moreover, the kinase activity of Mps1 appears to be dispensable for nuclear translocation, as inhibition of Mps1 by a highly specific small-molecule inhibitor did not perturb its nuclear entry. Remarkably, translocation-deficient Mps1 can mediate activation of spindle assembly checkpoint response; however, it fails to support a sustained mitotic arrest upon prolonged treatment with nocodazole. The mitotic slippage can be attributed to precocious degradation of Mps1 in the arrested cells. Our studies reveal a novel cell cycle-dependent nuclear translocation signal in the N terminus of Mps1 and suggest that timely nuclear entry could be important for sustaining spindle assembly checkpoint responses.
Two LXXLL motifs in the N terminus of Mps1 are required for Mps1 nuclear import during G2/M transition and sustained spindle checkpoint responses

PubMed Central

Zhang, Xiaojuan; Yin, Qingqing; Ling, Youguo; Zhang, Yanhong; Ma, Runlin; Ma, Qingjun; Cao, Cheng; Zhong, Hui

2011-01-01

Spindle assembly checkpoint kinase Mps1 is spatially and temporally regulated during cell cycle progression. Mps1 is predominately localized to the cytosol in interphase cells, whereas it is concentrated on kinetochores in prophase and prometaphase cells. The timing and mechanism of Mps1 redistribution during cell cycle transition is currently poorly understood. Here, we show that Mps1 relocates from the cytosol to the nucleus at the G2/M boundary prior to nuclear envelope breakdown (NEB). This timely translocation depends on two tandem LXXLL motifs in the N terminus of Mps1, and mutations in either motif abolish Mps1 nuclear accumulation. Furthermore, we found that phosphorylation of Mps1 Ser80 (which is located between the two LXXLL motifs) also plays a role in regulating timely nuclear entry of Mps1. Mps1 that is defective in LXXLL motifs has near wild-type kinase activity. Moreover, the kinase activity of Mps1 appears to be dispensable for nuclear translocation, as inhibition of Mps1 by a highly specific small-molecule inhibitor did not perturb its nuclear entry. Remarkably, translocation-deficient Mps1 can mediate activation of spindle assembly checkpoint response; however, it fails to support a sustained mitotic arrest upon prolonged treatment with nocodazole. The mitotic slippage can be attributed to precocious degradation of Mps1 in the arrested cells. Our studies reveal a novel cell cycle-dependent nuclear translocation signal in the N terminus of Mps1 and suggest that timely nuclear entry could be important for sustaining spindle assembly checkpoint responses. PMID:21778823
An online calculator for marine phytoplankton iron culturing experiments.

PubMed

Rivers, Adam R; Rose, Andrew L; Webb, Eric A

2013-10-01

Laboratory experiments with iron offer important insight into the physiology of marine phytoplankton and the biogeochemical cycles they influence. These experiments often rely on chelators to buffer the concentration of available iron, but the buffer can fail when cell density increases, causing the concentration of that iron to drop rapidly. To more easily determine the point when the iron concentration falls, we developed an online calculator to estimate the maximum phytoplankton density that a growth medium can support. The results of the calculator were compared to the numerical simulations of a Fe-limited culture of the diatom Thalassiosira weissflogii (Grunow) Fryxell and Hasle. Modeling reveals that the assumptions behind thermodynamic estimates of unchelated Fe concentration can fail before easily perceptible changes in growth rate, potentially causing physiological changes that could alter the conclusions of culture experiments. The calculator is available at http://www.marsci.uga.edu/fidoplankter. © 2013 Phycological Society of America.
Dermal papilla cell number specifies hair size, shape and cycling and its reduction causes follicular decline

PubMed Central

Chi, Woo; Wu, Eleanor; Morgan, Bruce A.

2013-01-01

Although the hair shaft is derived from the progeny of keratinocyte stem cells in the follicular epithelium, the growth and differentiation of follicular keratinocytes is guided by a specialized mesenchymal population, the dermal papilla (DP), that is embedded in the hair bulb. Here we show that the number of DP cells in the follicle correlates with the size and shape of the hair produced in the mouse pelage. The same stem cell pool gives rise to hairs of different sizes or types in successive hair cycles, and this shift is accompanied by a corresponding change in DP cell number. Using a mouse model that allows selective ablation of DP cells in vivo, we show that DP cell number dictates the size and shape of the hair. Furthermore, we confirm the hypothesis that the DP plays a crucial role in activating stem cells to initiate the formation of a new hair shaft. When DP cell number falls below a critical threshold, hair follicles with a normal keratinocyte compartment fail to generate new hairs. However, neighbouring follicles with a few more DP cells can re-enter the growth phase, and those that do exploit an intrinsic mechanism to restore both DP cell number and normal hair growth. These results demonstrate that the mesenchymal niche directs stem and progenitor cell behaviour to initiate regeneration and specify hair morphology. Degeneration of the DP population in mice leads to the types of hair thinning and loss observed during human aging, and the results reported here suggest novel approaches to reversing hair loss. PMID:23487317
Control of asgE Expression during Growth and Development of Myxococcus xanthus

PubMed Central

Garza, Anthony G.; Harris, Baruch Z.; Greenberg, Brandon M.; Singer, Mitchell

2000-01-01

One of the earliest events in the Myxococcus xanthus developmental cycle is production of an extracellular cell density signal called A-signal (or A-factor). Previously, we showed that cells carrying an insertion in the asgE gene fail to produce normal levels of this cell-cell signal. In this study we found that expression of asgE is growth phase regulated and developmentally regulated. Several lines of evidence indicate that asgE is cotranscribed with an upstream gene during development. Using primer extension analyses, we identified two 5′ ends for this developmental transcript. The DNA sequence upstream of one 5′ end has similarity to the promoter regions of several genes that are A-signal dependent, whereas sequences located upstream of the second 5′ end show similarity to promoter elements identified for genes that are C-signal dependent. Consistent with this result is our finding that mutants failing to produce A-signal or C-signal are defective for developmental expression of asgE. In contrast to developing cells, the large majority of the asgE transcript found in vegetative cells appears to be monocistronic. This finding suggests that asgE uses different promoters for expression during vegetative growth and development. Growth phase regulation of asgE is abolished in a relA mutant, indicating that this vegetative promoter is induced by starvation. The data presented here, in combination with our previous results, indicate that the level of AsgE in vegetative cells is sufficient for this protein to carry out its function during development. PMID:11073904
Cell-cycle arrest in mature adipocytes impairs BAT development but not WAT browning, and reduces adaptive thermogenesis in mice.

PubMed

Okamatsu-Ogura, Yuko; Fukano, Keigo; Tsubota, Ayumi; Nio-Kobayashi, Junko; Nakamura, Kyoko; Morimatsu, Masami; Sakaue, Hiroshi; Saito, Masayuki; Kimura, Kazuhiro

2017-07-27

We previously reported brown adipocytes can proliferate even after differentiation. To test the involvement of mature adipocyte proliferation in cell number control in fat tissue, we generated transgenic (Tg) mice over-expressing cell-cycle inhibitory protein p27 specifically in adipocytes, using the aP2 promoter. While there was no apparent difference in white adipose tissue (WAT) between wild-type (WT) and Tg mice, the amount of brown adipose tissue (BAT) was much smaller in Tg mice. Although BAT showed a normal cellular morphology, Tg mice had lower content of uncoupling protein 1 (UCP1) as a whole, and attenuated cold exposure- or β3-adrenergic receptor (AR) agonist-induced thermogenesis, with a decrease in the number of mature brown adipocytes expressing proliferation markers. An agonist for the β3-AR failed to increase the number of proliferating brown adipocytes, UCP1 content in BAT, and oxygen consumption in Tg mice, although the induction and the function of beige adipocytes in inguinal WAT from Tg mice were similar to WT mice. These results show that brown adipocyte proliferation significantly contributes to BAT development and adaptive thermogenesis in mice, but not to induction of beige adipocytes.
Conditioning monitoring by microstructural evaluation of cumulative fatigue damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukuoka, C.; Nakagawa, Y.G.; Lance, J.J.

1996-12-01

The objective of this work is to evaluate the damage induced below and above the fatigue limit ({Delta}{sigma}{sub t} = 360 MPa) in pressure vessel steels, such as SA508. Fatigue damage was induced in samples taken from an SA508 steel plate by various loading histories in order to examine the influence of prior cyclic loading below the fatigue limit. Cell-to-cell misorientation differences were measured by the selected area diffraction (SAD) method. Surface cracking was also studied by the replication method. Small cracks were observed after precycling both below and above the fatigue limit. It was, however, found that fatigue testmore » bars had a longer lifetime after precycling below the fatigue limit, while precycling above the fatigue limit caused other specimens to fail even when subsequently cycled below the fatigue limit. Cell-to-cell misorientation usually increases with accumulation of fatigue damage, but it was found that the misorientations measured after precycling below the fatigue limit decreased again at the beginning of the subsequent cycling above the fatigue limit. It should be noted that the misorientation at failure was always about 4 to 5 deg, regardless of loading histories. Misorientation showed good correlation with the fatigue lifetime of the samples.« less
Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry.

PubMed

Pesavento, James J; Bullock, Courtney R; LeDuc, Richard D; Mizzen, Craig A; Kelleher, Neil L

2008-05-30

Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >10(4) using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date.
Stromal-epithelial interaction mediates steroidal regulation of metalloproteinase expression in human endometrium.

PubMed Central

Osteen, K G; Rodgers, W H; Gaire, M; Hargrove, J T; Gorstein, F; Matrisian, L M

1994-01-01

The hallmark of the menstrual cycle is extensive steroid-dependent tissue turnover. Estrogen mediates endometrial cell growth and structural remodeling, whereas progesterone suppresses estrogen-dependent proliferation and promotes cellular differentiation. In nonfertile cycles, tissue degradation and menstruation occur as a consequence of steroidal deprivation as the ovarian corpus luteum fails. Stromal-epithelial interactions are recognized as a necessary component in mediating steroid-induced endometrial turnover. Specific mRNAs for metalloproteinases of the stromelysin family are expressed during endometrial growth and menstrual breakdown but are absent in the progestin-dominated secretory phase. This expression pattern suggests involvement of stromelysins in remodeling the extracellular matrix of the endometrium during tissue growth and breakdown and implicates progesterone in the suppression of these enzymes. We examined the regulation of endometrial stromelysins in explant cultures and found no acute effect of estradiol on their expression, whereas progesterone was a potent inhibitor of stromelysin expression. Progesterone also suppressed stromelysin expression in cultures of isolated stromal cells, but epithelial cells were progesterone insensitive. Coculture of recombined stromal and epithelial cells restored steroidal suppression of the epithelial-specific metalloproteinase. Our data confirm that progesterone inhibits endometrial stromelysins and further demonstrate the necessity for a stromal-derived factor(s) as a mediator of steroid suppression of an epithelial metalloproteinase. Images PMID:7937850
Uncoupling reproduction from metabolism extends chronological lifespan in yeast

PubMed Central

Nagarajan, Saisubramanian; Kruckeberg, Arthur L.; Schmidt, Karen H.; Kroll, Evgueny; Hamilton, Morgan; McInnerney, Kate; Summers, Ryan; Taylor, Timothy; Rosenzweig, Frank

2014-01-01

Studies of replicative and chronological lifespan in Saccharomyces cerevisiae have advanced understanding of longevity in all eukaryotes. Chronological lifespan in this species is defined as the age-dependent viability of nondividing cells. To date this parameter has only been estimated under calorie restriction, mimicked by starvation. Because postmitotic cells in higher eukaryotes often do not starve, we developed a model yeast system to study cells as they age in the absence of calorie restriction. Yeast cells were encapsulated in a matrix consisting of calcium alginate to form ∼3 mm beads that were packed into bioreactors and fed ad libitum. Under these conditions cells ceased to divide, became heat shock and zymolyase resistant, yet retained high fermentative capacity. Over the course of 17 d, immobilized yeast cells maintained >95% viability, whereas the viability of starving, freely suspended (planktonic) cells decreased to <10%. Immobilized cells exhibited a stable pattern of gene expression that differed markedly from growing or starving planktonic cells, highly expressing genes in glycolysis, cell wall remodeling, and stress resistance, but decreasing transcription of genes in the tricarboxylic acid cycle, and genes that regulate the cell cycle, including master cyclins CDC28 and CLN1. Stress resistance transcription factor MSN4 and its upstream effector RIM15 are conspicuously up-regulated in the immobilized state, and an immobilized rim15 knockout strain fails to exhibit the long-lived, growth-arrested phenotype, suggesting that altered regulation of the Rim15-mediated nutrient-sensing pathway plays an important role in extending yeast chronological lifespan under calorie-unrestricted conditions. PMID:24706810
DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jy-An John; Wang, Hong; Jiang, Hao

The objective of this project is to perform a systematic study of SNF/UNF (spent nuclear fuel/or used nuclear fuel) integrity under simulated transportation environments by using hot cell testing technology developed recently at Oak Ridge National Laboratory (ORNL), CIRFT (Cyclic Integrated Reversible-Bending Fatigue Tester). Under Nuclear Regulatory Commission (NRC) sponsorship, ORNL completed four benchmarking tests, four static tests, and twelve dynamic or cycle tests on H. B. Robinson (HBR) high burn-up (HBU) fuel. With support from the US Department of Energy and the NRC, CIRFT testing has been continued. The CIRFT testing was conducted on three HBR rods (R3, R4,more » and R5), with two specimens failed and one specimen un-failed. The total number of cycles in the test of un-failed specimens went over 2.23 107; the test was stopped as because the specimen did not show any sign of failure. The data analysis on all the HBR SNF rods demonstrated that it is necessary to characterize the fatigue life of used fuel rods in terms of both the curvature amplitude and the maximum of absolute of curvature extremes. The latter is significant because the maxima of extremes signify the maximum of tensile stress of the outer fiber of the bending rod. So far, a large variety of hydrogen contents has been covered in the CIRFT testing on HBR rods. It has been shown that the load amplitude is the dominant factor that controls the lifetime of bending rods, but the hydrogen content also has an important effect on the lifetime attained, according to the load range tested.« less
Selective dissolution of halide perovskites as a step towards recycling solar cells

PubMed Central

Kim, Byeong Jo; Kim, Dong Hoe; Kwon, Seung Lee; Park, So Yeon; Li, Zhen; Zhu, Kai; Jung, Hyun Suk

2016-01-01

Most research on perovskite solar cells has focused on improving power-conversion efficiency and stability. However, if one could refurbish perovskite solar cells, their stability might not be a critical issue. From the perspective of cost effectiveness, if failed, perovskite solar cells could be collected and recycled; reuse of their gold electrodes and transparent conducting glasses could reduce the price per watt of perovskite photovoltaic modules. Herein, we present a simple and effective method for removing the perovskite layer and reusing the mesoporous TiO2-coated transparent conducting glass substrate via selective dissolution. We find that the perovskite layer can be easily decomposed in polar aprotic solvents because of the reaction between polar aprotic solvents and Pb2+ cations. After 10 cycles of recycling, a mesoporous TiO2-coated transparent conducting glass substrate-based perovskite solar cell still shows a constant power-conversion efficiency, thereby demonstrating the possibility of recycling perovskite solar cells. PMID:27211006
Selective dissolution of halide perovskites as a step towards recycling solar cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Byeong Jo; Kim, Dong Hoe; Kwon, Seung Lee

Most research on perovskite solar cells has focused on improving power-conversion efficiency and stability. However, if one could refurbish perovskite solar cells, their stability might not be a critical issue. From the perspective of cost effectiveness, if failed, perovskite solar cells could be collected and recycled; reuse of their gold electrodes and transparent conducting glasses could reduce the price per watt of perovskite photovoltaic modules. Here, we present a simple and effective method for removing the perovskite layer and reusing the mesoporous TiO 2-coated transparent conducting glass substrate via selective dissolution. We find that the perovskite layer can be easilymore » decomposed in polar aprotic solvents because of the reaction between polar aprotic solvents and Pb 2+ cations. After 10 cycles of recycling, a mesoporous TiO 2-coated transparent conducting glass substrate-based perovskite solar cell still shows a constant power-conversion efficiency, thereby demonstrating the possibility of recycling perovskite solar cells.« less
Selective dissolution of halide perovskites as a step towards recycling solar cells

DOE PAGES

Kim, Byeong Jo; Kim, Dong Hoe; Kwon, Seung Lee; ...

2016-05-23

Most research on perovskite solar cells has focused on improving power-conversion efficiency and stability. However, if one could refurbish perovskite solar cells, their stability might not be a critical issue. From the perspective of cost effectiveness, if failed, perovskite solar cells could be collected and recycled; reuse of their gold electrodes and transparent conducting glasses could reduce the price per watt of perovskite photovoltaic modules. Here, we present a simple and effective method for removing the perovskite layer and reusing the mesoporous TiO 2-coated transparent conducting glass substrate via selective dissolution. We find that the perovskite layer can be easilymore » decomposed in polar aprotic solvents because of the reaction between polar aprotic solvents and Pb 2+ cations. After 10 cycles of recycling, a mesoporous TiO 2-coated transparent conducting glass substrate-based perovskite solar cell still shows a constant power-conversion efficiency, thereby demonstrating the possibility of recycling perovskite solar cells.« less
Gene Expression Analysis Reveals the Concurrent Activation of Proapoptotic and Antioxidant-Defensive Mechanisms in Flavokawain B–Treated Cervical Cancer HeLa Cells

PubMed Central

Yeap, Swee Keong; Abu, Nadiah; Akthar, Nadeem; Ho, Wan Yong; Ky, Huynh; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Kamarul, Tunku

2016-01-01

Flavokawain B (FKB) is known to possess promising anticancer abilities. This is demonstrated in various cancer cell lines including HeLa cells. Cervical cancer is among the most widely diagnosed cancer among women today. Though FKB has been shown to be effective in treating cancer cells, the exact molecular mechanism is still unknown. This study is aimed at understanding the effects of FKB on HeLa cells using a microarray-based mRNA expression profiling and proteome profiling of stress-related proteins. The results of this study suggest that FKB induced cell death through p21-mediated cell cycle arrest and activation of p38. However, concurrent activation of antioxidant-related pathways and iron sequestration pathway followed by activation of ER-resident stress proteins clearly indicate that FKB failed to induce apoptosis in HeLa cells via oxidative stress. This effect implies that the protection of HeLa cells by FKB from H2O2–induced cell death is via neutralization of reactive oxygen species. PMID:27458249
Gene Expression Analysis Reveals the Concurrent Activation of Proapoptotic and Antioxidant-Defensive Mechanisms in Flavokawain B-Treated Cervical Cancer HeLa Cells.

PubMed

Yeap, Swee Keong; Abu, Nadiah; Akthar, Nadeem; Ho, Wan Yong; Ky, Huynh; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Kamarul, Tunku

2017-09-01

Flavokawain B (FKB) is known to possess promising anticancer abilities. This is demonstrated in various cancer cell lines including HeLa cells. Cervical cancer is among the most widely diagnosed cancer among women today. Though FKB has been shown to be effective in treating cancer cells, the exact molecular mechanism is still unknown. This study is aimed at understanding the effects of FKB on HeLa cells using a microarray-based mRNA expression profiling and proteome profiling of stress-related proteins. The results of this study suggest that FKB induced cell death through p21-mediated cell cycle arrest and activation of p38. However, concurrent activation of antioxidant-related pathways and iron sequestration pathway followed by activation of ER-resident stress proteins clearly indicate that FKB failed to induce apoptosis in HeLa cells via oxidative stress. This effect implies that the protection of HeLa cells by FKB from H 2 O 2 -induced cell death is via neutralization of reactive oxygen species.
Centrosome Clustering in the Development of Bovine Binucleate Trophoblast Giant Cells.

PubMed

Klisch, Karl; Schraner, Elisabeth M; Boos, Alois

2017-01-01

Binucleate trophoblast giant cells (BNC) are the characteristic feature of the ruminant placenta. During their development, BNC pass through 2 acytokinetic mitoses and become binucleate with 2 tetraploid nuclei. In this study, we investigate the number and location of centrosomes in bovine BNC. Centrosomes typically consist of 2 centrioles surrounded by electron-dense pericentriolar material. Duplication of centrosomes is tightly linked to the cell cycle, which ensures that the number of centrosomes remains constant in proliferating diploid cells. Alterations of the cell cycle, which affect the number of chromosome sets, also affect the number of centrosomes. In this study, we use placentomal tissue from pregnant cows (gestational days 80-230) for immunohistochemical staining of γ-tubulin (n = 3) and transmission electron microscopy (n = 3). We show that mature BNC have 4 centrosomes with 8 centrioles, clustered in the angle between the 2 cell nuclei. During the second acytokinetic mitosis, the centrosomes must be clustered to form the poles of a bipolar spindle. In rare cases, centrosome clustering fails and tripolar mitosis leads to the formation of trinucleate "BNC". Generally, centrosome clustering occurs in polyploid tumor cells, which have an increased number of centrioles, but it is absent in proliferating diploid cells. Thus, inhibition of centrosome clustering in tumor cells is a novel promising strategy for cancer treatment. BNC are a cell population in which centrosome clustering occurs as part of the normal life history. Thus, they might be a good model for the study of the molecular mechanisms of centrosome clustering. © 2016 S. Karger AG, Basel.
Restoration of C/EBPα in dedifferentiated liposarcoma induces G2/M cell cycle arrest and apoptosis

PubMed Central

Wu, Yuhsin V.; Okada, Tomoyo; DeCarolis, Penelope; Socci, Nicholas; O’Connor, Rachael; Geha, Rula C.; Somberg, C. Joy; Antonescu, Cristina; Singer, Samuel

2012-01-01

Well differentiated liposarcoma (WDLS) and dedifferentiated liposarcoma (DDLS) represent the most common biological group of liposarcoma, and there is a pressing need to develop targeted therapies for patients with advanced disease. To identify potential therapeutic targets, we sought to identify differences in the adipogenic pathways between DDLS, WDLS, and normal adipose tissue. In a microarray analysis of DDLS (n=84), WDLS (n=79), and normal fat (n=23), C/EBPα, a transcription factor involved in cell cycle regulation and differentiation, was underexpressed in DDLS compared to both WDLS and normal fat (15.2 fold and 27.8 fold, respectively). In normal adipose-derived stem cells, C/EBPα expression was strongly induced when cells were cultured in differentiation media, but in three DDLS cell lines, this induction was nearly absent. We restored C/EBPα expression in one of the cell lines (DDLS8817) by transfection of an inducible C/EBα expression vector. Inducing C/EBPα expression reduced proliferation and caused cells to accumulate in G2/M. Under differentiation conditions, the cell proliferation was reduced further, and 66% of the DDLS cells containing the inducible C/EBPα expression vector underwent apoptosis as demonstrated by annexin V staining. These cells in differentiation conditions expressed early adipocyte-specific mRNAs such as LPL and FABP4, but they failed to accumulate intracellular lipid droplets, a characteristic of mature adipocytes. These results demonstrate that loss of C/EBPα is an important factor in suppressing apoptosis and maintaining the dedifferentiated state in DDLS. Restoring C/EBPα may be a useful therapeutic approach for dedifferentiated liposarcomas. PMID:22170698
Antitumor Effect of KX-01 through Inhibiting Src Family Kinases and Mitosis.

PubMed

Kim, Seongyeong; Min, Ahrum; Lee, Kyung-Hun; Yang, Yaewon; Kim, Tae-Yong; Lim, Jee Min; Park, So Jung; Nam, Hyun-Jin; Kim, Jung Eun; Song, Sang-Hyun; Han, Sae-Won; Oh, Do-Youn; Kim, Jee Hyun; Kim, Tae-You; Hangauer, David; Lau, Johnson Yiu-Nam; Im, Kyongok; Lee, Dong Soon; Bang, Yung-Jue; Im, Seock-Ah

2017-07-01

KX-01 is a novel dual inhibitor of Src and tubulin. Unlike previous Src inhibitors that failed to show clinical benefit during treatment of breast cancer, KX-01 can potentially overcome the therapeutic limitations of current Src inhibitors through inhibition of both Src and tubulin. The present study further evaluates the activity and mechanism of KX-01 in vitro and in vivo . The antitumor effect of KX-01 in triple negative breast cancer (TNBC) cell lines was determined by MTT assay. Wound healing and immunofluorescence assays were performed to evaluate the action mechanisms of KX-01. Changes in the cell cycle and molecular changes induced by KX-01 were also evaluated. A MDA-MB-231 mouse xenograft model was used to demonstrate the in vivo effects. KX-01 effectively inhibited the growth of breast cancer cell lines. The expression of phospho-Src and proliferative-signaling molecules were down-regulated in KX-01-sensitive TNBC cell lines. In addition, migration inhibition was observed by wound healing assay. KX-01-induced G2/M cell cycle arrest and increased the aneuploid cell population in KX-01-sensitive cell lines. Multi-nucleated cells were significantly increased after KX-01 treatment. Furthermore, KX-01 effectively delayed tumor growth in a MDA-MB-231 mouse xenograft model. KX-01 effectively inhibited cell growth and migration of TNBC cells. Moreover, this study demonstrated that KX-01 showed antitumor effects through the inhibition of Src signaling and the induction of mitotic catastrophe. The antitumor effects of KX-01 were also demonstrated in vivo using a mouse xenograft model.
Successful synthesis of active human coagulation factor VII by co-expression of mammalian gamma-glutamyl carboxylase and modification of vit.K cycle in Drosophila Schneider S2 cells.

PubMed

Nagahashi, Kotomi; Umemura, Kazuo; Kanayama, Naohiro; Iwaki, Takayuki

2017-04-01

Mammalian gamma-glutamyl carboxylase and reduced vitamin K are indispensable for synthesis of mature mammalian vitamin K dependent proteins including some of blood coagulation factors (factors II, VII, IX, and X). It was well known that Drosophila melanogaster expressed gamma-glutamyl carboxylase and possessed a vit.K cycle although native substrates for them have not been identified yet. Despite the potential capability of gamma carboxylation in D. melanogaster derived cells such as S2 cells, Drosophila gamma-glutamyl carboxylase failed to gamma carboxylate a peptide fused to the human coagulation factor IX propeptide. Thus, it had been believed that the Drosophila system was not adequate to synthesize mammalian vit.K dependent proteins. Indeed, we previously attempted to synthesize biologically active factor VII in S2 cells although we were not able to obtain it. However, recently, a successful transient expression of biologically active human factor IX from S2 cells was reported. In the present study, several expression vectors which enable expressing mammalian GGCX, VKORC1, and/or PDIA2 along with F7 were developed. S2 cells transfected with pMKA85, pMAK86, and pMAK219 successfully synthesized active FVII. Thus, mammalian GGCX was indispensable to synthesize active FVII while mammalian VKORC1 and PDIA2 were not critical but supportive factors for S2 cells.

The timing of pronuclear formation, DNA synthesis and cleavage in the human 1-cell embryo.

PubMed

Capmany, G; Taylor, A; Braude, P R; Bolton, V N

1996-05-01

The timing of pronuclear formation and breakdown, DNA synthesis and cleavage during the first cell cycle of human embryogenesis are described. Pronuclei formed between 3 and 10 h post-insemination (hpi; median 8 hpi). S-phase commenced between 8 and 14 hpi, and was completed between 10 and 18 hpi. M-phase was observed between 22 and 31 hpi (median duration 3 h), and cleavage to the 2-cell stage took place between 25 and 33 hpi. The timing of the same events was determined in 1-cell embryos derived from re-inseminated human oocytes that had failed to fertilize during therapeutic in-vitro fertilization (IVF). In these embryos, pronuclei formed between 3 and 8 h post-re-insemination (hpr-i), coinciding with the beginning of S-phase. While S-phase was completed as early as 10 hpr-i in some embryos, it extended until at least 16 hpr-i in others. Pronuclear breakdown and cleavage occurred from 23 and 26 hpr-i respectively; however, they did not occur in some embryos until after 46 hpr-i. The results demonstrate a markedly greater degree of variation in the timing of these events in embryos derived from re-inseminated oocytes compared with embryos derived from conventional IVF, and thus throw into question the validity of using the former as models for studies of the first cell cycle of human embryogenesis.
G protein-coupled receptor 30 ligand G-1 increases aryl hydrocarbon receptor signalling by inhibition of tubulin assembly and cell cycle arrest in human MCF-7 cells.

PubMed

Tarnow, Patrick; Tralau, Tewes; Luch, Andreas

2016-08-01

Regulatory crosstalk between the aryl hydrocarbon receptor (AHR) and oestrogen receptor α (ERα) is well established. Apart from the nuclear receptors ERα and ERβ, oestrogen signalling further involves an unrelated G protein-coupled receptor termed GPR30. In order to investigate potential regulatory crosstalk, this study investigated the influence of G-1 as one of the few GPR30-specific ligands on the AHR regulon in MCF-7 cells. As a well-characterised model system, these human mammary carcinoma cells co-express all three receptors (AHR, ERα and GPR30) and are thus ideally suited to study corresponding regulatory pathway interactions on transcript level. Indeed, treatment with micromolar concentrations of the GPR30-specific agonist G-1 resulted in up-regulation of AHR as well as the transcripts for cytochromes P450 1A1 and 1B1, two well-known targets of the AHR regulon. While this was partly attributable to G-1-mediated inhibition of tubulin assembly and subsequent cell cycle arrest in the G2/M phase, the effects nevertheless required functional AHR. However, G-1-induced up-regulation of CYP 1A1 was not mediated by GPR30, as G15 antagonist treatment as well as a knockdown of GPR30 and AHR failed to inhibit this effect.
Investigating the cellular fate of a DNA-targeted platinum-based anticancer agent by orthogonal double-click chemistry

PubMed Central

Qiao, Xin; Ding, Song; Liu, Fang; Kucera, Gregory L.

2014-01-01

Confocal fluorescence microscopy was used to study a platinum-based anticancer agent in intact NCI-H460 lung cancer cells. Orthogonal copper-catalyzed azide–alkyne cycloaddition (click) reactions were used to simultaneously determine the cell-cycle-specific localization of the azide-functionalized platinum–acridine agent 1 and monitor its effects on nucleic acid metabolism. Copper-catalyzed postlabeling showed advantages over copper-free click chemistry using a dibenzocyclooctyne (DIBO)-modified reporter dye, which produced high background levels in microscopic images and failed to efficiently label platinum adducts in chromatin. Compound 1 was successfully labeled with the fluorophore DIBO to yield 1* (characterized by in-line high-performance liquid chromatography/electrospray mass spectrometry). 1 and 1* show a high degree of colocalization in the confocal images, but the ability of 1* to target the (compacted) chromatin was markedly reduced, most likely owing to the steric bulk introduced by the DIBO tag. Nuclear platinum levels correlated inversely with the ability of the cells to synthesize DNA and cause cell cycle arrest, as confirmed by bivariate flow cytometry analysis. In addition, a decrease in the level of cellular transcription, shrinkage of the nucleolar regions, and redistribution of RNA into the cytosol were observed. Postlabeling in conjunction with colocalization experiments is a useful tool for studying the cell killing mechanism of this type of DNA-targeted agent. PMID:24407462
Age-Dependent Netrin-1 Signaling Regulates NG2+ Glial Cell Spatial Homeostasis in Normal Adult Gray Matter

PubMed Central

Birey, Fikri

2015-01-01

Neuron–glial antigen 2-positive (NG2+) glial cells are the most proliferative glia type in the adult CNS, and their tile-like arrangement in adult gray matter is under tight regulation. However, little is known about the cues that govern this unique distribution. To this end, using a NG2+ glial cell ablation model in mice, we examined the repopulation dynamics of NG2+ glial cells in the mature and aged mice gray matter. We found that some resident NG2+ glial cells that escaped depletion rapidly enter the cell cycle to repopulate the cortex with altered spatial distribution. We reveal that netrin-1 signaling is involved in the NG2+ glial cell early proliferative, late repopulation, and distribution response after ablation in the gray matter. However, ablation of NG2+ glial cell in older animals failed to stimulate a similar repopulation response, possibly because of a decrease in the sensitivity to netrin-1. Our findings indicate that endogenous netrin-1 plays a role in NG2+ glial cell homeostasis that is distinct from its role in myelination. PMID:25926469
Coregulation of FANCA and BRCA1 in human cells.

PubMed

Haitjema, Anneke; Mol, Berber M; Kooi, Irsan E; Massink, Maarten Pg; Jørgensen, Jens Al; Rockx, Davy Ap; Rooimans, Martin A; de Winter, Johan P; Meijers-Heijboer, Hanne; Joenje, Hans; Dorsman, Josephine C

2014-01-01

Fanconi anemia (FA) is a genetically heterogeneous syndrome associated with increased cancer predisposition. The underlying genes govern the FA pathway which functions to protect the genome during the S-phase of the cell cycle. While upregulation of FA genes has been linked to chemotherapy resistance, little is known about their regulation in response to proliferative stimuli. The purpose of this study was to examine how FA genes are regulated, especially in relation to the cell cycle, in order to reveal their possible participation in biochemical networks. Expression of 14 FA genes was monitored in two human cell-cycle models and in two RB1/E2F pathway-associated primary cancers, retinoblastoma and basal breast cancer. In silico studies were performed to further evaluate coregulation and identify connected networks and diseases. Only FANCA was consistently induced over 2-fold; FANCF failed to exhibit any regulatory fluctuations. Two tools exploiting public data sets indicated coregulation of FANCA with BRCA1. Upregulation of FANCA and BRCA1 correlated with upregulation of E2F3. Genes coregulated with both FANCA and BRCA1 were enriched for MeSH-Term id(s) genomic instability, microcephaly, and Bloom syndrome, and enriched for the cellular component centrosome. The regulation of FA genes appears highly divergent. In RB1-linked tumors, upregulation of FA network genes was associated with reduced expression of FANCF. FANCA and BRCA1 may jointly act in a subnetwork - supporting vital function(s) at the subcellular level (centrosome) as well as at the level of embryonic development (mechanisms controlling head circumference).
Analysis of the Borrelia burgdorferi Cyclic-di-GMP-Binding Protein PlzA Reveals a Role in Motility and Virulence ▿

PubMed Central

Pitzer, Joshua E.; Sultan, Syed Z.; Hayakawa, Yoshihiro; Hobbs, Gerry; Miller, Michael R.; Motaleb, Md A.

2011-01-01

The cyclic-dimeric-GMP (c-di-GMP)-binding protein PilZ has been implicated in bacterial motility and pathogenesis. Although BB0733 (PlzA), the only PilZ domain-containing protein in Borrelia burgdorferi, was reported to bind c-di-GMP, neither its role in motility or virulence nor it's affinity for c-di-GMP has been reported. We determined that PlzA specifically binds c-di-GMP with high affinity (dissociation constant [Kd], 1.25 μM), consistent with Kd values reported for c-di-GMP-binding proteins from other bacteria. Inactivation of the monocistronically transcribed plzA resulted in an opaque/solid colony morphology, whereas the wild-type colonies were translucent. While the swimming pattern of mutant cells appeared normal, on swarm plates, mutant cells exhibited a significantly reduced swarm diameter, demonstrating a role of plzA in motility. Furthermore, the plzA mutant cells were significantly less infectious in experimental mice (as determined by 50% infectious dose [ID50]) relative to wild-type spirochetes. The mutant also had survival rates in fed ticks lower than those of the wild type. Consequently, plzA mutant cells failed to complete the mouse-tick-mouse infection cycle, indicating plzA is essential for the enzootic life cycle of B. burgdorferi. All of these defects were corrected when the mutant was complemented in cis. We propose that failure of plzA mutant cells to infect mice was due to altered motility; however, the possibility that an unidentified factor(s) contributed to interruption of the B. burgdorferi enzootic life cycle cannot yet be excluded. PMID:21357718
The impact of constant light on the estrous cycle of the rat.

PubMed

Campbell, C S; Schwartz, N B

1980-04-01

The initial effects of constant bright light on the events of the rat estrous cycle were monitored in order to examine the interdependence of the hormonal and behavioral rhythms which comprise the cycle. Females exposed to constant bright light for only one cycle either failed to ovulate or showed a delay in the hormonal and behavioral events of the cycle as well as in ovulation. Females exposed to constant light for two cycles 1) failed to ovulate, 2) showed an advancement, or 3) showed a delay in the hormonal events of the estrous cycle and ovulation. Vaginal cytology and the onset of locomotor activity did not maintain their normal temporal relationships with the other events of the estrous cycle in constant light. In spite of the absence of an external timing signal, the majority of hormonal rhythms maintained their normal phase relationships and showed little sign of internal desynchrony. Ovaries in many animals showed high rates of follicular atresia early in the cycle, suggesting that the effects of bright constant light are far more complex than can be attributed to a simple absence of an external timing signal.
Integrated Metrics for Improving the Life Cycle Approach to Assessing Product System Sustainability

EPA Science Inventory

Life cycle approaches are critical for identifying and managing to reduce burdens in the sustainability of product systems. While these methods can indicate potential environmental impacts of a product, current Life Cycle Assessment (LCA) methods fail to integrate the multiple im...
Ste12/Fab1 phosphatidylinositol-3-phosphate 5-kinase is required for nitrogen-regulated mitotic commitment and cell size control

PubMed Central

Schauries, Marie; Kaczmarek, Adrian; Franz-Wachtel, Mirita; Du, Wei; Krug, Karsten; Maček, Boris; Petersen, Janni

2017-01-01

Tight coupling of cell growth and cell cycle progression enable cells to adjust their rate of division, and therefore size, to the demands of proliferation in varying nutritional environments. Nutrient stress promotes inhibition of Target Of Rapamycin Complex 1 (TORC1) activity. In fission yeast, reduced TORC1 activity advances mitotic onset and switches growth to a sustained proliferation at reduced cell size. A screen for mutants, that failed to advance mitosis upon nitrogen stress, identified a mutant in the PIKFYVE 1-phosphatidylinositol-3-phosphate 5-kinase fission yeast homolog Ste12. Ste12PIKFYVE deficient mutants were unable to advance the cell cycle to reduce cell size after a nitrogen downshift to poor nitrogen (proline) growth conditions. While it is well established that PI(3,5)P2 signalling is required for autophagy and that Ste12PIKFYVE mutants have enlarged vacuoles (yeast lysosomes), neither a block to autophagy or mutants that independently have enlarged vacuoles had any impact upon nitrogen control of mitotic commitment. The addition of rapamycin to Ste12PIKFYVE deficient mutants reduced cell size at division to suggest that Ste12PIKFYVE possibly functions upstream of TORC1. ste12 mutants display increased Torin1 (TOR inhibitor) sensitivity. However, no major impact on TORC1 or TORC2 activity was observed in the ste12 deficient mutants. In summary, Ste12PIKFYVE is required for nitrogen-stress mediated advancement of mitosis to reduce cell size at division. PMID:28273166
Transcriptome profiling of the honeybee parasite Varroa destructor provides new biological insights into the mite adult life cycle.

PubMed

Mondet, Fanny; Rau, Andrea; Klopp, Christophe; Rohmer, Marine; Severac, Dany; Le Conte, Yves; Alaux, Cedric

2018-05-04

The parasite Varroa destructor represents a significant threat to honeybee colonies. Indeed, development of Varroa infestation within colonies, if left untreated, often leads to the death of the colony. Although its impact on bees has been extensively studied, less is known about its biology and the functional processes governing its adult life cycle and adaptation to its host. We therefore developed a full life cycle transcriptomic catalogue in adult Varroa females and included pairwise comparisons with males, artificially-reared and non-reproducing females (10 life cycle stages and conditions in total). Extensive remodeling of the Varroa transcriptome was observed, with an upregulation of energetic and chitin metabolic processes during the initial and final phases of the life cycle (e.g. phoretic and post-oviposition stages), whereas during reproductive stages in brood cells genes showing functions related to transcriptional regulation were overexpressed. Several neurotransmitter and neuropeptide receptors involved in behavioural regulation, as well as active compounds of salivary glands, were also expressed at a higher level outside the reproductive stages. No difference was detected between artificially-reared phoretic females and their counterparts in colonies, or between females who failed to reproduce and females who successfully reproduced, indicating that phoretic individuals can be reared outside host colonies without impacting their physiology and that mechanisms underlying reproductive failure occur before oogenesis. We discuss how these new findings reveal the remarkable adaptation of Varroa to its host biology and notably to the switch from living on adults to reproducing in sealed brood cells. By spanning the entire adult life cycle, our work captures the dynamic changes in the parasite gene expression and serves as a unique resource for deciphering Varroa biology and identifying new targets for mite control.
Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint.

PubMed

Yamamori, Tohru; Yasui, Hironobu; Yamazumi, Masayuki; Wada, Yusuke; Nakamura, Yoshinari; Nakamura, Hideo; Inanami, Osamu

2012-07-15

Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that Ir-induced G2/M arrest contributed to the increase in the mitochondrial ROS level by accumulating cells in the G2/M phase. Copyright © 2012 Elsevier Inc. All rights reserved.
Epidermal cell-shape regulation and subpopulation kinetics during butyrate-induced terminal maturation of normal and SV40-transformed human keratinocytes: epithelial models of differentiation therapy.

PubMed

Staiano-Coico, L; Steinberg, M; Higgins, P J

1990-10-15

Recent data indicate that malignant human epidermal cells may be appropriate targets for sodium butyrate (NaB)-mediated differentiation therapy. The response of pre- and post-crisis populations of SV40-transformed human keratinocytes (SVKs) to this differentiation-inducing agent was assessed, therefore, within the framework of NaB-directed normal human keratinocyte (NHK) maturation. NaB augmented cornified envelope (CE) production in NHK and pre-crisis SVK cultures; the time-course and efficiency of induced maturation were similar in the 2 cell systems. In NHKs, the percentage of amplifying ("B" substate) cells decreased with time in NaB correlating with increases in both "C" stage keratinocytes and CEs. The latter formed over one or 2 layers of nucleated basal-like cells. Inductions were accompanied by immediate cell cycle blocks (in both the G1 and G2/M phases), reorganization within the actin cytoskeleton, and transient early increases in cellular actin content. Increased NHK and pre-crisis SVK cytoskeletal-associated actin reached a maximum approximately 48 hr after NaB addition and preceded development of CEs. The CE precursors, thus, probably reside in the "B" substate. Post-crisis SVKs, in contrast, were refractive to NaB-induced terminal maturation or cell-cycle perturbation, failed to initiate actin filament rearrangements, and retained a basal cell-like phenotype. Stable transformation of human SVKs in post-crisis phase, therefore, appears to be associated with loss of maturation "competence" within the "B" keratinocyte subpopulation.
The Role of a Novel Long Noncoding RNA TUC40- in Cardiomyocyte Induction and Maturation in P19 Cells.

PubMed

Li, Huijuan; Jiang, Li; Yu, Zhangbin; Han, Shuping; Liu, Xuehua; Li, Mengmeng; Zhu, Chun; Qiao, Lixing; Huang, Li

2017-12-01

In previous studies, TUC40-, a new long noncoding RNA, was found to be overexpressed in human ventricular septal defect (VSD) embryonic heart samples. In this article, we carried out experiments on the P19 cell line to elucidate the effects of TUC40- overexpression on cardiomyocyte development relevant to VSD pathogenesis. We established the overexpression cell model by plasmid transfection, and explored the expression profile of Pbx1, the sense gene of TUC40-, and the marker genes of cardiomyocyte linage commitment (Nkx2.5 and GATA4) and maturation (cardiac troponin T). In addition, we combined cell cycle and Cell Counting Kit-8 analysis to detect cell proliferation and used flow cytometry and caspase-3 assays to test apoptosis. At last, bioinformatics analysis was performed to show the possible role of TUC40-. In the control group, Pbx1 elevated steadily during cardiomyocyte induction; whereas in the overexpression group, it showed significantly lower expression at day 6, 8 and 10 of induction. Cells in the overexpression group failed to induce cardiomyocytes indicated by GATA4 and cardiac troponin T. Proliferation was inhibited possibly owing to G2/M cell cycle arrest and the induced apoptosis rate was higher in the overexpression group. TUC40- overexpression reduced Pbx1 expression, cardiomyocyte induction and differentiation, inhibited proliferation and promoted apoptosis. Combining the results and previous studies, we propose TUC40- as a potential pathologic factor for VSD. Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.
Expanded CAG/CTG Repeat DNA Induces a Checkpoint Response That Impacts Cell Proliferation in Saccharomyces cerevisiae

PubMed Central

Sundararajan, Rangapriya; Freudenreich, Catherine H.

2011-01-01

Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2), a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases. PMID:21437275
Failed landings after laying hen flight in a commercial aviary over two flock cycles.

PubMed

Campbell, D L M; Goodwin, S L; Makagon, M M; Swanson, J C; Siegford, J M

2016-01-01

Many egg producers are adopting alternative housing systems such as aviaries that provide hens a tiered cage and a litter-covered open floor area. This larger, more complex environment permits expression of behaviors not seen in space-limited cages, such as flight. Flight is an exercise important for strengthening bones; but domestic hens might display imperfect flight landings due to poor flight control. To assess the potential implications of open space, we evaluated the landing success of Lohmann white laying hens in a commercial aviary. Video recordings of hens were taken from 4 aviary sections at peak lay, mid lay and end lay across two flock cycles. Observations were made in each focal section of all flights throughout the day noting flight origin and landing location (outer perch or litter) and landing success or failure. In Flock 1, 9.1% of all flights failed and 21% failed in Flock 2. The number of flights decreased across the laying cycle for both flocks. Proportionally more failed landings were observed in the double row sections in Flock 2. Collisions with other hens were more common than slipping on the ground or colliding with aviary structures across sections and flocks. More hens slipped on the ground and collided with physical structures at peak lay for Flock 2 than at other time points. More collisions with other hens were seen at mid and end lay than at peak lay for Flock 2. Landings ending on perches failed more often than landings on litter. These results indicate potential for flight-related hen injuries in aviary systems resulting from failed landings, which may have implications for hen welfare and optimal system design and management.
The Frank-Starling mechanism involves deceleration of cross-bridge kinetics and is preserved in failing human right ventricular myocardium.

PubMed

Milani-Nejad, Nima; Canan, Benjamin D; Elnakish, Mohammad T; Davis, Jonathan P; Chung, Jae-Hoon; Fedorov, Vadim V; Binkley, Philip F; Higgins, Robert S D; Kilic, Ahmet; Mohler, Peter J; Janssen, Paul M L

2015-12-15

Cross-bridge cycling rate is an important determinant of cardiac output, and its alteration can potentially contribute to reduced output in heart failure patients. Additionally, animal studies suggest that this rate can be regulated by muscle length. The purpose of this study was to investigate cross-bridge cycling rate and its regulation by muscle length under near-physiological conditions in intact right ventricular muscles of nonfailing and failing human hearts. We acquired freshly explanted nonfailing (n = 9) and failing (n = 10) human hearts. All experiments were performed on intact right ventricular cardiac trabeculae (n = 40) at physiological temperature and near the normal heart rate range. The failing myocardium showed the typical heart failure phenotype: a negative force-frequency relationship and β-adrenergic desensitization (P < 0.05), indicating the expected pathological myocardium in the right ventricles. We found that there exists a length-dependent regulation of cross-bridge cycling kinetics in human myocardium. Decreasing muscle length accelerated the rate of cross-bridge reattachment (ktr) in both nonfailing and failing myocardium (P < 0.05) equally; there were no major differences between nonfailing and failing myocardium at each respective length (P > 0.05), indicating that this regulatory mechanism is preserved in heart failure. Length-dependent assessment of twitch kinetics mirrored these findings; normalized dF/dt slowed down with increasing length of the muscle and was virtually identical in diseased tissue. This study shows for the first time that muscle length regulates cross-bridge kinetics in human myocardium under near-physiological conditions and that those kinetics are preserved in the right ventricular tissues of heart failure patients. Copyright © 2015 the American Physiological Society.
Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb.

PubMed

Neuveut, C; Low, K G; Maldarelli, F; Schmitt, I; Majone, F; Grassmann, R; Jeang, K T

1998-06-01

Human T-cell leukemia virus type 1 is etiologically linked to the development of adult T-cell leukemia and various human neuropathies. The Tax protein of human T-cell leukemia virus type I has been implicated in cellular transformation. Like other oncoproteins, such as Myc, Jun, and Fos, Tax is a transcriptional activator. How it mechanistically dysregulates the cell cycle is unclear. Previously, it was suggested that Tax affects cell-phase transition by forming a direct protein-protein complex with p16(INK4a), thereby inactivating an inhibitor of G1-to-S-phase progression. Here we show that, in T cells deleted for p16(INK4a), Tax can compel an egress of cells from G0/G1 into S despite the absence of serum. We also show that in undifferentiated myocytes, expression of Tax represses cellular differentiation. In both settings, Tax expression was found to increase cyclin D-cdk activity and to enhance pRb phosphorylation. In T cells, a Tax-associated increase in steady-state E2F2 protein was also documented. In searching for a molecular explanation for these observations, we found that Tax forms a protein-protein complex with cyclin D3, whereas a point-mutated and transcriptionally inert Tax mutant failed to form such a complex. Interestingly, expression of wild-type Tax protein in cells was also correlated with the induction of a novel hyperphosphorylated cyclin D3 protein. Taken together, these findings suggest that Tax might directly influence cyclin D-cdk activity and function, perhaps by a route independent of cdk inhibitors such as p16(INK4a).
Chlorpyrifos promotes colorectal adenocarcinoma H508 cell growth through the activation of EGFR/ERK1/2 signaling pathway but not cholinergic pathway.

PubMed

Suriyo, Tawit; Tachachartvanich, Phum; Visitnonthachai, Daranee; Watcharasit, Piyajit; Satayavivad, Jutamaad

2015-12-02

Aside from the effects on neuronal cholinergic system, epidemiological studies suggest an association between chlorpyrifos (CPF) exposure and cancer risk. This in vitro study examined the effects of CPF and its toxic metabolite, chlorpyrifos oxon (CPF-O), on the growth of human colorectal adenocarcinoma H508, colorectal adenocarcinoma HT-29, normal colon epithelial CCD841, liver hepatocellular carcinoma HepG2, and normal liver hepatocyte THLE-3 cells. The results showed that CPF (5-100 μM) concentration-dependently increased viability of H508 and CCD841 cells in serum-free conditions. This increasing trend was not found in HT-29, HepG2 and THLE-3 cells. In contrast, CPF-O (50-100 μM) reduced the viability of all cell lines. Cell cycle analysis showed the induction of cells in the S phase, and EdU incorporation assay revealed the induction of DNA synthesis in CPF-treated H508 cells indicating that CPF promotes cell cycle progression. Despite the observation of acetylcholinesterase activity inhibition and reactive oxygen species (ROS) generation, atropine (a non-selective muscarinic acetylcholine receptor antagonist) and N-acetylcysteine (a potent antioxidant) failed to inhibit the growth-promoting effect of CPF. CPF increased the phosphorylation of epidermal growth factor receptor (EGFR) and its downstream effector, extracellular signal regulated kinase (ERK1/2), in H508 cells. AG-1478 (a specific EGFR tyrosine kinase inhibitor) and U0126 (a specific MEK inhibitor) completely mitigated the growth promoting effect of CPF. Altogether, these results suggest that EGFR/ERK1/2 signaling pathway but not cholinergic pathway involves in CPF-induced colorectal adenocarcinoma H508 cell growth. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
BRCA1 and FancJ cooperatively promote interstrand crosslinker induced centrosome amplification through the activation of polo-like kinase 1

PubMed Central

Zou, Jianqiu; Zhang, Deli; Qin, Guang; Chen, Xiangming; Wang, Hongmin; Zhang, Dong

2014-01-01

DNA damage response (DDR) and the centrosome cycle are 2 of the most critical cellular processes affecting the genome stability in animal cells. Yet the cross-talks between DDR and the centrosome are poorly understood. Here we showed that deficiency of the breast cancer 1, early onset gene (BRCA1) induces centrosome amplification in non-stressed cells as previously reported while attenuating DNA damage-induced centrosome amplification (DDICA) in cells experiencing prolonged genotoxic stress. Mechanistically, the function of BRCA1 in promoting DDICA is through binding and recruiting polo-like kinase 1 (PLK1) to the centrosome. In a recent study, we showed that FancJ also suppresses centrosome amplification in non-stressed cells while promoting DDICA in both hydroxyurea and mitomycin C treated cells. FancJ is a key component of the BRCA1 B-complex. Here, we further demonstrated that, in coordination with BRCA1, FancJ promotes DDICA by recruiting both BRCA1 and PLK1 to the centrosome in the DNA damaged cells. Thus, we have uncovered a novel role of BRCA1 and FancJ in the regulation of DDICA. Dysregulation of DDR or centrosome cycle leads to aneuploidy, which is frequently seen in both solid and hematological cancers. BRCA1 and FancJ are known tumor suppressors and have well-recognized functions in DNA damage checkpoint and DNA repair. Together with our recent findings, we demonstrated here that BRCA1 and FancJ also play an important role in centrosome cycle especially in DDICA. DDICA is thought to be an alternative fail-safe mechanism to prevent cells experiencing severe DNA damage from becoming carcinogenic. Therefore, BRCA1 and FancJ are potential liaisons linking early DDR with the DDICA. We propose that together with their functions in DDR, the role of BRCA1 and FancJ in the activation of DDICA is also crucial for their tumor suppression functions in vivo. PMID:25483079
MAPK signaling pathways and HDAC3 activity are disrupted during differentiation of emerin-null myogenic progenitor cells

PubMed Central

Collins, Carol M.; Ellis, Joseph A.

2017-01-01

ABSTRACT Mutations in the gene encoding emerin cause Emery–Dreifuss muscular dystrophy (EDMD). Emerin is an integral inner nuclear membrane protein and a component of the nuclear lamina. EDMD is characterized by skeletal muscle wasting, cardiac conduction defects and tendon contractures. The failure to regenerate skeletal muscle is predicted to contribute to the skeletal muscle pathology of EDMD. We hypothesize that muscle regeneration defects are caused by impaired muscle stem cell differentiation. Myogenic progenitors derived from emerin-null mice were used to confirm their impaired differentiation and analyze selected myogenic molecular pathways. Emerin-null progenitors were delayed in their cell cycle exit, had decreased myosin heavy chain (MyHC) expression and formed fewer myotubes. Emerin binds to and activates histone deacetylase 3 (HDAC3). Here, we show that theophylline, an HDAC3-specific activator, improved myotube formation in emerin-null cells. Addition of the HDAC3-specific inhibitor RGFP966 blocked myotube formation and MyHC expression in wild-type and emerin-null myogenic progenitors, but did not affect cell cycle exit. Downregulation of emerin was previously shown to affect the p38 MAPK and ERK/MAPK pathways in C2C12 myoblast differentiation. Using a pure population of myogenic progenitors completely lacking emerin expression, we show that these pathways are also disrupted. ERK inhibition improved MyHC expression in emerin-null cells, but failed to rescue myotube formation or cell cycle exit. Inhibition of p38 MAPK prevented differentiation in both wild-type and emerin-null progenitors. These results show that each of these molecular pathways specifically regulates a particular stage of myogenic differentiation in an emerin-dependent manner. Thus, pharmacological targeting of multiple pathways acting at specific differentiation stages may be a better therapeutic approach in the future to rescue muscle regeneration in vivo. PMID:28188262

New therapeutic possibilities for vein graft disease in the post-edifoligide era.

PubMed

Cai, Xinjiang; Freedman, Neil J

2006-07-01

Vein graft neointimal hyperplasia involves proliferation and migration of vascular smooth muscle cells into the vessel intima, and ultimately engenders accelerated atherosclerosis and vein graft failure. Since a myriad of stimuli provoke smooth muscle cell proliferation, molecular therapies for vein graft disease have targeted mechanisms fundamental to all cell proliferation - the 'cell-cycle' machinery. Preclinically, the most successful of these therapies has been edifoligide (E2F decoy), a double-stranded oligodeoxynucleotide that binds to the transcription factor known as E2F. Recently, PRoject of Ex vivo vein GRaft Engineering via Transfection (PREVENT) III and IV demonstrated that edifoligide failed to benefit human vein grafts employed to treat lower-extremity ischemia and coronary heart disease, respectively. The clinical failure of edifoligide calls into question previous models of vein graft disease and lends credence to recent animal studies demonstrating that vein graft arterialization substantially involves the immigration into the vein graft of a variety of vascular progenitor cells. Future vein graft disease therapies will likely target not only proliferation of graft-intrinsic cells, but also immigration of graft-extrinsic cells.
Toward scalable parts families for predictable design of biological circuits.

PubMed

Lucks, Julius B; Qi, Lei; Whitaker, Weston R; Arkin, Adam P

2008-12-01

Our current ability to engineer biological circuits is hindered by design cycles that are costly in terms of time and money, with constructs failing to operate as desired, or evolving away from the desired function once deployed. Synthetic biologists seek to understand biological design principles and use them to create technologies that increase the efficiency of the genetic engineering design cycle. Central to the approach is the creation of biological parts--encapsulated functions that can be composited together to create new pathways with predictable behaviors. We define five desirable characteristics of biological parts--independence, reliability, tunability, orthogonality and composability, and review studies of small natural and synthetic biological circuits that provide insights into each of these characteristics. We propose that the creation of appropriate sets of families of parts with these properties is a prerequisite for efficient, predictable engineering of new function in cells and will enable a large increase in the sophistication of genetic engineering applications.
Metformin attenuates ovarian cancer cell growth in an AMP-kinase dispensable manner

PubMed Central

Rattan, R; Giri, S; Hartmann, LC; Shridhar, V

2011-01-01

Abstract Metformin, the most widely used drug for type 2 diabetes activates 59 adenosine monophosphate (AMP)-activated protein kinase (AMPK), which regulates cellular energy metabolism. Here, we report that ovarian cell lines VOSE, A2780, CP70, C200, OV202, OVCAR3, SKOV3ip, PE01 and PE04 predominantly express -α1, -β1, -γ1 and -γ2 isoforms of AMPK subunits. Our studies show that metformin treatment (1) significantly inhibited proliferation of diverse chemo-responsive and -resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3, SKVO3ip, PE01 and PE04), (2) caused cell cycle arrest accompanied by decreased cyclin D1 and increased p21 protein expression, (3) activated AMPK in various ovarian cancer cell lines as evident from increased phosphorylation of AMPKα and its downstream substrate; acetyl co-carboxylase (ACC) and enhanced β-oxidation of fatty acid and (4) attenuated mTOR-S6RP phosphorylation, inhibited protein translational and lipid biosynthetic pathways, thus implicating metformin as a growth inhibitor of ovarian cancer cells. We also show that metformin-mediated effect on AMPK is dependent on liver kinase B1 (LKB1) as it failed to activate AMPK-ACC pathway and cell cycle arrest in LKB1 null mouse embryo fibroblasts (mefs). This observation was further supported by using siRNA approach to down-regulate LKB1 in ovarian cancer cells. In contrast, met formin inhibited cell proliferation in both wild-type and AMPKα1/2 null mefs as well as in AMPK silenced ovarian cancer cells. Collectively, these results provide evidence on the role of metformin as an anti-proliferative therapeutic that can act through both AMPK-dependent as well as AMPK-independent pathways. PMID:19874425
VMY-1-103, a dansylated analog of purvalanol B, induces caspase-3-dependent apoptosis in LNCaP prostate cancer cells

PubMed Central

Yenugonda, Venkata Mahidhar; Ghosh, Anup; Divito, Kyle; Trabosh, Valerie; Patel, Yesha; Brophy, Amanda; Grindrod, Scott; Lisanti, Michael P; Rosenthal, Dean; Brown, Milton L; Avantaggiati, Maria Laura; Rodriguez, Olga

2010-01-01

The 2,6,9-trisubstituted purine group of cyclin dependent kinase inhibitors have the potential to be clinically relevant inhibitors of cancer cell proliferation. We have recently designed and synthesized a novel dansylated analog of purvalanol B, termed VMY-1-103, that inhibited cell cycle progression in breast cancer cell lines more effectively than did purvalanol B and allowed for uptake analyses by fluorescence microscopy. ErbB-2 plays an important role in the regulation of signal transduction cascades in a number of epithelial tumors, including prostate cancer (PCa). Our previous studies demonstrated that transgenic expression of activated ErbB-2 in the mouse prostate initiated PCa and either the overexpression of ErbB-2 or the addition of the ErbB-2/ErbB-3 ligand, heregulin (HRG), induced cell cycle progression in the androgen-responsive prostate cancer cell line, LNCaP. In the present study, we tested the efficacy of VMY-1-103 in inhibiting HRG-induced cell proliferation in LNCaP prostate cancer cells. At concentrations as low as 1 µM, VMY-1-103 increased both the proportion of cells in G1 and p21CIP1 protein levels. At higher concentrations (5 µM or 10 µM), VMY-1-103 induced apoptosis via decreased mitochondrial membrane polarity and induction of p53 phosphorylation, caspase-3 activity and PARP cleavage. Treatment with 10 µM Purvalanol B failed to either influence proliferation or induce apoptosis. Our results demonstrate that VMY-1-103 was more effective in inducing apoptosis in PCa cells than its parent compound, purvalanol B, and support the testing of VMY-1-103 as a potential small molecule inhibitor of prostate cancer in vivo. PMID:20574155
Restoration of C/EBPα in dedifferentiated liposarcoma induces G2/M cell cycle arrest and apoptosis.

PubMed

Wu, Yuhsin V; Okada, Tomoyo; DeCarolis, Penelope; Socci, Nicholas; O'Connor, Rachael; Geha, Rula C; Joy Somberg, C; Antonescu, Cristina; Singer, Samuel

2012-04-01

Well-differentiated liposarcoma (WDLS) and dedifferentiated liposarcoma (DDLS) represent the most common biological group of liposarcoma, and there is a pressing need to develop targeted therapies for patients with advanced disease. To identify potential therapeutic targets, we sought to identify differences in the adipogenic pathways between DDLS, WDLS, and normal adipose tissue. In a microarray analysis of DDLS (n = 84), WDLS (n = 79), and normal fat (n = 23), C/EBPα, a transcription factor involved in cell cycle regulation and differentiation, was underexpressed in DDLS when compared to both WDLS and normal fat (15.2- and 27.8-fold, respectively). In normal adipose-derived stem cells, C/EBPα expression was strongly induced when cells were cultured in differentiation media, but in three DDLS cell lines, this induction was nearly absent. We restored C/EBPα expression in one of the cell lines (DDLS8817) by transfection of an inducible C/EBPα expression vector. Inducing C/EBPα expression reduced proliferation and caused cells to accumulate in G2/M. Under differentiation conditions, the cell proliferation was reduced further, and 66% of the DDLS cells containing the inducible C/EBPα expression vector underwent apoptosis as demonstrated by annexin V staining. These cells in differentiation conditions expressed early adipocyte-specific mRNAs such as LPL and FABP4, but they failed to accumulate intracellular lipid droplets, a characteristic of mature adipocytes. These results demonstrate that loss of C/EBPα is an important factor in suppressing apoptosis and maintaining the dedifferentiated state in DDLS. Restoring C/EBPα may be a useful therapeutic approach for DDLS. Copyright © 2011 Wiley Periodicals, Inc.
The Cellular Phenotype of Roberts Syndrome Fibroblasts as Revealed by Ectopic Expression of ESCO2

PubMed Central

van der Lelij, Petra; van Gosliga, Djoke; Oostra, Anneke B.; Steltenpool, Jûrgen; de Groot, Jan; Scheper, Rik J.; Wolthuis, Rob M.; Waisfisz, Quinten; Darroudi, Firouz; Joenje, Hans; de Winter, Johan P.

2009-01-01

Cohesion between sister chromatids is essential for faithful chromosome segregation. In budding yeast, the acetyltransferase Eco1/Ctf7 establishes cohesion during DNA replication in S phase and in response to DNA double strand breaks in G2/M phase. In humans two Eco1 orthologs exist: ESCO1 and ESCO2. Both proteins are required for proper sister chromatid cohesion, but their exact function is unclear at present. Since ESCO2 has been identified as the gene defective in the rare autosomal recessive cohesinopathy Roberts syndrome (RBS), cells from RBS patients can be used to elucidate the role of ESCO2. We investigated for the first time RBS cells in comparison to isogenic controls that stably express V5- or GFP-tagged ESCO2. We show that the sister chromatid cohesion defect in the transfected cell lines is rescued and suggest that ESCO2 is regulated by proteasomal degradation in a cell cycle-dependent manner. In comparison to the corrected cells RBS cells were hypersensitive to the DNA-damaging agents mitomycin C, camptothecin and etoposide, while no particular sensitivity to UV, ionizing radiation, hydroxyurea or aphidicolin was found. The cohesion defect of RBS cells and their hypersensitivity to DNA-damaging agents were not corrected by a patient-derived ESCO2 acetyltransferase mutant (W539G), indicating that the acetyltransferase activity of ESCO2 is essential for its function. In contrast to a previous study on cells from patients with Cornelia de Lange syndrome, another cohesinopathy, RBS cells failed to exhibit excessive chromosome aberrations after irradiation in G2 phase of the cell cycle. Our results point at an S phase-specific role for ESCO2 in the maintenance of genome stability. PMID:19738907
Biphasic Incorporation of Centromeric Histone CENP-A in Fission Yeast

PubMed Central

Takayama, Yuko; Sato, Hiroshi; Saitoh, Shigeaki; Ogiyama, Yuki; Masuda, Fumie

2008-01-01

CENP-A is a centromere-specific histone H3 variant that is essential for kinetochore formation. Here, we report that the fission yeast Schizosaccharomyces pombe has at least two distinct CENP-A deposition phases across the cell cycle: S and G2. The S phase deposition requires Ams2 GATA factor, which promotes histone gene activation. In Δams2, CENP-A fails to retain during S, but it reaccumulates onto centromeres via the G2 deposition pathway, which is down-regulated by Hip1, a homologue of HIRA histone chaperon. Reducing the length of G2 in Δams2 results in failure of CENP-A accumulation, leading to chromosome missegregation. N-terminal green fluorescent protein-tagging reduces the centromeric association of CENP-A, causing cell death in Δams2 but not in wild-type cells, suggesting that the N-terminal tail of CENP-A may play a pivotal role in the formation of centromeric nucleosomes at G2. These observations imply that CENP-A is normally localized to centromeres in S phase in an Ams2-dependent manner and that the G2 pathway may salvage CENP-A assembly to promote genome stability. The flexibility of CENP-A incorporation during the cell cycle may account for the plasticity of kinetochore formation when the authentic centromere is damaged. PMID:18077559
CDC-25.1 controls the rate of germline mitotic cell cycle by counteracting WEE-1.3 and by positively regulating CDK-1 in Caenorhabditis elegans.

PubMed

Yoon, Sunghee; Kawasaki, Ichiro; Shim, Yhong-Hee

2012-04-01

In Caenorhabditis elegans, cdc-25.1 loss-of-function mutants display a lack of germline proliferation. We found that the proliferation defect of cdc-25.1 mutants was suppressed by wee-1.3 RNAi. Further, among the seven cdk and seven cyclin homologs examined, cdk-1 and cyb-3 RNAi treatment caused the most severe germline proliferation defects in an rrf-1 mutant background, which were similar to those of the cdc-25.1 mutants. In addition, while RNAi of cyd-1 and cye-1 caused significant germline proliferation defects, RNAi of cdk-2 and cdk-4 did not. Compared with the number of germ nuclei in wee-1.3(RNAi) worms, the number in wee-1.3(RNAi);cdk-1(RNAi) and wee-1.3(RNAi);cyb-3(RNAi) worms further decreased to the level of cdk-1(RNAi) and cyb-3(RNAi) worms, respectively, indicating that cdk-1 and cyb-3 are epistatic and function downstream of cdc-25.1 and wee-1.3 in the control of the cell cycle. BrdU labeling of adult worms showed that, while 100% of the wild-type germ nuclei in the mitotic region incorporated BrdU when labeled for more than 12 h at 20°C, a small fraction of the cdc-25.1 mutant germ nuclei failed to incorporate BrdU even when labeled for 68 h. These results indicate that CDC-25.1 is required for maintaining proper rate of germline mitotic cell cycle. We propose that CDC-25.1 regulates the rate of germline mitotic cell cycle by counteracting WEE-1.3 and by positively controlling CDK-1, which forms a complex primarily with CYB-3, but also possibly with CYD-1 and CYE-1.
Life cycle monitoring of lithium-ion polymer batteries using cost-effective thermal infrared sensors with applications for lifetime prediction

NASA Astrophysics Data System (ADS)

Zhou, Xunfei; Malik, Anav; Hsieh, Sheng-Jen

2017-05-01

Lithium-ion batteries have become indispensable parts of our lives for their high-energy density and long lifespan. However, failure due to from abusive usage conditions, flawed manufacturing processes, and aging and adversely affect battery performance and even endanger people and property. Therefore, battery cells that are failing or reaching their end-of-life need to be replaced. Traditionally, battery lifetime prediction is achieved by analyzing data from current, voltage and impedance sensors. However, such a prognostic system is expensive to implement and requires direct contact. In this study, low-cost thermal infrared sensors were used to acquire thermographic images throughout the entire lifetime of small scale lithium-ion polymer batteries (410 cycles). The infrared system (non-destructive) took temperature readings from multiple batteries during charging and discharging cycles of 1C. Thermal characteristics of the batteries were derived from the thermographic images. A time-dependent and spatially resolved temperature mapping was obtained and quantitatively analyzed. The developed model can predict cycle number using the first 10 minutes of surface temperature data acquired through infrared imaging at the beginning of the cycle, with an average error rate of less than 10%. This approach can be used to correlate thermal characteristics of the batteries with life cycles, and to propose cost-effective thermal infrared imaging applications in battery prognostic systems.
Processing of DNA double strand breaks by alternative non-homologous end-joining in hyperacetylated chromatin.

PubMed

Manova, Vasilissa; Singh, Satyendra K; Iliakis, George

2012-08-22

Mammalian cells employ at least two subpathways of non-homologous end-joining for the repair of ionizing radiation induced DNA double strand breaks: The canonical DNA-PK-dependent form of non-homologous end-joining (D-NHEJ) and an alternative, slowly operating, error-prone backup pathway (B-NHEJ). In contrast to D-NHEJ, which operates with similar efficiency throughout the cell cycle, B-NHEJ operates more efficiently in G2-phase. Notably, B-NHEJ also shows strong and as of yet unexplained dependency on growth activity and is markedly compromised in serum-deprived cells, or in cells that enter the plateau-phase of growth. The molecular mechanisms underpinning this response remain unknown. Since chromatin structure or changes in chromatin structure are prime candidate-B-NHEJ-modulators, we study here the role of chromatin hyperacetylation, either by HDAC2 knockdown or treatment with the HDAC inhibitor TSA, on the repair by B-NHEJ of IR-induced DSBs. siRNA-mediated knockdown of HDAC2 fails to provoke histone hyperacetylation in Lig4-/- MEFs and has no detectable effect on B-NHEJ function. Treatment with TSA that inhibits multiple HDACs causes efficient, reversible chromatin hyperacetylation in Lig4-/- MEFs, as well as in human HCT116 Lig4-/- cells and the human glioma cell line M059K. The IR yield of DSBs in TSA-treated cells remains similar to that of untreated cells despite the expected chromatin relaxation. In addition, chromatin hyperacetylation leaves unchanged repair of DSBs by B-NHEJ in irradiated exponentially growing, or plateau-phase cells. Notably, under the experimental conditions employed here, chromatin hyperacetylation fails to detectably modulate B-NHEJ in M059K cells as well. In summary, the results show that chromatin acetylation or deacetylation does not affect the kinetics of alternative NHEJ in all types of cells examined both in exponentially growing and serum deprived cultures. We conclude that parameters beyond chromatin acetylation determine B-NHEJ efficiency in the plateau-phase of growth.
p53 protects against genome instability following centriole duplication failure

PubMed Central

Lambrus, Bramwell G.; Uetake, Yumi; Clutario, Kevin M.; Daggubati, Vikas; Snyder, Michael; Sluder, Greenfield

2015-01-01

Centriole function has been difficult to study because of a lack of specific tools that allow persistent and reversible centriole depletion. Here we combined gene targeting with an auxin-inducible degradation system to achieve rapid, titratable, and reversible control of Polo-like kinase 4 (Plk4), a master regulator of centriole biogenesis. Depletion of Plk4 led to a failure of centriole duplication that produced an irreversible cell cycle arrest within a few divisions. This arrest was not a result of a prolonged mitosis, chromosome segregation errors, or cytokinesis failure. Depleting p53 allowed cells that fail centriole duplication to proliferate indefinitely. Washout of auxin and restoration of endogenous Plk4 levels in cells that lack centrioles led to the penetrant formation of de novo centrioles that gained the ability to organize microtubules and duplicate. In summary, we uncover a p53-dependent surveillance mechanism that protects against genome instability by preventing cell growth after centriole duplication failure. PMID:26150389
A Proteasome Cap Subunit Required for Spindle Pole Body Duplication in Yeast

PubMed Central

McDonald, Heather B.; Byers, Breck

1997-01-01

Proteasome-mediated protein degradation is a key regulatory mechanism in a diversity of complex processes, including the control of cell cycle progression. The selection of substrates for degradation clearly depends on the specificity of ubiquitination mechanisms, but further regulation may occur within the proteasomal 19S cap complexes, which attach to the ends of the 20S proteolytic core and are thought to control entry of substrates into the core. We have characterized a gene from Saccharomyces cerevisiae that displays extensive sequence similarity to members of a family of ATPases that are components of the 19S complex, including human subunit p42 and S. cerevisiae SUG1/ CIM3 and CIM5 products. This gene, termed PCS1 (for proteasomal cap subunit), is identical to the recently described SUG2 gene (Russell, S.J., U.G. Sathyanarayana, and S.A. Johnston. 1996. J. Biol. Chem. 271:32810– 32817). We have shown that PCS1 function is essential for viability. A temperature-sensitive pcs1 strain arrests principally in the second cycle after transfer to the restrictive temperature, blocking as large-budded cells with a G2 content of unsegregated DNA. EM reveals that each arrested pcs1 cell has failed to duplicate its spindle pole body (SPB), which becomes enlarged as in other monopolar mutants. Additionally, we have shown localization of a functional Pcs1–green fluorescent protein fusion to the nucleus throughout the cell cycle. We hypothesize that Pcs1p plays a role in the degradation of certain potentially nuclear component(s) in a manner that specifically is required for SPB duplication. PMID:9151663
HU-331, a novel cannabinoid-based anticancer topoisomerase II inhibitor.

PubMed

Kogan, Natalya M; Schlesinger, Michael; Priel, Esther; Rabinowitz, Ruth; Berenshtein, Eduard; Chevion, Mordechai; Mechoulam, Raphael

2007-01-01

Anthracyclines, a large group of quinonoid compounds, are used to treat some forms of cancer. Although highly effective in cancer therapy, the mechanism of action of these compounds is not specific; they act on cancer and other cells by numerous mechanisms. A new anticancer quinone (HU-331) was synthesized from cannabidiol. It shows significant high efficacy against human cancer cell lines in vitro and against in vivo tumor grafts in nude mice. In this study, we investigated its mode of action and present evidence on its unique mechanism. HU-331 does not cause cancer cell cycle arrest, cell apoptosis, or caspase activation. HU-331-caused cell death of human cancer cell lines is not mediated by reactive oxygen intermediates/species, as exposure to HU-331 failed to elicit the generation of reactive oxygen species. HU-331 inhibits DNA topoisomerase II even at nanomolar concentrations but has only a slight nonsignificant effect on DNA topoisomerase I action. The cannabinoid quinone HU-331 is a highly specific inhibitor of topoisomerase II, compared with most known anticancer quinones. It might represent a new potent anticancer drug.
[Cell cycle and apoptosis mechanisms implicated in intravesical chemotherapy resistances in superficial bladder cancer].

PubMed

Burgués Gasión, J P; Pontones Moreno, J L; Vera Donoso, C D; Jiménez Cruz, J F; Ozonas Moragues, M

2005-10-01

It is well documented the effectiveness of intravesical chemotherapy following transurethral resection to prevent recurrences of superficial bladder cancer. But it is also known that efficacy may be limited by tumour cell resistance to one or several of the drugs available for instillation. In addition to the genetically determined unicellular mechanisms classically described in the literature such as glycoprotein P-170 expression (mdr-1), overexpression of Bcl-2 or glutation S-transferase activity, it has been recently shown that multicellular mechanisms may also be involved in drug resistance. Multicellular resistance can only be demonstrated in three-dimensional cultures and fails to be shown in monolayers or cell suspensions. This is explained by the fact that cell-to-cell and cell-to-stroma adhesion limits drug penetration and by the variable susceptibility to cytotoxicity determined by oxygen and tissue proliferation gradients. A better understanding of the molecular mechanisms involved in drug resistance is necessary to increase intravesical chemotherapy effectiveness. Current research includes improving drug penetration, searching resistance reversing agents and developing in vitro chemosensitivity tests to identify drug resistance.
Hsp90 inhibitors sensitise human colon cancer cells to topoisomerase I poisons by depletion of key anti-apoptotic and cell cycle checkpoint proteins.

PubMed

McNamara, Anne V; Barclay, Monica; Watson, Alastair J M; Jenkins, John R

2012-02-01

Hsp90 and topoisomerase I are both targets for chemotherapeutic agents. Topoisomerase I poisons are standard clinical treatments, whilst Hsp90 inhibitors are progressing through clinical trials. We have demonstrated that when an Hsp90 inhibitor and topoisomerase I poison are combined they produce a synergistic increase in apoptosis in both p53⁺/⁺ and p53⁻/⁻ HCT116 human colon cancer cells. Lack of p53 is associated with an increase in sensitivity to the combination treatment; p53⁺/⁺ cells treated with the topoisomerase I poison topotecan (TPT) arrest at G2, whereas in p53⁻/⁻ cells the additional presence of the Hsp90 inhibitor geldanamycin (GA) selectively abrogates the G2M checkpoint. More importantly we report that there is a common underlying p53-independent mechanism behind the observed synergistic combined drug effect. We show that concurrent treatment with GA and TPT is able to reverse TPT induced up-regulation of the anti-apoptotic protein Bcl2 in both p53⁺/⁺ and p53⁻/⁻ HCT116 cells. The data suggests that inhibition of Hsp90 mediates down-regulation of Bcl2 following the combination treatment and cause a synergistic increase in apoptosis in both p53⁺/⁺ and p53⁻/⁻ HCT116 cells; p53⁻/⁻ HCT116 cells are more sensitive to the treatment because they also fail to arrest at G2 in the cell cycle. Copyright © 2011 Elsevier Inc. All rights reserved.
Multiscale/Multifunctional Probabilistic Composite Fatigue

NASA Technical Reports Server (NTRS)

Chamis, Christos C.

2010-01-01

A multilevel (multiscale/multifunctional) evaluation is demonstrated by applying it to three different sample problems. These problems include the probabilistic evaluation of a space shuttle main engine blade, an engine rotor and an aircraft wing. The results demonstrate that the blade will fail at the highest probability path, the engine two-stage rotor will fail by fracture at the rim and the aircraft wing will fail at 109 fatigue cycles with a probability of 0.9967.
Investigation of Failed Components from a 7-in High Altitude Research Program (HARP) Cannon

DTIC Science & Technology

2011-11-01

figure 39. The crack directions were determined from the oblique lighting fractography . Although there were three primary origins, there is redundancy...photograph in the collage of figure 39). The fractography supports that this part failed under two loading cycles. An initial high load cracked it
Main propulsion system design recommendations for an advanced Orbit Transfer Vehicle

NASA Technical Reports Server (NTRS)

Redd, L.

1985-01-01

Various main propulsion system configurations of an advanced OTV are evaluated with respect to the probability of nonindependent failures, i.e., engine failures that disable the entire main propulsion system. Analysis of the life-cycle cost (LCC) indicates that LCC is sensitive to the main propulsion system reliability, vehicle dry weight, and propellant cost; it is relatively insensitive to the number of missions/overhaul, failures per mission, and EVA and IVA cost. In conclusion, two or three engines are recommended in view of their highest reliability, minimum life-cycle cost, and fail operational/fail safe capability.
Age-Dependent Netrin-1 Signaling Regulates NG2+ Glial Cell Spatial Homeostasis in Normal Adult Gray Matter.

PubMed

Birey, Fikri; Aguirre, Adan

2015-04-29

Neuron-glial antigen 2-positive (NG2(+)) glial cells are the most proliferative glia type in the adult CNS, and their tile-like arrangement in adult gray matter is under tight regulation. However, little is known about the cues that govern this unique distribution. To this end, using a NG2(+) glial cell ablation model in mice, we examined the repopulation dynamics of NG2(+) glial cells in the mature and aged mice gray matter. We found that some resident NG2(+) glial cells that escaped depletion rapidly enter the cell cycle to repopulate the cortex with altered spatial distribution. We reveal that netrin-1 signaling is involved in the NG2(+) glial cell early proliferative, late repopulation, and distribution response after ablation in the gray matter. However, ablation of NG2(+) glial cell in older animals failed to stimulate a similar repopulation response, possibly because of a decrease in the sensitivity to netrin-1. Our findings indicate that endogenous netrin-1 plays a role in NG2(+) glial cell homeostasis that is distinct from its role in myelination. Copyright © 2015 the authors 0270-6474/15/356946-06$15.00/0.
[A case of 63,X/64,XX mosaicism in a subfertile pony mare].

PubMed

Pieńkowska-Schelling, A; Handler, J; Neuhauser, S; Schelling, C

2016-04-01

The present case report describes a 6-year old subfertile pony mare, which became pregnant after the eleventh artificial insemination. The examination of the ovaries and the uterus did not reveal any abnormal clinical findings and the mare showed a regular oestrous cycle. Based on cytogenetic and molecular genetic analyses it became possible to elucidate the observed subfertility. The mosaic karyotype of the mare consisted of 63,X (20%) and 64,XX (80%) cells. A PCR analysis failed to amplify sequences from the equine SRY gene. The observed classic 63,X/64,XX mosaicism is a plausible explanation for the subfertility of the mare.

Suspended animation in C. elegans requires the spindle checkpoint.

PubMed

Nystul, Todd G; Goldmark, Jesse P; Padilla, Pamela A; Roth, Mark B

2003-11-07

In response to environmental signals such as anoxia, many organisms enter a state of suspended animation, an extreme form of quiescence in which microscopically visible movement ceases. We have identified a gene, san-1, that is required for suspended animation in Caenorhabditis elegans embryos. We show that san-1 functions as a spindle checkpoint component in C. elegans. During anoxia-induced suspended animation, embryos lacking functional SAN-1 or a second spindle checkpoint component, MDF-2, failed to arrest the cell cycle, exhibited chromosome missegregation, and showed reduced viability. These data provide a model for how a dynamic biological process is arrested in suspended animation.
Prolonged early G(1) arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle-coupled loss of IRF4.

PubMed

Huang, Xiangao; Di Liberto, Maurizio; Jayabalan, David; Liang, Jun; Ely, Scott; Bretz, Jamieson; Shaffer, Arthur L; Louie, Tracey; Chen, Isan; Randolph, Sophia; Hahn, William C; Staudt, Louis M; Niesvizky, Ruben; Moore, Malcolm A S; Chen-Kiang, Selina

2012-08-02

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G(1) arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G(1) and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G(1) block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy.
Multi-Scale/Multi-Functional Probabilistic Composite Fatigue

NASA Technical Reports Server (NTRS)

Chamis, Christos C.

2008-01-01

A multi-level (multi-scale/multi-functional) evaluation is demonstrated by applying it to three different sample problems. These problems include the probabilistic evaluation of a space shuttle main engine blade, an engine rotor and an aircraft wing. The results demonstrate that the blade will fail at the highest probability path, the engine two-stage rotor will fail by fracture at the rim and the aircraft wing will fail at 109 fatigue cycles with a probability of 0.9967.
Deficiency of Huntingtin Has Pleiotropic Effects in the Social Amoeba Dictyostelium discoideum

PubMed Central

Myre, Michael A.; Lumsden, Amanda L.; Thompson, Morgan N.; Wasco, Wilma; MacDonald, Marcy E.; Gusella, James F.

2011-01-01

Huntingtin is a large HEAT repeat protein first identified in humans, where a polyglutamine tract expansion near the amino terminus causes a gain-of-function mechanism that leads to selective neuronal loss in Huntington's disease (HD). Genetic evidence in humans and knock-in mouse models suggests that this gain-of-function involves an increase or deregulation of some aspect of huntingtin's normal function(s), which remains poorly understood. As huntingtin shows evolutionary conservation, a powerful approach to discovering its normal biochemical role(s) is to study the effects caused by its deficiency in a model organism with a short life-cycle that comprises both cellular and multicellular developmental stages. To facilitate studies aimed at detailed knowledge of huntingtin's normal function(s), we generated a null mutant of hd, the HD ortholog in Dictyostelium discoideum. Dictyostelium cells lacking endogenous huntingtin were viable but during development did not exhibit the typical polarized morphology of Dictyostelium cells, streamed poorly to form aggregates by accretion rather than chemotaxis, showed disorganized F-actin staining, exhibited extreme sensitivity to hypoosmotic stress, and failed to form EDTA-resistant cell–cell contacts. Surprisingly, chemotactic streaming could be rescued in the presence of the bivalent cations Ca2+ or Mg2+ but not pulses of cAMP. Although hd − cells completed development, it was delayed and proceeded asynchronously, producing small fruiting bodies with round, defective spores that germinated spontaneously within a glassy sorus. When developed as chimeras with wild-type cells, hd − cells failed to populate the pre-spore region of the slug. In Dictyostelium, huntingtin deficiency is compatible with survival of the organism but renders cells sensitive to low osmolarity, which produces pleiotropic cell autonomous defects that affect cAMP signaling and as a consequence development. Thus, Dictyostelium provides a novel haploid organism model for genetic, cell biological, and biochemical studies to delineate the functions of the HD protein. PMID:21552328
The Immortal Senescence.

PubMed

Bianchi-Smiraglia, Anna; Lipchick, Brittany C; Nikiforov, Mikhail A

2017-01-01

Activation of oncogenic signaling paradoxically results in the permanent withdrawal from cell cycle and induction of senescence (oncogene-induced senescence (OIS)). OIS is a fail-safe mechanism used by the cells to prevent uncontrolled tumor growth, and, as such, it is considered as the first barrier against cancer. In order to progress, tumor cells thus need to first overcome the senescent phenotype. Despite the increasing attention gained by OIS in the past 20 years, this field is still rather young due to continuous emergence of novel pathways and processes involved in OIS. Among the many factors contributing to incomplete understanding of OIS are the lack of unequivocal markers for senescence and the complexity of the phenotypes revealed by senescent cells in vivo and in vitro. OIS has been shown to play major roles at both the cellular and organismal levels in biological processes ranging from embryonic development to barrier to cancer progression. Here we will briefly outline major advances in methodologies that are being utilized for induction, identification, and characterization of molecular processes in cells undergoing oncogene-induced senescence. The full description of such methodologies is provided in the corresponding chapters of the book.
Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief

PubMed Central

Harshman, Dustin K.; Rao, Brianna M.; McLain, Jean E.; Watts, George S.; Yoon, Jeong-Yeol

2015-01-01

Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections. PMID:26601245
Capacitor bonding techniques and reliability. [thermal cycling tests

NASA Technical Reports Server (NTRS)

Kinser, D. L.; Graff, S. M.; Allen, R. V.; Caruso, S. V.

1974-01-01

The effect of thermal cycling on the mechanical failure of bonded ceramic chip capacitors mounted on alumina substrates is studied. It is shown that differential thermal expansion is responsible for the cumulative effects which lead to delayed failure of the capacitors. Harder or higher melting solders are found to be less susceptible to thermal cycling effects, although they are more likely to fail during initial processing operations.
75 FR 27972 - Airworthiness Directives; Pratt & Whitney JT8D-9, -9A, -11, -15, -17, and -17R Turbofan Engines

Federal Register 2010, 2011, 2012, 2013, 2014

2010-05-19

... cycles-in-service (CIS) since the last total fan blade overhaul was performed. This proposed AD results from reports of failed fan blades. We are proposing this AD to prevent high-cycle fatigue cracking at...-cycle fatigue cracking at the blade root, which could result in uncontained failures of first stage fan...
The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Han-Soo; Kim, Ju Won; Gang, Jingu

2006-09-15

LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21{sup CIP1/WAF1}, RhoB and EGFR, that can explain themore » differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21{sup CIP1/WAF1} and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21{sup CIP1/WAF1} and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells.« less
DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Xuemei; Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022, Hubei Province; Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province

Thyroid cancer is a common malignancy of the endocrine system. Although radioiodine {sup 131}I treatment on differentiated thyroid cancer is widely used, many patients still fail to benefit from {sup 131}I therapy. Therefore, exploration of novel targeted therapies to suppress tumor growth and improve radioiodine uptake remains necessary. Bromodomain-containing protein 4 (BRD4) is an important member of the bromodomain and extra terminal domain family that influences transcription of downstream genes by binding to acetylated histones. In the present study, we found that BRD4 was up-regulated in thyroid cancer tissues and cell lines. Inhibition of BRD4 in thyroid cancer cells bymore » JQ1 resulted in cell cycle arrest at G0/G1 phase and enhanced {sup 131}I uptake in vitro and suppressed tumor growth in vivo. Moreover, JQ1 treatment suppressed C-MYC but enhanced NIS expression. We further demonstrated that BRD4 was enriched in the promoter region of C-MYC, which could be markedly blocked by JQ1 treatment. In conclusion, our findings revealed that the aberrant expression of BRD4 in thyroid cancer is possibly involved in tumor progression, and JQ1 is potentially an effective chemotherapeutic agent against human thyroid cancer. - Highlights: • BRD4 is upregulated in thyroid cancer tissues and cell lines. • Inhibition of BRD4 induced cell cycle arrest and enhanced radioiodine uptake in vitro and impaired tumor growth in vivo. • JQ1 suppressed the expression of C-MYC and promoted the expression of NIS and P21. • JQ1 attenuated the recruitment of BRD4 to MYC promoter in thyroid cancer.« less
Deficiency in DGCR8-dependent canonical microRNAs causes infertility due to multiple abnormalities during uterine development in mice.

PubMed

Kim, Yeon Sun; Kim, Hye-Ryun; Kim, Hyongbum; Yang, Seung Chel; Park, Mira; Yoon, Jung Ah; Lim, Hyunjung J; Hong, Seok-Ho; DeMayo, Francesco J; Lydon, John P; Choi, Youngsok; Lee, Dong Ryul; Song, Haengseok

2016-02-02

DGCR8 is an RNA-binding protein that interacts with DROSHA to produce pre-microRNA in the nucleus, while DICER generates not only mature microRNA, but also endogenous small interfering RNAs in the cytoplasm. Here, we produced Dgcr8 conditional knock-out mice using progesterone receptor (PR)-Cre (Dgcr8(d/d)) and demonstrated that canonical microRNAs dependent on the DROSHA-DGCR8 complex are required for uterine development as well as female fertility in mice. Adult Dgcr8(d/d) females neither underwent regular reproductive cycles nor produced pups, whereas administration of exogenous gonadotropins induced normal ovulation in these mice. Interestingly, immune cells associated with acute inflammation aberrantly infiltrated into reproductive organs of pregnant Dgcr8(d/d) mice. Regarding uterine development, multiple uterine abnormalities were noticeable at 4 weeks of age when PR is significantly increased, and the severity of these deformities increased over time. Gland formation and myometrial layers were significantly reduced, and the stromal cell compartment did not expand and became atrophic during uterine development in these mice. These results were consistent with aberrantly reduced stromal cell proliferation and completely failed decidualization. Collectively, we suggest that DGCR8-dependent canonical microRNAs are essential for uterine development and physiological processes such as proper immune modulation, reproductive cycle, and steroid hormone responsiveness in mice.
Role of non-receptor protein kinases in spermatid transport during spermatogenesis*

PubMed Central

Wan, H. T.; Mruk, Dolores D.; Tang, Elizabeth I.; Xiao, Xiang; Cheng, Yan-ho; Wong, Elissa W.P.; Wong, Chris K. C.; Cheng, C. Yan

2014-01-01

Non-receptor protein tyrosine kinases are cytoplasmic kinases that activate proteins by phosphorylating target protein tyrosine residues, in turn affecting multiple functions in eukaryotic cells. Herein, we focus on the role of non-receptor protein tyrosine kinases, most notably, FAK, c-Yes and c-Src, in the transport of spermatids across the seminiferous epithelium during spermatogenesis. Since spermatids, which are formed from spermatocytes via meiosis, are immotile haploid cells, they must be transported by Sertoli cells across the seminiferous epithelium during the epithelial cycle of spermatogenesis. Without the timely transport of spermatids across the epithelium, the release of sperms at spermiation fails to occur, leading to infertility. Thus, the molecular event pertinent to spermatid transport is crucial to spermatogenesis. Herein, we provide a critical discussion based on recent findings in the field. We also provide a hypothetical model on spermatid transport, and the role of non-receptor protein tyrosine kinases in this event. We also highlight areas of research that deserve attention by investigators in the field. PMID:24727349
A PAIR OF TRANSMEMBRANE RECEPTORS ESSENTIAL FOR THE RETENTION AND PIGMENTATION OF HAIR

PubMed Central

Han, Rong; Beppu, Hideyuki; Lee, Yun-Kyoung; Georgopoulos, Katia; Larue, Lionel; Li, En; Weiner, Lorin; Brissette, Janice L.

2012-01-01

Hair follicles are simple, accessible models for many developmental processes. Here, using mutant mice, we show that Bmpr2, a known receptor for bone morphogenetic proteins (Bmps), and Acvr2a, a known receptor for Bmps and activins, are individually redundant but together essential for multiple follicular traits. When Bmpr2/Acvr2a function is reduced in cutaneous epithelium, hair follicles undergo rapid cycles of hair generation and loss. Alopecia results from a failure to terminate hair development properly, as hair clubs never form, and follicular retraction is slowed. Hair regeneration is rapid due to premature activation of new hair-production programs. Hair shafts differentiate aberrantly due to impaired arrest of medullary-cell proliferation. When Bmpr2/Acvr2a function is reduced in melanocytes, gray hair develops, as melanosomes differentiate but fail to grow, resulting in organelle miniaturization. We conclude that Bmpr2 and Acvr2a normally play cell-type-specific, necessary roles in organelle biogenesis and the shutdown of developmental programs and cell division. PMID:22611050
Synergistic effect of low-dose cucurbitacin B and low-dose methotrexate for treatment of human osteosarcoma

PubMed Central

Goff, Catherine; Iwanski, Gabriela B.; Forscher, Charles; Doan, Ngan B.; Said, Jonathan W.; Koeffler, H. Phillip

2016-01-01

We investigated the use of cucurbitacin B, a plant-derived tetracyclic triterpenoid, as a single agent or in combination with methotrexate (MTX) for human osteosarcoma (OS) treatment. Cucurbitacin B showed antiproliferative activity against seven human OS cell lines in vitro accompanying G2/M cell cycle arrest, apoptosis, and inhibition of ERK, Akt, and mTOR proteins. Cucurbitacin B in combination with MTX synergistically inhibited OS cell growth in vitro. Low-dose cucurbitacin B (LD-CuB, 0.5 mg/kg body weight) or low-dose MTX (LD-MTX, 150 mg/kg) failed to decrease the size of human OS xenografts in nude mice. However, combined therapy at identical concentrations inhibited tumor growth by 62% vs. LD-CuB and 81% vs. LD-MTX (p < 0.001). Strikingly, the effect persisted even when the dose of MTX was decreased by two thirds (VLD-MTX, 50 mg/kg). In conclusion, cucurbitacin B alone or in combination with MTX shows promising antiproliferative activity against human OS. PMID:21440986
Combination chemotherapy containing mitoguazone, ifosfamide, methotrexate, etoposide (MIME) and G-CSF efficiently mobilize peripheral blood progenitor cells in heavily pre-treated relapsed lymphoma patients.

PubMed

Aurlien, E; Holte, H; Kvaløy, S; Jakobsen, E; Rusten, L S; Kvalheim, G

2001-07-01

In this study we explored whether a standard chemotherapy regimen consisting of mitoguazone, ifosfamide, methotrexate and etoposide (MIME) combined with 5 micrograms/kg or 10 micrograms/kg G-CSF was capable of mobilizing peripheral blood progenitor cells (PBPC) in lymphoma patients. Thirty-three patients with Hodgkin's disease (HD) and 108 patients with non-Hodgkin's lymphoma (NHL) were mobilized with MIME/G-CSF. Most patients were heavily treated with different chemotherapy regimens receiving a median of 11 cycles (range 3-40) of chemotherapy prior to mobilization. Eight of 141 patients failed to mobilize PBPC and bone marrow was harvested. In addition, 10 patients obtained a harvest of < 2.0 x 10(6) CD34+ cells/kg. More than 2.0 x 10(6) CD34+ cells/kg were achieved in all HD patients and in 83% of the NHL patients. Fifty-eight per cent of the patients harvested > or = 5 x 10(6) CD34+ cells/kg. Eleven per cent of the patients developed neutropenic fever during the mobilization and 3% had nadir platelet values below 20 x 10(9)/L. An inverse correlation was observed in high-grade NHL (H-NHL) patients between the number of chemotherapy cycles given before mobilization and yield of CD34+ cells. Such an association was not seen among patients with HD, transformed and low-grade NHL. When G-CSF 10 micrograms/kg was used in combination with MIME, this correlation was no longer seen in patients with H-NHL. There was also association between CD34+ cell yield and prior radiotherapy in patients with HD or transformed NHL or low-grade NHL. These results demonstrate that an ordinary salvage chemotherapy regimen, such as MIME combined with G-CSF, can be successfully used to mobilize PBPC. Although no significant effect of increased dose of G-CSF was found, our data suggest that MIME/G-CSF 10 micrograms/kg should preferentially be used to mobilize PBPC in H-NHL patients pre-treated with > or = 12 cycles of chemotherapy, in irradiated HD patients and in all low-grade and transformed lymphomas.
[RESULTS OF PREPARATION AND IMPLEMENTATION OF IVF PROGRAM IN PATIENTS WITH THROMBOPHILIA AND HISTORY OF FAILED IVF].

PubMed

Abrahamyan, G

2017-01-01

The problem of infertility and reproductive losses maintains its urgency, as well as medical and social significance. Frequency of infertility in overall population, according to the data from different authors, varies from 9 to 18 per cent. Methods of aided reproductive technologies (ART) opened a new era in the field of correction of infertile marriage. As a result, more and more couples choose to solve this problem by means of aided reproductive technologies (ART): in-vitro fertilization (IVF) and embryo transfer (ET). However, despite of all achievements, the frequency of pregnancy development remains relatively low and makes 25-30% per treatment cycle, furthermore, during the last decade this value did not change to any significant extent. Analysis of literature sources revealed that genetic, acquired and combined forms of thrombophilia, which often cause severe complications at ART, are among main causes of IVF failures. The aim of the research was to develop and to introduce main principles of prophylaxis of repeated IVF failures in women with thrombophilia and history of failed IVF. In order to achieve the goal we have examined 80 patients (main group) with genetic, acquired or combined thrombophilia, identified on the first stage of standard examination. One of the main reasons of IVF failure is genetic, acquired or combined thrombophilia. Delivery of pathogenetically justified antithrombotic prophylaxis (75 mg. of aspirin and low molecular heparin - enoxaparinum) in patients with thrombophilia and history of failed IVFs allowed improvement of hemostasiogram profile and efficiency of IVF. Frequency of pregnancy in patients with history of failed IVF after the therapy made 31,3% in the first cycle of simulation (in 25 women), 20,0% in the second cycle of simulation (in 16 women) and 11,3% (9 women) in the third cycle. Due to justified antithrombotic prophylaxis 50 cases of pregnancy was registered (62,5%). Introduction of long-term therapy with application of antithrombotic preparations and vitamins in continuous mode promoted successful course of pregnancy, occurred as a result of IVF in patients with history of failed IVF.
40 CFR 86.1439 - Certification Short Test emission test procedures-EPA.

Code of Federal Regulations, 2010 CFR

2010-07-01

... (CONTINUED) Emission Regulations for New Gasoline-Fueled Otto-Cycle Light-Duty Vehicles and New Gasoline-Fueled Otto-Cycle Light-Duty Trucks; Certification Short Test Procedures § 86.1439 Certification Short... seconds (mt=30), whichever comes second. (B) The vehicle fails the idle mode and the test is immediately...
40 CFR 86.1439 - Certification Short Test emission test procedures-EPA.

Code of Federal Regulations, 2011 CFR

2011-07-01

... (CONTINUED) Emission Regulations for New Gasoline-Fueled Otto-Cycle Light-Duty Vehicles and New Gasoline-Fueled Otto-Cycle Light-Duty Trucks; Certification Short Test Procedures § 86.1439 Certification Short... seconds (mt=30), whichever comes second. (B) The vehicle fails the idle mode and the test is immediately...
The Novel IκB Kinase β Inhibitor, IMD-0560, Has Potent Therapeutic Efficacy in Ovarian Cancer Xenograft Model Mice.

PubMed

Sawada, Ikuko; Hashimoto, Kae; Sawada, Kenjiro; Kinose, Yasuto; Nakamura, Koji; Toda, Aska; Nakatsuka, Erika; Yoshimura, Akihiko; Mabuchi, Seiji; Fujikawa, Tomoyuki; Itai, Akiko; Kimura, Tadashi

2016-05-01

Aberrant activation of nuclear factor-kappa β (NF-κB) signaling has been correlated with poor outcome among patients with ovarian cancer. Although the therapeutic potential of NF-κB pathway disruption in cancers has been extensively studied, most classical NF-κB inhibitors are poorly selective, exhibit off-target effects, and have failed to be applied in clinical use. IMD-0560, N-[2,5-bis (trifluoromethyl) phenyl]-5-bromo-2-hydroxybenzamide, is a novel low-molecular-weight compound that selectively inhibits the IκB kinase complex and works as an inhibitor of NF-κB signaling. The aim of this study was to assess the therapeutic potential of IMD-0560 against ovarian cancer in vitro and in vivo. NF-κB activity (phosphorylation) was determined in 9 ovarian cancer cell lines and the inhibitory effect of IMD-0560 on NF-κB activation was analyzed by Western blotting. Cell viability, cell cycle, vascular endothelial growth factor (VEGF) expression, and angiogenesis were assessed in vitro to evaluate the effect of IMD-0560 on ovarian cancer cells. In vivo efficacy of IMD-0560 was also investigated using an ovarian cancer xenograft mouse model. The NF-κB signaling pathway was constitutively activated in 8 of 9 ovarian cancer cell lines. IMD-0560 inhibited NF-κB activation and suppressed ovarian cancer cell proliferation by inducing G1 phase arrest. IMD-0560 decreased VEGF secretion from cancer cells and inhibited the tube formation of human umbilical vein endothelial cells. IMD-0560 significantly inhibited peritoneal metastasis and prolonged the survival in an ovarian cancer xenograft mice model. Immunohistochemical staining of excised tumors revealed that IMD-0560 suppressed VEGF expression, tumor angiogenesis, and cancer cell proliferation. IMD-0560 showed promising therapeutic efficacy against ovarian cancer xenograft mice by inducing cell cycle arrest and suppressing VEGF production from cancer cells. IMD-0560 may be a potential future option in regimens for the treatment of ovarian cancer.
Cytokinetic Failure-induced Tetraploidy Develops into Aneuploidy, Triggering Skin Aging in Phosphovimentin-deficient Mice.

PubMed

Tanaka, Hiroki; Goto, Hidemasa; Inoko, Akihito; Makihara, Hiroyuki; Enomoto, Atsushi; Horimoto, Katsuhisa; Matsuyama, Makoto; Kurita, Kenichi; Izawa, Ichiro; Inagaki, Masaki

2015-05-22

Tetraploidy, a state in which cells have doubled chromosomal sets, is observed in ∼20% of solid tumors and is considered to frequently precede aneuploidy in carcinogenesis. Tetraploidy is also detected during terminal differentiation and represents a hallmark of aging. Most tetraploid cultured cells are arrested by p53 stabilization. However, the fate of tetraploid cells in vivo remains largely unknown. Here, we analyze the ability to repair wounds in the skin of phosphovimentin-deficient (VIM(SA/SA)) mice. Early into wound healing, subcutaneous fibroblasts failed to undergo cytokinesis, resulting in binucleate tetraploidy. Accordingly, the mRNA level of p21 (a p53-responsive gene) was elevated in a VIM(SA/SA)-specific manner. Disappearance of tetraploidy coincided with an increase in aneuploidy. Thereafter, senescence-related markers were significantly elevated in VIM(SA/SA) mice. Because our tetraploidy-prone mouse model also exhibited subcutaneous fat loss at the age of 14 months, another premature aging phenotype, our data suggest that following cytokinetic failure, a subset of tetraploid cells enters a new cell cycle and develops into aneuploid cells in vivo, which promote premature aging. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A sex difference in oxidative stress and behavioral suppression induced by ethanol withdrawal in rats

PubMed Central

Jung, Marianna E.; Metzger, Daniel B.

2016-01-01

Ethanol withdrawal (EW) is referred to the abrupt termination of long-term heavy drinking, and provokes oxidative brain damage. Here, we investigated whether the cerebellum and hippocampus of female rats are less affected by prooxidant EW than male rats due to the antioxidant effect of 17β-estradiol (E2). Female and male rats received a four-week ethanol diet and three-week withdrawal per cycle for two cycles. Some female rats were ovariectomized with E2 or antioxidant (Vitamin E+Co-Q10) treatment. Measurements were cerebellum (Rotarod) and hippocampus (water-maze)-related behaviors, oxidative markers (O2•−, malondialdehyde, protein carbonyls), mitochondrial membrane swelling, and a key mitochondrial enzyme, cytochrome c oxidase (CcO). Separately, HT22 (hippocampal) cells were subjected to ethanol-exposure and withdrawal for two cycles to assess the effect of a CcO inhibitor on E2’s protection for mitochondrial respiration and cell viability. Ethanol-withdrawn female rats showed a smaller increase in oxidative markers in cerebellum and hippocampus than male rats, and E2 treatment decreased the oxidative markers. Compared to male counterparts, ethanol-withdrawn female rats showed better Rotarod but poorer water-maze performance, accompanied by more severe mitochondrial membrane swelling and CcO suppression in hippocampus. E2 or antioxidant treatment improved Rotarod but not water-maze performance. In the presence of a CcO inhibitor, E2 treatment failed to protect mitochondrial respiration and cell viability from EW. These data suggest that antioxidant E2 contributes to smaller oxidative stress in ethanol-withdrawn female than male rats. They also suggest that EW-induced severe mitochondrial damage in hippocampus may blunt E2’s antioxidant protection for hippocampus-related behavior. PMID:27503149
Reversible Age-Related Phenotypes Induced during Larval Quiescence in C. elegans

PubMed Central

Roux, Antoine E.; Langhans, Kelley; Huynh, Walter; Kenyon, Cynthia

2017-01-01

Summary Cells can enter quiescent states in which cell cycling and growth are suspended. We find that during a long developmental arrest (quiescence) induced by starvation, newly-hatched C. elegans acquire features associated with impaired proteostasis and aging: mitochondrial fission, ROS production, protein aggregation, decreased proteotoxic-stress resistance, and at the organismal level, decline of mobility and high mortality. All signs of aging but one, the presence of protein aggregates, were reversed upon return to development induced by feeding. The endoplasmic reticulum receptor IRE-1 is completely required for recovery, and the downstream transcription factor XBP-1, as well as a protein kinase, KGB-1, are partially required. Interestingly, kgb-1(−) mutants that do recover fail to reverse aging-like mitochondrial phenotypes and have a short adult lifespan. Our study describes the first pathway that reverses phenotypes of aging at the exit of prolonged quiescence. PMID:27304510
Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone

PubMed Central

Shuhaibar, Leia C.; Egbert, Jeremy R.; Edmund, Aaron B.; Uliasz, Tracy F.; Dickey, Deborah M.; Yee, Siu-Pok; Potter, Lincoln R.; Jaffe, Laurinda A.

2016-01-01

The meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to restart the cell cycle. An essential event in this process is a decrease in cyclic GMP in the granulosa cells, and part of the cGMP decrease results from dephosphorylation and inactivation of the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase, also known as guanylyl cyclase B. However, it is unknown whether NPR2 dephosphorylation is essential for LH-induced meiotic resumption. Here, we prevented NPR2 dephosphorylation by generating a mouse line in which the seven regulatory serines and threonines of NPR2 were changed to the phosphomimetic amino acid glutamate (Npr2–7E). Npr2–7E/7E follicles failed to show a decrease in enzyme activity in response to LH, and the cGMP decrease was attenuated; correspondingly, LH-induced meiotic resumption was delayed. Meiotic resumption in response to EGF receptor activation was likewise delayed, indicating that NPR2 dephosphorylation is a component of the pathway by which EGF receptor activation mediates LH signaling. We also found that most of the NPR2 protein in the follicle was present in the mural granulosa cells. These findings indicate that NPR2 dephosphorylation in the mural granulosa cells is essential for the normal progression of meiosis in response to LH and EGF receptor activation. In addition, these studies provide the first demonstration that a change in phosphorylation of a transmembrane guanylyl cyclase regulates a physiological process, a mechanism that may also control other developmental events. PMID:26522847
Single-cell high resolution melting analysis: A novel, generic, pre-implantation genetic diagnosis (PGD) method applied to cystic fibrosis (HRMA CF-PGD).

PubMed

Destouni, A; Poulou, M; Kakourou, G; Vrettou, C; Tzetis, M; Traeger-Synodinos, J; Kitsiou-Tzeli, S

2016-03-01

Institutions offering CF-PGD face the challenge of developing and optimizing single cell genotyping protocols that should cover for the extremely heterogeneous CF mutation spectrum. Here we report the development and successful clinical application of a generic CF-PGD protocol to facilitate direct detection of any CFTR nucleotide variation(s) by HRMA and simultaneous confirmation of diagnosis through haplotype analysis. A multiplex PCR was optimized supporting co-amplification of any CFTR exon-region, along with 6 closely linked STRs. Single cell genotypes were established through HRM analysis following melting of the 2nd round PCR products and were confirmed by STR haplotype analysis of the 1st PCR products. The protocol was validated pre-clinically, by testing 208 single lymphocytes, isolated from whole blood samples from 4 validation family trios. Fifteen PGD cycles were performed and 103 embryos were biopsied. In 15 clinical PGD cycles, genotypes were achieved in 88/93 (94.6%) embryo biopsy samples, of which 57/88 (64.8%) were deemed genetically suitable for embryo transfer. Amplification failed at all loci for 10/103 blastomeres biopsied from poor quality embryos. Six clinical pregnancies were achieved (2 twin, 4 singletons). PGD genotypes were confirmed following conventional amniocentesis or chorionic villus sampling in all achieved pregnancies. The single cell HRMA CF-PGD protocol described herein is a flexible, generic, low cost and robust genotyping method, which facilitates the analysis of any CFTR genotype combination. Single-cell HRMA can be beneficial to other clinical settings, for example the detection of single nucleotide variants in single cells derived from clinical tumor samples. Copyright © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
Simulation of Arrhythmogenic Effect of Rogue RyRs in Failing Heart by Using a Coupled Model

PubMed Central

Lu, Luyao; Xia, Ling; Zhu, Xiuwei

2012-01-01

Cardiac cells with heart failure are usually characterized by impairment of Ca2+ handling with smaller SR Ca2+ store and high risk of triggered activities. In this study, we developed a coupled model by integrating the spatiotemporal Ca2+ reaction-diffusion system into the cellular electrophysiological model. With the coupled model, the subcellular Ca2+ dynamics and global cellular electrophysiology could be simultaneously traced. The proposed coupled model was then applied to study the effects of rogue RyRs on Ca2+ cycling and membrane potential in failing heart. The simulation results suggested that, in the presence of rogue RyRs, Ca2+ dynamics is unstable and Ca2+ waves are prone to be initiated spontaneously. These release events would elevate the membrane potential substantially which might induce delayed afterdepolarizations or triggered action potentials. Moreover, the variation of membrane potential depolarization is indicated to be dependent on the distribution density of rogue RyR channels. This study provides a new possible arrhythmogenic mechanism for heart failure from subcellular to cellular level. PMID:23056145
Processing of DNA double strand breaks by alternative non-homologous end-joining in hyperacetylated chromatin

PubMed Central

2012-01-01

Background Mammalian cells employ at least two subpathways of non-homologous end-joining for the repair of ionizing radiation induced DNA double strand breaks: The canonical DNA-PK-dependent form of non-homologous end-joining (D-NHEJ) and an alternative, slowly operating, error-prone backup pathway (B-NHEJ). In contrast to D-NHEJ, which operates with similar efficiency throughout the cell cycle, B-NHEJ operates more efficiently in G2-phase. Notably, B-NHEJ also shows strong and as of yet unexplained dependency on growth activity and is markedly compromised in serum-deprived cells, or in cells that enter the plateau-phase of growth. The molecular mechanisms underpinning this response remain unknown. Since chromatin structure or changes in chromatin structure are prime candidate-B-NHEJ-modulators, we study here the role of chromatin hyperacetylation, either by HDAC2 knockdown or treatment with the HDAC inhibitor TSA, on the repair by B-NHEJ of IR-induced DSBs. Results siRNA-mediated knockdown of HDAC2 fails to provoke histone hyperacetylation in Lig4-/- MEFs and has no detectable effect on B-NHEJ function. Treatment with TSA that inhibits multiple HDACs causes efficient, reversible chromatin hyperacetylation in Lig4-/- MEFs, as well as in human HCT116 Lig4-/- cells and the human glioma cell line M059K. The IR yield of DSBs in TSA-treated cells remains similar to that of untreated cells despite the expected chromatin relaxation. In addition, chromatin hyperacetylation leaves unchanged repair of DSBs by B-NHEJ in irradiated exponentially growing, or plateau-phase cells. Notably, under the experimental conditions employed here, chromatin hyperacetylation fails to detectably modulate B-NHEJ in M059K cells as well. Conclusions In summary, the results show that chromatin acetylation or deacetylation does not affect the kinetics of alternative NHEJ in all types of cells examined both in exponentially growing and serum deprived cultures. We conclude that parameters beyond chromatin acetylation determine B-NHEJ efficiency in the plateau-phase of growth. PMID:22908892
Fatigue and failure responses of lead zirconate titanate multilayer actuator under unipolar high-field electric cycling

NASA Astrophysics Data System (ADS)

Zeng, Fan Wen; Wang, Hong; Lin, Hua-Tay

2013-07-01

Lead zirconate titanate (PZT) multilayer actuators with an interdigital electrode design were studied under high electric fields (3 and 6 kV/mm) in a unipolar cycling mode. A 100 Hz sine wave was used in cycling. Five specimens tested under 6 kV/mm failed from 3.8 × 105 to 7 × 105 cycles, whereas three other specimens tested under 3 kV/mm were found to be still functional after 108 cycles. Variations in piezoelectric and dielectric responses of the tested specimens were observed during the fatigue test, depending on the measuring and cycling conditions. Selected fatigued and damaged actuators were characterized using an impedance analyzer or small signal measurement. Furthermore, involved fatigue and failure mechanisms were investigated using scanning acoustic microscope and scanning electron microscope. The extensive cracks and porous regions were revealed across the PZT layers on the cross sections of a failed actuator. The results from this study have demonstrated that the high-field cycling can accelerate the fatigue of PZT stacks as long as the partial discharge is controlled. The small signal measurement can also be integrated into the large signal measurement to characterize the fatigue response of PZT stacks in a more comprehensive basis. The former can further serve as an experimental method to test and monitor the behavior of PZT stacks.
Cytogenetic damage, oncogenic transformation and p53 induction in human epithelial cells in response to irradiation

NASA Astrophysics Data System (ADS)

Armitage, Mark

Ionizing radiation can have several different effects on cells, some are almost instantaneous such as the generation of DNA damage, other cellular responses take a matter of minutes or hours - DNA repair protein induction/activation, and others may take months or even years to be manifested - carcinogenesis. Human epithelial cell lines derived from both normal, non-neoplastic tissues and from a malignant source were cultured in order to examine several effects of ionizing radiation on such cell types. Cells not from a malignant source were previously immortalized by viral infection or by transfection with viral sequences. Simian virus 40 immortalised uroepithelial cells (SV-HUC) were found to be approximately a factor of two fold more radioresistant than cells of malignant origin (T24) in terms of unrepaired clastogenic damage i.e. assessment of micronuclei levels following irradiation. SV-HUC lines unlike T24 cells are non-tumourigenic when inoculated into nude athymic mice. SV-HUC lines proved very resistant to full oncogenic transformation using radiation and chemical carcinogens. However, morphological alterations and decreased anchorage dependant growth was observed in post carcinogen treated cells after appropriate cell culture conditions were utilized. The progression from this phenotype to a fully tumourigenic one was not recorded in this study. The ability of ionizing radiation to induce increased levels of the nuclear phosphoprotein p53 was also assessed using several different cell lines. SV- HUC and T24 cell lines failed to exhibit any increased p53 stabilization following irradiation. One cell line, a human papilloma virus transformed line (HPV) did show an approximate two fold increase of the wild type p53 protein after treatment with radiation. Only the cell line HPV showed any cell cycle delay, resulting in accumulation of cells in the G2/M compartment in post irradiation cell cycle analysis. The status of p53 was also assessed i.e. wild type or mutant conformation in all the above cells lines and two other control lines HOS (a human osteosarcoma cell line) and H Tori-3 (SV40 immortalised thyroid epithelial cells).
Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

PubMed Central

Warleta, Fernando; Quesada, Cristina Sánchez; Campos, María; Allouche, Yosra; Beltrán, Gabriel; Gaforio, José J.

2011-01-01

Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A) or breast cancer cells (MDA-MB-231 and MCF7). We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS) level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells. PMID:22254082
Loss of the Mammalian DREAM Complex Deregulates Chondrocyte Proliferation

PubMed Central

Forristal, Chantal; Henley, Shauna A.; MacDonald, James I.; Bush, Jason R.; Ort, Carley; Passos, Daniel T.; Talluri, Srikanth; Ishak, Charles A.; Thwaites, Michael J.; Norley, Chris J.; Litovchick, Larisa; DeCaprio, James A.; DiMattia, Gabriel; Holdsworth, David W.; Beier, Frank

2014-01-01

Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes. PMID:24710275
Current trends of reproductive immunology practices in in vitro fertilization (IVF) - a first world survey using IVF-Worldwide.com.

PubMed

Kwak-Kim, Joanne; Han, Ae Ra; Gilman-Sachs, Alice; Fishel, Simon; Leong, Milton; Shoham, Zeev

2013-01-01

Reproductive immunology has evolved from basic research studies to clinical applications. In this study, we aim to investigate the actual application of reproductive immunology concepts and findings in clinical reproductive medicine such as recurrent pregnancy losses (RPL), repeated implantation failures (RIF), and failed in vitro fertilization (IVF) cycles. A web-based survey was performed on IVF-Worldwide.com. Collected data were analyzed by the computerized software. A significant proportion of physicians recommend thrombophilia workups (86%), parental genetic study (79%), and immunologic evaluations (69%) to IVF candidates who have a history of RPL or chemical pregnancy losses. IVF physicians consider an immunologic workup when patients have two (30%) or three (21%) failed IVF cycles. Assays for anticardiolipin antibody, lupus anticoagulant, thyroid peroxidase antibody, and antinuclear antibody are the four most commonly ordered immunologic tests for RPL (88, 84, 50, 47% each) and RIF (68, 63, 38, 38% each). Cellular immune evaluations, such as NK assay, human leukocyte antigen study, Th1/Th2 study or immunophenotype assay, are less commonly ordered. Reproductive immunology principles have been applied to the clinical management of RPL, RIF, and failed IVF cycles, and a significant proportion of IVF physicians acknowledge the importance of immunologic alterations with reproductive outcomes. © 2012 John Wiley & Sons A/S.
Rocket Fuel R and D at AFRL: Recent Activities and Future Direction

DTIC Science & Technology

2017-04-12

Clearance Number 17163 Rocket Cycles and Environments SpaceX Merlin 1D 190 klbf Russian RD-180 860 klbf Gas Generator Cycle Ox-Rich Staged Combustion...affordability & reusability • Modeling & Simulation • Key to development • Requires accurate models “CFD simulations… shorten the test-fail-fix loop” SpaceX
The binding of histone deacetylases and the integrity of zinc finger-like motifs of the E7 protein are essential for the life cycle of human papillomavirus type 31.

PubMed

Longworth, Michelle S; Laimins, Laimonis A

2004-04-01

The E7 oncoprotein of high-risk human papillomaviruses (HPVs) binds to and alters the action of cell cycle regulatory proteins such as members of the retinoblastoma (Rb) family of proteins as well as the histone deacetylases (HDACs). To examine the significance of the binding of E7 to HDACs in the viral life cycle, a mutational analysis of the E7 open reading frame was performed in the context of the complete HPV type 31 (HPV-31) genome. Human foreskin keratinocytes were transfected with wild-type HPV-31 genomes or HPV-31 genomes containing mutations in HDAC binding sequences as well as in the C-terminal zinc finger-like domain, and stable cell lines were isolated. All mutant genomes, except those with E7 mutations in the HDAC binding site, were found to be stably maintained extrachromosomally at an early passage following transfection. Upon further passage in culture, genomes containing mutations to the Rb binding domain as well as the zinc finger-like region quickly lost the ability to maintain episomal genomes. Genomes containing mutations abolishing E7 binding to HDACs or to Rb or mutations to the zinc finger-like motifs failed to extend the life span of transfected keratinocytes and caused cells to arrest at the same time as the untransfected keratinocytes. When induced to differentiate by suspension in methylcellulose, cells maintaining genomes with mutations in the Rb binding domain or the zinc finger-like motifs were impaired in their abilities to activate late viral functions. This study demonstrates that the interaction of E7 with HDACs and the integrity of the zinc finger-like motifs are essential for extending the life span of keratinocytes and for stable maintenance of viral genomes.
Cell-free DNA and telomere length among women undergoing in vitro fertilization treatment.

PubMed

Czamanski-Cohen, J; Sarid, O; Cwikel, J; Douvdevani, A; Levitas, E; Lunenfeld, E; Har-Vardi, I

2015-11-01

The current research is aimed at finding potential non-invasive bio-markers that will help us learn more about the mechanisms at play in failed assisted reproduction treatment. This exploratory pilot study examined the relationship between cell-free DNA (CFD) in plasma and telomere length in lymphocytes among women undergoing in vitro fertilization (IVF) and compared telomere length and CFD levels to a healthy control group. Blood of 20 women undergoing IVF was collected at three time points during the IVF cycle. We assessed the relationship between CFD and telomere length as well as controlling for morning cortisol levels. We also collected blood of 10 healthy controls at two time points (luteal and follicular phases of the menstrual cycle) and compared mean telomere length, CFD, and cortisol levels between the IVF patients and healthy controls. The results revealed an inverse relationship between CFD levels and telomere lengths at several time points that remained significant even after controlling for cortisol levels. Women undergoing IVF had statistically significant higher levels of CFD and shorter telomeres compared to healthy controls. The relationship between telomere length and CFD should be further explored in larger studies in order to uncover potential mechanisms that cause both shortened telomere length and elevated CFD in women undergoing IVF.
Gravity independence of seed-to-seed cycling in Brassica rapa

NASA Technical Reports Server (NTRS)

Musgrave, M. E.; Kuang, A.; Xiao, Y.; Stout, S. C.; Bingham, G. E.; Briarty, L. G.; Levenskikh, M. A.; Sychev, V. N.; Podolski, I. G.

2000-01-01

Growth of higher plants in the microgravity environment of orbital platforms has been problematic. Plants typically developed more slowly in space and often failed at the reproductive phase. Short-duration experiments on the Space Shuttle showed that early stages in the reproductive process could occur normally in microgravity, so we sought a long-duration opportunity to test gravity's role throughout the complete life cycle. During a 122-d opportunity on the Mir space station, full life cycles were completed in microgravity with Brassica rapa L. in a series of three experiments in the Svet greenhouse. Plant material was preserved in space by chemical fixation, freezing, and drying, and then compared to material preserved in the same way during a high-fidelity ground control. At sampling times 13 d after planting, plants on Mir were the same size and had the same number of flower buds as ground control plants. Following hand-pollination of the flowers by the astronaut, siliques formed. In microgravity, siliques ripened basipetally and contained smaller seeds with less than 20% of the cotyledon cells found in the seeds harvested from the ground control. Cytochemical localization of storage reserves in the mature embryos showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in the ground control seeds. While these successful seed-to-seed cycles show that gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity.
Characterization and functional analysis of a slow-cycling subpopulation in colorectal cancer enriched by cell cycle inducer combined chemotherapy.

PubMed

Wu, Feng-Hua; Mu, Lei; Li, Xiao-Lan; Hu, Yi-Bing; Liu, Hui; Han, Lin-Tao; Gong, Jian-Ping

2017-10-03

The concept of cancer stem cells has been proposed in various malignancies including colorectal cancer. Recent studies show direct evidence for quiescence slow-cycling cells playing a role in cancer stem cells. There exists an urgent need to isolate and better characterize these slow-cycling cells. In this study, we developed a new model to enrich slow-cycling tumor cells using cell-cycle inducer combined with cell cycle-dependent chemotherapy in vitro and in vivo . Our results show that Short-term exposure of colorectal cancer cells to chemotherapy combined with cell-cycle inducer enriches for a cell-cycle quiescent tumor cell population. Specifically, these slow-cycling tumor cells exhibit increased chemotherapy resistance in vitro and tumorigenicity in vivo . Notably, these cells are stem-cell like and participate in metastatic dormancy. Further exploration indicates that slow-cycling colorectal cancer cells in our model are less sensitive to cytokine-induced-killer cell mediated cytotoxic killing in vivo and in vitro . Collectively, our cell cycle inducer combined chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that are resistant to cytokine induced killer cell based immunotherapy. Studying unique signaling pathways in dormant tumor cells enriched by cell cycle inducer combined chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.
Characterization and functional analysis of a slow-cycling subpopulation in colorectal cancer enriched by cell cycle inducer combined chemotherapy

PubMed Central

Wu, Feng-Hua; Mu, Lei; Li, Xiao-Lan; Hu, Yi-Bing; Liu, Hui; Han, Lin-Tao; Gong, Jian-Ping

2017-01-01

The concept of cancer stem cells has been proposed in various malignancies including colorectal cancer. Recent studies show direct evidence for quiescence slow-cycling cells playing a role in cancer stem cells. There exists an urgent need to isolate and better characterize these slow-cycling cells. In this study, we developed a new model to enrich slow-cycling tumor cells using cell-cycle inducer combined with cell cycle-dependent chemotherapy in vitro and in vivo. Our results show that Short-term exposure of colorectal cancer cells to chemotherapy combined with cell-cycle inducer enriches for a cell-cycle quiescent tumor cell population. Specifically, these slow-cycling tumor cells exhibit increased chemotherapy resistance in vitro and tumorigenicity in vivo. Notably, these cells are stem-cell like and participate in metastatic dormancy. Further exploration indicates that slow-cycling colorectal cancer cells in our model are less sensitive to cytokine-induced-killer cell mediated cytotoxic killing in vivo and in vitro. Collectively, our cell cycle inducer combined chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that are resistant to cytokine induced killer cell based immunotherapy. Studying unique signaling pathways in dormant tumor cells enriched by cell cycle inducer combined chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence. PMID:29108242
Failed CTL/NK cell killing and cytokine hypersecretion are directly linked through prolonged synapse time

PubMed Central

Rudd-Schmidt, Jesse A.; Lopez, Jamie A.; Ramsbottom, Kelly M.; Mannering, Stuart I.; Andrews, Daniel M.; Voskoboinik, Ilia

2015-01-01

Failure of cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells to kill target cells by perforin (Prf)/granzyme (Gzm)-induced apoptosis causes severe immune dysregulation. In familial hemophagocytic lymphohistiocytosis, Prf-deficient infants suffer a fatal “cytokine storm” resulting from macrophage overactivation, but the link to failed target cell death is not understood. We show that prolonged target cell survival greatly amplifies the quanta of inflammatory cytokines secreted by CTLs/NK cells and that interferon-γ (IFN-γ) directly invokes the activation and secondary overproduction of proinflammatory IL-6 from naive macrophages. Furthermore, using live cell microscopy to visualize hundreds of synapses formed between wild-type, Prf-null, or GzmA/B-null CTLs/NK cells and their targets in real time, we show that hypersecretion of IL-2, TNF, IFN-γ, and various chemokines is linked to failed disengagement of Prf- or Gzm-deficient lymphocytes from their targets, with mean synapse time increased fivefold, from ∼8 to >40 min. Surprisingly, the signal for detachment arose from the dying target cell and was caspase dependent, as delaying target cell death with various forms of caspase blockade also prevented their disengagement from fully competent CTLs/NK cells and caused cytokine hypersecretion. Our findings provide the cellular mechanism through which failed killing by lymphocytes causes systemic inflammation involving recruitment and activation of myeloid cells. PMID:25732304
Clinical significance of intercellular contact at the four-cell stage of human embryos, and the use of abnormal cleavage patterns to identify embryos with low implantation potential: a time-lapse study.

PubMed

Liu, Yanhe; Chapple, Vincent; Feenan, Katie; Roberts, Peter; Matson, Phillip

2015-06-01

To investigate the clinical significance of intercellular contact point (ICCP) in four-cell stage human embryos and the effectiveness of morphology and abnormal cleavage patterns in identifying embryos with low implantation potential. Retrospective cohort study. Private IVF center. A total of 223 consecutive IVF and intracytoplasmic sperm injection treatment cycles, with all resulting embryos cultured in the Embryoscope, and a subset of 207 cycles analyzed for ICCP number where good-quality four-cell embryos were available on day 2 (n = 373 IVF and n = 392 intracytoplasmic sperm injection embryos). None. Morphologic score on day 3, embryo morphokinetic parameters, incidence of abnormal biological events, and known implantation results. Of 765 good-quality four-cell embryos, 89 (11.6%) failed to achieve six ICCPs; 166 of 765 (21.7%) initially had fewer than six ICCPs but were able to establish six ICCPs before subsequent division. Embryos with fewer than six ICCPs at the end of four-cell stage had a lower implantation rate (5.0% vs. 38.5%), with lower embryology performance in both conventional and morphokinetic assessments, compared with embryos achieving six ICCPs by the end of four-cell stage. Deselecting embryos with poor morphology, direct cleavage, reverse cleavage, and fewer than six ICCPs at the four-cell stage led to a significantly improved implantation rate (33.6% vs. 22.4%). Embryos with fewer than six ICCPs at the end of the four-cell stage show compromised subsequent development and reduced implantation potential. Deselection of embryos with poor morphology and abnormal cleavage revealed via time-lapse imaging could provide the basis of a qualitative algorithm for embryo selection. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
Fractographic evaluation of creep effects on strain-controlled fatigue-cracking of AISI 304LC and 316 stainless steel

NASA Technical Reports Server (NTRS)

Oldrieve, R. E.

1978-01-01

Analysis of high temperature low cycle fatigue of AISI 304LC and 316 stainless steels by the method of strainrange partitioning results in four separate strainrange versus life relationships, depending upon the way in which creep-strain and plastic strain are combined within a cycle. Fractography is used in this investigation of the creep-fatigue interaction associated with these cycles. The PP and PC-cycle fractures were transgranular. The PC-cycle resulted in fewer cycles of initiation and shorter total cyclic life for the same applied inelastic strainrange. The CC-cycle had mixed transgranular and intergranular fracture, fewer cycles of initiation and shorter cycle life than PP or PC. The CP-cycle had fully integranular cracking, and failed in fewer cycles than were required for cracks to initate for PP,PC, and CC.

The Family Life Cycle and Critical Transitions: Utilizing Cinematherapy to Facilitate Understanding and Increase Communication

ERIC Educational Resources Information Center

Ballard, Mary B.

2012-01-01

Transitioning successfully from one stage of development to the next in the family life cycle requires the accomplishment of certain developmental tasks. Couples and families who fail to accomplish these tasks often become "stuck" and unable to move forward. This impasse frequently leads to heightened stress reactions and crippled channels of…
Evaluating Managerial Styles for System Development Life Cycle Stages to Ensure Software Project Success

ERIC Educational Resources Information Center

Kocherla, Showry

2012-01-01

Information technology (IT) projects are considered successful if they are completed on time, within budget, and within scope. Even though, the required tools and methodologies are in place, IT projects continue to fail at a higher rate. Current literature lacks explanation for success within the stages of system development life-cycle (SDLC) such…
Implementing Army Training Programs: An Overview for Managers. Research Report 1382.

ERIC Educational Resources Information Center

Gray, Wayne D.

The place and importance of implementation in the life cycle of Army training programs is frequently misunderstood. Typically, a program's life cycle is thought of as research, development, and use. If implementation is thought of at all, it is regarded as an event, not a process. Many worthwhile programs have failed because the implementation…
Pemetrexed plus dendritic cells as second-line treatment for patients with stage IIIB/IV non-small cell lung cancer who had treatment with TKI.

PubMed

Hu, Rong-Hang; Shi, Sheng-Bin; Qi, Jie-Lin; Tian, Jing; Tang, Xiao-Yong; Liu, Guo-Fang; Chang, Chun-Xiao

2014-08-01

The aim of this study was to determine the efficacy and toxicity of pemetrexed plus dendritic cells (DCs) in patients suffering from stage IIIB or IV lung adenocarcinoma, who had undergone maintenance treatment with gefitinib or erlotinib. Patients who had failed gefitinib or erlotinib maintenance treatment had ECOG performance statuses ranging from 0 to 2.27 patients received pemetrexed plus DCs as second-line treatment. Dosage: 500 mg/m(2) pemetrexed was administered on day 1 of a 21-day cycle. DCs were given for one cycle of 21 days. Three patients (11.1 %) experienced a partial response and 14 patients (51.9 %) showed stable disease. Ten patients (37.0 %) had progressive disease. The median time to progression-free survival (PFS) was 4.8 months [95 % confidence interval (CI) 4.4-5.2], and the median overall survival was 10.7 months (95 % CI 10.3-11.2). In the subgroup analysis, PFS had a significant difference between the low ratio of CD4/CD8 and normal ratio of CD4/CD8, with 4.5 months (95 % CI 4.2-4.9) and 5.0 months (95 % CI 4.5-5.7), (Log Rank = 0.039), respectively. No one patient experienced grade 4 toxicity. A regimen of pemetrexed combined with DCs is marginally effective and well tolerated in patients with stage IIIB or IV lung adenocarcinoma who had received gefitinib or erlotinib first-line treatment.
Reciprocal expression of integration host factor and HU in the developmental cycle and infectivity of Legionella pneumophila.

PubMed

Morash, Michael G; Brassinga, Ann Karen C; Warthan, Michelle; Gourabathini, Poornima; Garduño, Rafael A; Goodman, Steven D; Hoffman, Paul S

2009-04-01

Legionella pneumophila is an intracellular parasite of protozoa that differentiates late in infection into metabolically dormant cysts that are highly infectious. Regulation of this process is poorly understood. Here we report that the small DNA binding regulatory proteins integration host factor (IHF) and HU are reciprocally expressed over the developmental cycle, with HU expressed during exponential phase and IHF expressed postexponentially. To assess the role of these regulatory proteins in development, chromosomal deletions were constructed. Single (ihfA or ihfB) and double deletion (Deltaihf) IHF mutants failed to grow in Acanthamoeba castellanii unless complemented in trans when expressed temporally from the ihfA promoter but not under P(tac) (isopropyl-beta-d-thiogalactopyranoside). In contrast, IHF mutants were infectious for HeLa cells, though electron microscopic examination revealed defects in late-stage cyst morphogenesis (thickened cell wall, intracytoplasmic membranes, and inclusions of poly-beta-hydroxybutyrate), and were depressed for the developmental marker MagA. Green fluorescent protein promoter fusion assays indicated that IHF and the stationary-phase sigma factor RpoS were required for full postexponential expression of magA. Finally, defects in cyst morphogenesis noted for Deltaihf mutants in HeLa cells correlated with a loss of both detergent resistance and hyperinfectivity compared with results for wild-type cysts. These studies establish IHF and HU as markers of developmental stages and show that IHF function is required for both differentiation and full virulence of L. pneumophila in natural amoebic hosts.
Efficacy and safety of Apatinib in stage IV sarcomas: experience of a major sarcoma center in China.

PubMed

Li, Feng; Liao, Zhichao; Zhao, Jun; Zhao, Gang; Li, Xubin; Du, Xiaoling; Yang, Yun; Yang, Jilong

2017-09-08

This study was conducted to review the efficacy and safety of Apatinib in stage IV sarcoma patients who failed previous chemotherapy. The clinical information on 16 patients with stage IV sarcomas who failed in prior chemotherapy and subsequently received Apatinib treatment was collected. Apatinib was given 500mg/daily and 4 weeks as a cycle. All patients had at least one measurable extracranial tumor according to Response Evaluation Criteria In Solid Tumors 1.0 criteria. Progression free survival (PFS), overall survival (OS), objective response rate (ORR), disease control rate (DCR) and treatment-related adverse effects (AEs) were reviewed and evaluated. Patients was administered Apatinib for 0 to 9 cycles with the median of 3.2 cycles. Median follow-up time was 8.4 months (1 to 12 months). Ten of 16 patients received at least 1 complete cycle of Apatinib treatment were eligible for the efficacy analysis. The median PFS was 8.84 months. Two patients achieved partial response (PR) and 6 patients achieved stable disease (SD). Two patients were evaluated as progression disease (PD) and one patient died of disease progression. The ORR was 20.0% (2/10) and the DCR was 80.0% (8/10). The most common grade 3/4 treatment-related AEs were hypertension (18.7%), hand-foot syndrome (12.5%) and proteinuria (6.3%). No drug-related severe AEs occurred. CApatinib treatment in this exploratory study exhibited objective efficacy and manageable toxicity in stage IV sarcoma patients who failed in chemotherapy. This result supports future random controlled trial to further define Apatinib activity in stage IV sarcomas.
Exploring the life cycles of three South American rusts that have potential as biocontrol agents of the stipoid grass Nassella neesiana in Australasia.

PubMed

Anderson, Freda E; Díaz, Marina L; Barton, Jane; Flemmer, Andrea C; Hansen, Paula V; McLaren, David A

2011-01-01

Three rusts, Puccinia nassellae, Uromyces pencanus, and Puccinia graminella, are being studied as potential biological control agents for Nassella neesiana (Chilean needle grass) in Australia and New Zealand. An understanding of the life cycle of a pathogen is desirable before its release as a biocontrol agent is considered, to enable the assessment of the risks involved in such a release. Field observations and experiments have been carried out to elucidate the life cycles of these three pathogens. Puccinia nassellae cycles as urediniospores and produces dormant teliospores. Dormancy of teliospores has been broken through manipulation in the laboratory, but resulting basidiospores have failed to infect N. neesiana plants under the conditions tested. Uromyces pencanus cycles as urediniospores and its telia appear to have lost the capacity to produce basidiospores. Aecia have been reported for this rust in the literature. However, evidence is provided that these aecia in fact belong to the life cycle of P. graminella. Puccinia graminella produces only aecia and telia. The aeciospores have been shown to be repetitive (aecidioid urediniospores). Teliospores germinate directly without a dormant phase, but resulting basidiospores failed to infect N. neesiana plants under the conditions tested. The role of teliospores in the life cycle of all three rusts remains unknown. Although the autoecious nature of their life cycles has not been proven experimentally, neither is there any evidence that they are heteroecious. Practical and theoretical implications of these findings are discussed. Copyright © 2011 The British Mycological Society. All rights reserved.
Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process.

PubMed

Laranjeiro, Ricardo; Tamai, T Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-04-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles.
Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process

PubMed Central

Tamai, T. Katherine; Letton, William; Hamilton, Noémie; Whitmore, David

2018-01-01

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles. PMID:29444612
Process of Infection with Bacteriophage φX174

PubMed Central

Dalgarno, L.; Sinsheimer, Robert L.

1968-01-01

A group of temperature-sensitive mutants of φX174 has been isolated which can go through a single, normal one-step growth cycle at 40 C but fail to form plaques at this temperature. Such mutants fail to initiate a second cycle at 40 C; however they can gain the capacity to infect at 40 C, upon incubation for 10 min in broth at 30 C. In regaining the ability to infect, the phage appear to undergo a temperature-dependent conformational alteration. The inverse process, a reversible loss of ability to infect at 40 C, is observed when such phage produced at 30 C are incubated for 2 hr at 40 C. The defect in initiation of a second cycle of infection appears to be in the injection of viral deoxyribonucleic acid. A two-step complementation test has been used to identify the cistron coding for the affected function. Such mutants are also unusually sensitive to an irreversible thermal inactivation when incubated at 40 C. PMID:4883013
Reactive oxygen species activate differentiation gene transcription of acute myeloid leukemia cells via the JNK/c-JUN signaling pathway.

PubMed

Lam, Chung Fan; Yeung, Hoi Ting; Lam, Yuk Man; Ng, Ray Kit

2018-05-01

Reactive oxygen species (ROS) and altered cellular redox status are associated with many malignancies. Acute myeloid leukemia (AML) cells are maintained at immature state by differentiation blockade, which involves deregulation of transcription factors in myeloid differentiation. AML cells can be induced to differentiate by phorbol-12-myristate-13-acetate (PMA), which possesses pro-oxidative activity. However, the signaling events mediated by ROS in the activation of transcriptional program during AML differentiation has not been fully elucidated. Here, we investigated AML cell differentiation by treatment with PMA and ROS scavenger N-acetyl-l-cysteine (NAC). We observed elevation of intracellular ROS level in the PMA-treated AML cells, which correlated with differentiated cell morphology and increased CD11b + mature cell population. The effect of PMA can be abolished by NAC co-treatment, supporting the involvement of ROS in the process. Moreover, we demonstrated that short ROS elevation mediated cell cycle arrest, but failed to activate myeloid gene transcription; whereas prolonged ROS elevation activated JNK/c-JUN signaling pathway. Inhibition of JNK suppressed the expression of key myeloid transcriptional regulators c-JUN, SPI-1 and MAFB, and prevented AML cells from undergoing terminal differentiation. These findings provide new insights into the crucial role of JNK/c-Jun signaling pathway in the activation of transcriptional program during ROS-mediated AML differentiation. Copyright © 2018 Elsevier Ltd. All rights reserved.
Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells.

PubMed

Carlson, Morgan E; Hsu, Michael; Conboy, Irina M

2008-07-24

Adult skeletal muscle robustly regenerates throughout an organism's life, but as the muscle ages, its ability to repair diminishes and eventually fails. Previous work suggests that the regenerative potential of muscle stem cells (satellite cells) is not triggered in the old muscle because of a decline in Notch activation, and that it can be rejuvenated by forced local activation of Notch. Here we report that, in addition to the loss of Notch activation, old muscle produces excessive transforming growth factor (TGF)-beta (but not myostatin), which induces unusually high levels of TGF-beta pSmad3 in resident satellite cells and interferes with their regenerative capacity. Importantly, endogenous Notch and pSmad3 antagonize each other in the control of satellite-cell proliferation, such that activation of Notch blocks the TGF-beta-dependent upregulation of the cyclin-dependent kinase (CDK) inhibitors p15, p16, p21 and p27, whereas inhibition of Notch induces them. Furthermore, in muscle stem cells, Notch activity determines the binding of pSmad3 to the promoters of these negative regulators of cell-cycle progression. Attenuation of TGF-beta/pSmad3 in old, injured muscle restores regeneration to satellite cells in vivo. Thus a balance between endogenous pSmad3 and active Notch controls the regenerative competence of muscle stem cells, and deregulation of this balance in the old muscle microniche interferes with regeneration.
HES6 enhances the motility of alveolar rhabdomyosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wickramasinghe, Caroline M; MRC Laboratory of Molecular Biology, Addenbrooke's Hospital Cambridge, CB2 0QH; Domaschenz, Renae

Absract: HES6, a member of the hairy-enhancer-of-split family of transcription factors, plays multiple roles in myogenesis. It is a direct target of the myogenic transcription factor MyoD and has been shown to regulate the formation of the myotome in development, myoblast cell cycle exit and the organization of the actin cytoskeleton during terminal differentiation. Here we investigate the expression and function of HES6 in rhabdomyosarcoma, a soft tissue tumor which expresses myogenic genes but fails to differentiate into muscle. We show that HES6 is expressed at high levels in the subset of alveolar rhabdomyosarcomas expressing PAX/FOXO1 fusion genes (ARMSp). Knockdownmore » of HES6 mRNA in the ARMSp cell line RH30 reduces proliferation and cell motility. This phenotype is rescued by expression of mouse Hes6 which is insensitive to HES6 siRNA. Furthermore, expression microarray analysis indicates that the HES6 knockdown is associated with a decrease in the levels of Transgelin, (TAGLN), a regulator of the actin cytoskeleton. Knockdown of TAGLN decreases cell motility, whilst TAGLN overexpression rescues the motility defect resulting from HES6 knockdown. These findings indicate HES6 contributes to the pathogenesis of ARMSp by enhancing both proliferation and cell motility.« less
Inhibition of mutant BRAF splice variant signaling by next-generation, selective RAF inhibitors.

PubMed

Basile, Kevin J; Le, Kaitlyn; Hartsough, Edward J; Aplin, Andrew E

2014-05-01

Vemurafenib and dabrafenib block MEK-ERK1/2 signaling and cause tumor regression in the majority of advanced-stage BRAF(V600E) melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next-generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAF(V600E) melanoma cells without paradoxical effects in wild-type cells. Furthermore, PLX7904 blocks the growth of vemurafenib-resistant BRAF(V600E) cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAF(V600E) splice variant-mediated vemurafenib-resistant cells. We show that paradox-breaker RAF inhibitors potently block MEK-ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720-resistant cells harboring distinct BRAF(V600E) splice variants. These data support the further investigation of paradox-breaker RAF inhibitors as a second-line treatment option for patients failing on vemurafenib or dabrafenib. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Determinants of orofacial clefting I: Effects of 5-Aza-2'-deoxycytidine on cellular processes and gene expression during development of the first branchial arch.

PubMed

Mukhopadhyay, Partha; Seelan, Ratnam S; Rezzoug, Francine; Warner, Dennis R; Smolenkova, Irina A; Brock, Guy; Pisano, M Michele; Greene, Robert M

2017-01-01

In this study, we identify gene targets and cellular events mediating the teratogenic action(s) of 5-Aza-2'-deoxycytidine (AzaD), an inhibitor of DNA methylation, on secondary palate development. Exposure of pregnant mice (on gestation day (GD) 9.5) to AzaD for 12h resulted in the complete penetrance of cleft palate (CP) in fetuses. Analysis of cells of the embryonic first branchial arch (1-BA), in fetuses exposed to AzaD, revealed: 1) significant alteration in expression of genes encoding several morphogenetic factors, cell cycle inhibitors and regulators of apoptosis; 2) a decrease in cell proliferation; and, 3) an increase in apoptosis. Pyrosequencing of selected genes, displaying pronounced differential expression in AzaD-exposed 1-BAs, failed to reveal significant alterations in CpG methylation levels in their putative promoters or gene bodies. CpG methylation analysis suggested that the effects of AzaD on gene expression were likely indirect. Copyright © 2016 Elsevier Inc. All rights reserved.
Cdc20 hypomorphic mice fail to counteract de novo synthesis of cyclin B1 in mitosis

PubMed Central

Malureanu, Liviu; Jeganathan, Karthik B.; Jin, Fang; Baker, Darren J.; van Ree, Janine H.; Gullon, Oliver; Chen, Zheyan; Henley, John R.

2010-01-01

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome–microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element–binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset. PMID:20956380
Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

PubMed

Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

2013-03-01

Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.
From overload to failure: what happens inside the myocyte.

PubMed

Harding, S E; Davia, K; Davies, C H; del Monte, F; Money-Kyrle, A R; Poole-Wilson, P A

1998-08-01

To determine whether there is a defect in the surviving muscle cells of the failing human heart, studies have been performed on individual myocytes isolated from normal and failing human myocardium. Myocytes from the failing ventricle contract and relax more slowly, and have a reduced contraction amplitude at physiological (but not low) stimulation frequencies. Slow relaxation is seen irrespective of the aetiology of the heart disease studied, and is more pronounced in myocytes from hypertrophied ventricles. Myocytes from hypertrophied ventricles are larger than normal, but the relaxation deficit is independent of cell size. Beta-adrenoceptor desensitization is evident in myocytes and it varies according to the severity of disease and with the age of the patient. Action potentials are longer in myocytes from failing human heart, probably because of an alteration in K+ current density. Many of the functional changes identified in failing human myocardium are seen at the level of the single cardiac myocyte, which implies that pharmacological or genetic manipulation of surviving cells is a logical therapeutic strategy.
In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas)

USGS Publications Warehouse

Work, Thierry M.; Dagenais, Julie; Balazs, George H.; Schumacher, Joanne; Lewis, Teresa D.; Leong, Jo-Ann C.; Casey, Rufina N.; Casey, James W.

2009-01-01

Fibropapillomatosis (FP) of green turtles has a global distribution and causes debilitating tumours of the skin and internal organs in several species of marine turtles. FP is associated with a presently non-cultivable alphaherpesvirus Chelonid fibropapilloma-associated herpesvirus (CFPHV). Our aims were to employ quantitative PCR targeted to pol DNA of CFPHV to determine (i) if DNA sequesters by tumour size and/or cell type, (ii) whether subculturing of cells is a viable strategy for isolating CFPHV and (iii) whether CFPHV can be induced to a lytic growth cycle in vitro using chemical modulators of replication (CMRs), temperature variation or co-cultivation. Additional objectives included determining whether non-tumour and tumour cells behave differently in vitro and confirming the phenotype of cultured cells using cell-type-specific antigens. CFPHV pol DNA was preferentially concentrated in dermal fibroblasts of skin tumours and the amount of viral DNA per cell was independent of tumour size. Copy number of CFPHV pol DNA per cell rapidly decreased with cell doubling of tumour-derived fibroblasts in culture. Attempts to induce viral replication in known CFPHV-DNA-positive cells using temperature or CMR failed. No significant differences were seen in in vitro morphology or growth characteristics of fibroblasts from tumour cells and paired normal skin, nor from CFPHV pol-DNA-positive intestinal tumour cells. Tumour cells were confirmed as fibroblasts or keratinocytes by positive staining with anti-vimentin and anti-pancytokeratin antibodies, respectively. CFPHV continues to be refractory to in vitro cultivation.
School Crisis Management: A Model of Dynamic Responsiveness to Crisis Life Cycle

ERIC Educational Resources Information Center

Liou, Yi-Hwa

2015-01-01

Purpose: This study aims to analyze a school's crisis management and explore emerging aspects of its response to a school crisis. Traditional linear modes of analysis often fail to address complex crisis situations. The present study applied a dynamic crisis life cycle model that draws on chaos and complexity theory to a crisis management case,…

A map of protein dynamics during cell-cycle progression and cell-cycle exit

PubMed Central

Gookin, Sara; Min, Mingwei; Phadke, Harsha; Chung, Mingyu; Moser, Justin; Miller, Iain; Carter, Dylan

2017-01-01

The cell-cycle field has identified the core regulators that drive the cell cycle, but we do not have a clear map of the dynamics of these regulators during cell-cycle progression versus cell-cycle exit. Here we use single-cell time-lapse microscopy of Cyclin-Dependent Kinase 2 (CDK2) activity followed by endpoint immunofluorescence and computational cell synchronization to determine the temporal dynamics of key cell-cycle proteins in asynchronously cycling human cells. We identify several unexpected patterns for core cell-cycle proteins in actively proliferating (CDK2-increasing) versus spontaneously quiescent (CDK2-low) cells, including Cyclin D1, the levels of which we find to be higher in spontaneously quiescent versus proliferating cells. We also identify proteins with concentrations that steadily increase or decrease the longer cells are in quiescence, suggesting the existence of a continuum of quiescence depths. Our single-cell measurements thus provide a rich resource for the field by characterizing protein dynamics during proliferation versus quiescence. PMID:28892491
Cell division cycle 45 promotes papillary thyroid cancer progression via regulating cell cycle.

PubMed

Sun, Jing; Shi, Run; Zhao, Sha; Li, Xiaona; Lu, Shan; Bu, Hemei; Ma, Xianghua

2017-05-01

Cell division cycle 45 was reported to be overexpressed in some cancer-derived cell lines and was predicted to be a candidate oncogene in cervical cancer. However, the clinical and biological significance of cell division cycle 45 in papillary thyroid cancer has never been investigated. We determined the expression level and clinical significance of cell division cycle 45 using The Cancer Genome Atlas, quantitative real-time polymerase chain reaction, and immunohistochemistry. A great upregulation of cell division cycle 45 was observed in papillary thyroid cancer tissues compared with adjacent normal tissues. Furthermore, overexpression of cell division cycle 45 positively correlates with more advanced clinical characteristics. Silence of cell division cycle 45 suppressed proliferation of papillary thyroid cancer cells via G1-phase arrest and inducing apoptosis. The oncogenic activity of cell division cycle 45 was also confirmed in vivo. In conclusion, cell division cycle 45 may serve as a novel biomarker and a potential therapeutic target for papillary thyroid cancer.
The E7 oncoprotein associates with Mi2 and histone deacetylase activity to promote cell growth.

PubMed

Brehm, A; Nielsen, S J; Miska, E A; McCance, D J; Reid, J L; Bannister, A J; Kouzarides, T

1999-05-04

E7 is the main transforming protein of human papilloma virus type 16 (HPV16) which is implicated in the formation of cervical cancer. The transforming activity of E7 has been attributed to its interaction with the retinoblastoma (Rb) tumour suppressor. However, Rb binding is not sufficient for transformation by E7. Mutations within a zinc finger domain, which is dispensable for Rb binding, also abolish E7 transformation functions. Here we show that HPV16 E7 associates with histone deacetylase in vitro and in vivo, via its zinc finger domain. Using a genetic screen, we identify Mi2beta, a component of the recently identified NURD histone deacetylase complex, as a protein that binds directly to the E7 zinc finger. A zinc finger point mutant which is unable to bind Mi2beta and histone deacetylase but is still able to bind Rb fails to overcome cell cycle arrest in osteosarcoma cells. Our results suggest that the binding to a histone deacetylase complex is an important parameter for the growthpromoting activity of the human papilloma virus E7 protein. This provides the first indication that viral oncoproteins control cell proliferation by targeting deacetylation pathways.
Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis.

PubMed

Hong, Kyung Uk; Choi, Yong-Bock; Lee, Jung-Hwa; Kim, Hyun-Jun; Kwon, Hye-Rim; Seong, Yeon-Sun; Kim, Heung Tae; Park, Joobae; Bae, Chang-Dae; Hong, Kyeong-Man

2008-08-31

Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 recognizes the amino acid sequence between 569 and 625 and that phosphorylation at T596 completely abolishes the reactivity of the antibody, suggesting that the differential reactivity originates from the phosphorylation status at T596. Immunofluorescence staining showed that mAb D-12-3 fails to detect TMAP/CKAP2 in mitotic cells between prophase and metaphase, but the staining becomes evident again in anaphase, suggesting that phosphorylation at T596 occurs transiently during early phases of mitosis. These results suggest that the cellular functions of TMAP/CKAP2 might be regulated by timely phosphorylation and dephosphorylation during the course of mitosis.
Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis

PubMed Central

Hong, Kyung Uk; Choi, Yong-Bock; Lee, Jung-Hwa; Kim, Hyun-Jun; Kwon, Hye-Rim; Seong, Yeon-Sun; Kim, Heung Tae; Park, Joobae

2008-01-01

Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 recognizes the amino acid sequence between 569 and 625 and that phosphorylation at T596 completely abolishes the reactivity of the antibody, suggesting that the differential reactivity originates from the phosphorylation status at T596. Immunofluorescence staining showed that mAb D-12-3 fails to detect TMAP/CKAP2 in mitotic cells between prophase and metaphase, but the staining becomes evident again in anaphase, suggesting that phosphorylation at T596 occurs transiently during early phases of mitosis. These results suggest that the cellular functions of TMAP/CKAP2 might be regulated by timely phosphorylation and dephosphorylation during the course of mitosis. PMID:18779650
Landscape and flux reveal a new global view and physical quantification of mammalian cell cycle

PubMed Central

Li, Chunhe; Wang, Jin

2014-01-01

Cell cycles, essential for biological function, have been investigated extensively. However, enabling a global understanding and defining a physical quantification of the stability and function of the cell cycle remains challenging. Based upon a mammalian cell cycle gene network, we uncovered the underlying Mexican hat landscape of the cell cycle. We found the emergence of three local basins of attraction and two major potential barriers along the cell cycle trajectory. The three local basins of attraction characterize the G1, S/G2, and M phases. The barriers characterize the G1 and S/G2 checkpoints, respectively, of the cell cycle, thus providing an explanation of the checkpoint mechanism for the cell cycle from the physical perspective. We found that the progression of a cell cycle is determined by two driving forces: curl flux for acceleration and potential barriers for deceleration along the cycle path. Therefore, the cell cycle can be promoted (suppressed), either by enhancing (suppressing) the flux (representing the energy input) or by lowering (increasing) the barrier along the cell cycle path. We found that both the entropy production rate and energy per cell cycle increase as the growth factor increases. This reflects that cell growth and division are driven by energy or nutrition supply. More energy input increases flux and decreases barrier along the cell cycle path, leading to faster oscillations. We also identified certain key genes and regulations for stability and progression of the cell cycle. Some of these findings were evidenced from experiments whereas others lead to predictions and potential anticancer strategies. PMID:25228772
Identification of Cell Cycle-Regulated Genes by Convolutional Neural Network.

PubMed

Liu, Chenglin; Cui, Peng; Huang, Tao

2017-01-01

The cell cycle-regulated genes express periodically with the cell cycle stages, and the identification and study of these genes can provide a deep understanding of the cell cycle process. Large false positives and low overlaps are big problems in cell cycle-regulated gene detection. Here, a computational framework called DLGene was proposed for cell cycle-regulated gene detection. It is based on the convolutional neural network, a deep learning algorithm representing raw form of data pattern without assumption of their distribution. First, the expression data was transformed to categorical state data to denote the changing state of gene expression, and four different expression patterns were revealed for the reported cell cycle-regulated genes. Then, DLGene was applied to discriminate the non-cell cycle gene and the four subtypes of cell cycle genes. Its performances were compared with six traditional machine learning methods. At last, the biological functions of representative cell cycle genes for each subtype are analyzed. Our method showed better and more balanced performance of sensitivity and specificity comparing to other machine learning algorithms. The cell cycle genes had very different expression pattern with non-cell cycle genes and among the cell-cycle genes, there were four subtypes. Our method not only detects the cell cycle genes, but also describes its expression pattern, such as when its highest expression level is reached and how it changes with time. For each type, we analyzed the biological functions of the representative genes and such results provided novel insight to the cell cycle mechanisms. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
What's Trust Got to Do with It? A Communications and Engagement Guide for School Leaders Tackling the Problem of Persistently Failing Schools

ERIC Educational Resources Information Center

Johnson, Jean

2011-01-01

The rationale for taking bold action on the nation's persistently failing schools can be summed up in one dramatic and disturbing statistic: half of the young Americans who drop out of high school attend just 12 percent of the nation's schools. Ending the cycle of failure at schools is a daunting challenge and a surprisingly controversial one.…
A fully integrated new paradigm for lithium's mode of action - lithium utilizes latent cellular fail-safe mechanisms.

PubMed

van Woerkom, Arthur Ernst

2017-01-01

It is proposed that lithium's therapeutic effects occur indirectly by augmenting a cascade of protective "fail-safe" pathways pre-configured to activate in response to a dangerous low cell [Mg ++ ] situation, eg, posttraumatic brain injury, alongside relative cell adenosine triphosphate depletion. Lithium activates cell protection, as it neatly mimics a lowered intracellular [Mg ++ ] level.
Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

PubMed

Brewer, B J; Chlebowicz-Sledziewska, E; Fangman, W L

1984-11-01

During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.
Altered myocardial force generation in end-stage human heart failure.

PubMed

Papp, Zoltán; van der Velden, Jolanda; Borbély, Attila; Édes, István; Stienen, Ger J M

2014-12-01

This study aimed to elucidate the molecular background of increased Ca 2+ sensitivity of force production in cardiomyocytes of end-stage human heart failure. Ca 2+ -activated isometric force and the cross-bridge specific rate of force redevelopment (k tr ) were determined in Triton-skinned myocytes from end-stage failing and non-failing donor hearts. Measurements (control: pH 7.2, 0 mM inorganic phosphate (P i )) were performed under test conditions that probed either the Ca 2+ -regulatory function of the thin filaments (pH 6.5), the kinetics of the actin-myosin cross-bridge cycle (10 mM P i ), or both (pH 6.5, 10 mM P i ). The control maximal Ca 2+ -activated force (F o ) and k trmax did not differ between failing and non-failing myocytes. At submaximal [Ca 2+ ], however, both force and k tr were higher in failing than in donor myocytes. The difference in the Ca 2+ sensitivities of force production was preserved when the thin filament regulatory function was perturbed by acidosis (pH 6.5) but was abolished by cross-bridge modulation (i.e. by P i ) both at pH 7.2 and at pH 6.5. P i induced a larger reduction in force but a smaller increase in k tr in the failing myocytes than in the non-failing myocytes at submaximal [Ca 2+ ]. The enhanced P i sensitivity of the actin-myosin interaction suggests that the P i release step of the actin-myosin cross-bridge cycle is modified during end-stage human heart failure. This might be of functional importance when P i accumulates (e.g. during cardiac ischaemia). Moreover, this alteration can influence cardiac energetics and the clinical efficacy of sarcomere targeted agents in human heart failure. © 2015 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.
Hair Follicle Bulge Stem Cells Appear Dispensable for the Acute Phase of Wound Re‐epithelialization

PubMed Central

Garcin, Clare L.; Ansell, David M.; Headon, Denis J.; Paus, Ralf

2016-01-01

Abstract The cutaneous healing response has evolved to occur rapidly, in order to minimize infection and to re‐establish epithelial homeostasis. Rapid healing is achieved through complex coordination of multiple cell types, which importantly includes specific cell populations within the hair follicle (HF). Under physiological conditions, the epithelial compartments of HF and interfollicular epidermis remain discrete, with K15+ve bulge stem cells contributing progeny for HF reconstruction during the hair cycle and as a basis for hair shaft production during anagen. Only upon wounding do HF cells migrate from the follicle to contribute to the neo‐epidermis. However, the identity of the first‐responding cells, and in particular whether this process involves a direct contribution of K15+ve bulge cells to the early stage of epidermal wound repair remains unclear. Here we demonstrate that epidermal injury in murine skin does not induce bulge activation during early epidermal wound repair. Specifically, bulge cells of uninjured HFs neither proliferate nor appear to migrate out of the bulge niche upon epidermal wounding. In support of these observations, Diphtheria toxin‐mediated partial ablation of K15+ve bulge cells fails to delay wound healing. Our data suggest that bulge cells only respond to epidermal wounding during later stages of repair. We discuss that this response may have evolved as a protective safeguarding mechanism against bulge stem cell exhaust and tumorigenesis. Stem Cells 2016;34:1377–1385 PMID:26756547
The Role of Notch Signaling Pathway in Breast Cancer Pathogenesis

DTIC Science & Technology

2005-07-01

breast cancer cells, I tested whether ErbB2 overexpression will cooperate with Notch in HMLE cells. While overexpression of activated Notch1 failed to...tyrosine kinase upstream of Ras normally found overexpressed in many breast cancers , also failed to transform HMLE cells. These observations suggested...cooperation between Notch1IC and ErbB2 signaling in transforming HMLE cells. Breast cancers typically do not harbor oncogenic Ras mutations; nevertheless
The Global Regulatory Architecture of Transcription during the Caulobacter Cell Cycle

PubMed Central

Zhou, Bo; Schrader, Jared M.; Kalogeraki, Virginia S.; Abeliuk, Eduardo; Dinh, Cong B.; Pham, James Q.; Cui, Zhongying Z.; Dill, David L.; McAdams, Harley H.; Shapiro, Lucy

2015-01-01

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5′ RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle. PMID:25569173
Indirect-fired gas turbine dual fuel cell power cycle

DOEpatents

Micheli, Paul L.; Williams, Mark C.; Sudhoff, Frederick A.

1996-01-01

A fuel cell and gas turbine combined cycle system which includes dual fuel cell cycles combined with a gas turbine cycle wherein a solid oxide fuel cell cycle operated at a pressure of between 6 to 15 atms tops the turbine cycle and is used to produce CO.sub.2 for a molten carbonate fuel cell cycle which bottoms the turbine and is operated at essentially atmospheric pressure. A high pressure combustor is used to combust the excess fuel from the topping fuel cell cycle to further heat the pressurized gas driving the turbine. A low pressure combustor is used to combust the excess fuel from the bottoming fuel cell to reheat the gas stream passing out of the turbine which is used to preheat the pressurized air stream entering the topping fuel cell before passing into the bottoming fuel cell cathode. The CO.sub.2 generated in the solid oxide fuel cell cycle cascades through the system to the molten carbonate fuel cell cycle cathode.
The global regulatory architecture of transcription during the Caulobacter cell cycle.

PubMed

Zhou, Bo; Schrader, Jared M; Kalogeraki, Virginia S; Abeliuk, Eduardo; Dinh, Cong B; Pham, James Q; Cui, Zhongying Z; Dill, David L; McAdams, Harley H; Shapiro, Lucy

2015-01-01

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5' RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle.
Measuring cell cycle progression kinetics with metabolic labeling and flow cytometry.

PubMed

Fleisig, Helen; Wong, Judy

2012-05-22

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments. Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.
The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

PubMed

El-Badawy, Ahmed; El-Badri, Nagwa

2016-01-13

The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.
Differential Activation of Cellular DNA Damage Responses by Replication-Defective and Replication-Competent Adenovirus Mutants

PubMed Central

Prakash, Anand; Jayaram, Sumithra

2012-01-01

Adenovirus (Ad) mutants that lack early region 4 (E4) activate the phosphorylation of cellular DNA damage response proteins. In wild-type Ad type 5 (Ad5) infections, E1b and E4 proteins target the cellular DNA repair protein Mre11 for redistribution and degradation, thereby interfering with its ability to activate phosphorylation cascades important during DNA repair. The characteristics of Ad infection that activate cellular DNA repair processes are not yet well understood. We investigated the activation of DNA damage responses by a replication-defective Ad vector (AdRSVβgal) that lacks E1 and fails to produce the immediate-early E1a protein. E1a is important for activating early gene expression from the other viral early transcription units, including E4. AdRSVβgal can deliver its genome to the cell, but it is subsequently deficient for viral early gene expression and DNA replication. We studied the ability of AdRSVβgal-infected cells to induce cellular DNA damage responses. AdRSVβgal infection does activate formation of foci containing the Mdc1 protein. However, AdRSVβgal fails to activate phosphorylation of the damage response proteins Nbs1 and Chk1. We found that viral DNA replication is important for Nbs1 phosphorylation, suggesting that this step in the viral life cycle may provide an important trigger for activating at least some DNA repair proteins. PMID:23015708
Comparative cell cycle transcriptomics reveals synchronization of developmental transcription factor networks in cancer cells

PubMed Central

Johard, Helena; Mahdessian, Diana; Fedr, Radek; Marks, Carolyn; Medalová, Jiřina; Souček, Karel; Lundberg, Emma; Linnarsson, Sten; Bryja, Vítězslav; Sekyrova, Petra; Altun, Mikael; Andäng, Michael

2017-01-01

The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development. PMID:29228002

Safety hazards associated with the charging of lithium/sulfur dioxide cells

NASA Technical Reports Server (NTRS)

Frank, H.; Halpert, G.; Lawson, D. D.; Barnes, J. A.; Bis, R. F.

1986-01-01

A continuing research program to assess the responses of spirally wound, lithium/sulfur dioxide cells to charging as functions of charging current, temperature, and cell condition prior to charging is described. Partially discharged cells that are charged at currents greater than one ampere explode with the time to explosion inversely proportional to the charging current. Cells charged at currents of less than one ampere may fail in one of several modes. The data allows an empirical prediction of when certain cells will fail given a constant charging current.
Experiences of young smokers in quitting smoking in twin cities of Pakistan: a phenomenological study.

PubMed

Shaheen, Kanwal; Oyebode, Oyinlola; Masud, Haleema

2018-04-10

Smoking is highly prevalent in Pakistan claiming the lives of over 100,000 individuals every year. A significant proportion of smokers (24.7%) make an attempt to quit each year but 97.4% fail to quit successfully. Little is known about the reasons for, and experiences of, failed quit attempts. This study was carried out to explore the experiences of young male smokers in quitting smoking in the twin cities of Pakistan METHOD: A qualitative study was carried out using a phenomenological approach in Rawalpindi and Islamabad. A total of 11 participants were interviewed. All study participants were male and had made at least one quit attempt. Study participants were a mix of smokers who failed to quit smoking, intermittent smokers and successful quitters. Streubert's (1991) method of phenomenology was followed during data analysis. The experiences of smokers while smoking "the smoking phase" have major effects on their journey towards quitting smoking. The smoking phase consists of three major stages: contact with initial smoking stimuli, the journey from first puff to enjoying smoking and then finally smoking becoming part of life. However, the journey towards quitting smoking is not as simple as the journey towards becoming a smoker. Instead, smokers get trapped in three overlapping cycles of smoking and quit attempts: smoking & forced quitting, smoking & intentional quitting, and smoking & intermittent smoking before successful quitting. Breaking the cycle is not easy in the presence of trapping factors (addiction, high availability, easy affordability, conducive social setup and low perceived risks of smoking). Three factors play a major role in breaking these cycles which are strong will power, continuous peer support and avoidance of smokers' company. A young smoker, during his experience of quitting smoking gets entrapped in several overlapping cycles of smoking & quit attempts before successful quitting. There are known entrapping factors as well as factors which help in breaking these cycles. Targeted interventions are needed to facilitate smoking cessation among young smokers in Pakistan.
Distinct prophase arrest mechanisms in human male meiosis.

PubMed

Jan, Sabrina Z; Jongejan, Aldo; Korver, Cindy M; van Daalen, Saskia K M; van Pelt, Ans M M; Repping, Sjoerd; Hamer, Geert

2018-04-16

To prevent chromosomal aberrations being transmitted to the offspring, strict meiotic checkpoints are in place to remove aberrant spermatocytes. However, in about 1% of males these checkpoints cause complete meiotic arrest leading to azoospermia and subsequent infertility. Here, we unravel two clearly distinct meiotic arrest mechanisms that occur during prophase of human male meiosis. Type I arrested spermatocytes display severe asynapsis of the homologous chromosomes, disturbed XY-body formation and increased expression of the Y chromosome-encoded gene ZFY and seem to activate a DNA damage pathway leading to induction of p63, possibly causing spermatocyte apoptosis. Type II arrested spermatocytes display normal chromosome synapsis, normal XY-body morphology and meiotic crossover formation but have a lowered expression of several cell cycle regulating genes and fail to silence the X chromosome-encoded gene ZFX Discovery and understanding of these meiotic arrest mechanisms increases our knowledge of how genomic stability is guarded during human germ cell development. © 2018. Published by The Company of Biologists Ltd.
The cell cycle.

PubMed

Singh, N; Lim, R B; Sawyer, M A

2000-07-01

The cell cycle and the cell cycle control system are the engines that drive life. They allow for the processes of cell renewal and the growth of organisms, under controlled conditions. The control system is essential for the monitoring of normal cell growth and replication of genetic material and to ensure that normal, functional daughter cells are produced at completion of each cell cycle. Although certain clinical applications exist which take advantage of the events of the cell cycle, our understanding of its mechanisms and how to manipulate them is infantile. The next decades will continue to see the effort of many researchers focused upon unlocking the mysteries of the cell cycle and the cell cycle control system.
Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage.

PubMed

Solozobova, Valeriya; Rolletschek, Alexandra; Blattner, Christine

2009-06-17

P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. In embryonic stem cells where (anti-proliferative) p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.
p14(ARF) nuclear overexpression in aggressive B-cell lymphomas is a sensor of malfunction of the common tumor suppressor pathways.

PubMed

Sánchez-Aguilera, Abel; Sánchez-Beato, Margarita; García, Juan F; Prieto, Ignacio; Pollan, Marina; Piris, Miguel A

2002-02-15

p14(ARF), the alternative product from the human INK4a/ARF locus, antagonizes Hdm2 and mediates p53 activation in response to oncogenic stimuli. An immunohistochemical study of p14(ARF) expression in 74 samples of aggressive B-cell lymphomas was performed, demonstrating an array of different abnormalities. A distinct nucleolar expression pattern was detected in nontumoral tissue and a subset of lymphomas (50/74). In contrast, a group of cases (8/74) showed absence of p14(ARF) expression, dependent either on promoter hypermethylation or gene loss. Additionally, 16 out of 74 cases displayed an abnormal nuclear p14(ARF) overexpression not confined to the nucleoli, as confirmed by confocal microscopy, and that was associated with high levels of p53 and Hdm2. A genetic study of these cases failed to show any alteration in the p14(ARF) gene, but revealed the presence of p53 mutations in over 50% of these cases. An increased growth fraction and a more aggressive clinical course, with a shortened survival time, also characterized the group of tumors with p14(ARF) nuclear overexpression. Moreover, this p14(ARF) expression pattern was more frequent in tumors displaying accumulated alterations in the p53, p16(INK4a), and p27(KIP1) tumor supressors. These observations, together with the consideration of the central role of p14(ARF) in cell cycle control, suggest that p14(ARF) abnormal nuclear overexpression is a sensor of malfunction of the major cell cycle regulatory pathways, and consequently a marker of a high tumor aggressivity.
Pathological implications of cell cycle re-entry in Alzheimer disease.

PubMed

Bonda, David J; Lee, Hyun-pil; Kudo, Wataru; Zhu, Xiongwei; Smith, Mark A; Lee, Hyoung-gon

2010-06-29

The complex neurodegeneration underlying Alzheimer disease (AD), although incompletely understood, is characterised by an aberrant re-entry into the cell cycle in neurons. Pathological evidence, in the form of cell cycle markers and regulatory proteins, suggests that cell cycle re-entry is an early event in AD, which precedes the formation of amyloid-beta plaques and neurofibrillary tangles (NFTs). Although the exact mechanisms that induce and mediate these cell cycle events in AD are not clear, significant advances have been made in further understanding the pathological role of cell cycle re-entry in AD. Importantly, recent studies indicate that cell cycle re-entry is not a consequence, but rather a cause, of neurodegeneration, suggesting that targeting of cell cycle re-entry may provide an opportunity for therapeutic intervention. Moreover, multiple inducers of cell cycle re-entry and their interactions in AD have been proposed. Here, we review the most recent advances in understanding the pathological implications of cell cycle re-entry in AD.
Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting.

PubMed

Kabani, Sarah; Waterfall, Martin; Matthews, Keith R

2010-01-01

Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase.
Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting

PubMed Central

Kabani, Sarah; Waterfall, Martin; Matthews, Keith R.

2010-01-01

Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase. PMID:19729042
Fail-safe designs for large capacity battery systems

DOEpatents

Kim, Gi-Heon; Smith, Kandler; Ireland, John; Pesaran, Ahmad A.; Neubauer, Jeremy

2016-05-17

Fail-safe systems and design methodologies for large capacity battery systems are disclosed. The disclosed systems and methodologies serve to locate a faulty cell in a large capacity battery, such as a cell having an internal short circuit, determine whether the fault is evolving, and electrically isolate the faulty cell from the rest of the battery, preventing further electrical energy from feeding into the fault.
Fractographic Analysis of High-Cycle Fatgue in Aircraft Engines

DTIC Science & Technology

2000-01-01

and indicated its variation with crack growth distance. 14. SUBJECT TERMS Fractography Failure Analysis Fourier Fracture Surface High-Cycle...hosted a two-day meeting of the project team at their West Palm Beach facility in which they described how they used fractography and discussed their...that failed in service, and very valuable technical advice throughout this program. IX INTRODUCTION Fractography , the art of interpreting markings
Enough is enough! Patients who do not conceive on 600IU/day of gonadotropins show no improvement from an additional 150IU of LH activity

PubMed Central

Maguire, Marcy; Csokmay, John; Segars, James; Payson, Mark; Armstrong, Alicia

2010-01-01

Studies have suggested that supplemental LH improves outcomes in ART cycles. In this retrospective review, an additional 150IU of LH activity did not improve ART outcomes in women undergoing a second round of IVF/ICSI following an initial failed cycle employing 600IU of gonadotropins. PMID:20850732
Facile synthesis of carbon dot and residual carbon nanobeads: Implications for ion sensing, medicinal and biological applications.

PubMed

Gaddam, Rohit Ranganathan; Mukherjee, Sudip; Punugupati, Neelambaram; Vasudevan, D; Patra, Chitta Ranjan; Narayan, Ramanuj; Vsn Kothapalli, Raju

2017-04-01

Synthesis of carbon dots (Cdots) via chemical route involves disintegration of carbon materials into nano-domains, wherein, after extraction of Cdots, the remaining carbon material is discarded. The present work focuses on studying even the leftover carbon residue namely, carbon nanobeads (CNBs) as an equally important material for applications on par with that of carbon dot. It employs oxidative treatment of carbonised gum olibanum resin (GOR) to produce the carbons namely Cdots and CNBs (as the residue). The Cdots (~5-10nm) exhibit blue-green fluorescence with an optical absorption at ~300nm unlike the CNBs (40-50nm) which fail to exhibit fluorescence. The fluorescence behaviour exhibited by Cdots were utilized for heavy metal ion sensing of Pb 2+ , Hg 2+ and Cd 2+ ions in aqueous media. Interestingly, both Cdots and CNBs are biocompatible to normal cell lines but cytotoxic to cancer cell lines, observed during several in vitro experiments (cell viability assay, cell cycle assay, apoptosis assay, ROS determination assay, caspase-9 activity assay). Additionally, Cdots exhibit bright green fluorescence in B16F10 cells. The Cdots and CNB's demonstrate multifunctional activities (sensor, cellular imaging and cancer therapy) in biomedical applications. Copyright © 2016 Elsevier B.V. All rights reserved.
Entry inhibitors: New advances in HCV treatment

PubMed Central

Qian, Xi-Jing; Zhu, Yong-Zhe; Zhao, Ping; Qi, Zhong-Tian

2016-01-01

Hepatitis C virus (HCV) infection affects approximately 3% of the world's population and causes chronic liver diseases, including liver fibrosis, cirrhosis, and hepatocellular carcinoma. Although current antiviral therapy comprising direct-acting antivirals (DAAs) can achieve a quite satisfying sustained virological response (SVR) rate, it is still limited by viral resistance, long treatment duration, combined adverse reactions, and high costs. Moreover, the currently marketed antivirals fail to prevent graft reinfections in HCV patients who receive liver transplantations, probably due to the cell-to-cell transmission of the virus, which is also one of the main reasons behind treatment failure. HCV entry is a highly orchestrated process involving initial attachment and binding, post-binding interactions with host cell factors, internalization, and fusion between the virion and the host cell membrane. Together, these processes provide multiple novel and promising targets for antiviral therapy. Most entry inhibitors target host cell components with high genetic barriers and eliminate viral infection from the very beginning of the viral life cycle. In future, the addition of entry inhibitors to a combination of treatment regimens might optimize and widen the prevention and treatment of HCV infection. This review summarizes the molecular mechanisms and prospects of the current preclinical and clinical development of antiviral agents targeting HCV entry. PMID:26733381
BTK inhibition results in impaired CXCR4 chemokine receptor surface expression, signaling and function in chronic lymphocytic leukemia.

PubMed

Chen, S-S; Chang, B Y; Chang, S; Tong, T; Ham, S; Sherry, B; Burger, J A; Rai, K R; Chiorazzi, N

2016-04-01

Bruton's tyrosine kinase (BTK) is involved in the regulation of B-cell growth, migration and adhesion. The importance of BTK in cell trafficking is emphasized by the clonal contraction proceeded by lymphocytosis typical for the enzyme inhibitor, ibrutinib, in B-cell malignancies, including chronic lymphocytic leukemia (CLL). Here, we investigated BTK regulation of leukemic B-cell trafficking in a mouse model of aggressive TCL1 CLL-like disease. Inhibiting BTK by ibrutinib reduced surface membrane (sm) levels of CXCR4 but not CXCR5, CD49d and other adhesion/homing receptors. Decreased smCXCR4 levels resulted from blocking receptor signal transduction, which in turn aborted cycling from and to the membrane. This resulted in rapid re-distribution of CLL cells from spleens and lymph nodes into the circulation. CLL cells with impaired smCXCR4 from BTK inhibition failed to home to spleens. These functional changes mainly resulted from inhibition of CXCR4 phosphorylation at Ser339, mediated directly by blocking BTK enzymatic activity and indirectly by affecting the function of downstream targets PLCγ2 and PKCμ, and eventually synthesis of PIM-1 and BTK itself. Our data identify CXCR4 as a key regulator in BTK-mediated CLL-cell retention and have elucidated a complex set of not previously described mechanisms responsible for these effects.
Amphiastral Mitotic Spindle Assembly in Vertebrate Cells Lacking Centrosomes

PubMed Central

Hornick, Jessica E.; Mader, Christopher C.; Tribble, Emily K.; Bagne, Cydney C.; Vaughan, Kevin T.; Shaw, Sidney L.; Hinchcliffe, Edward H.

2011-01-01

Summary The role of centrosomes/centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles [1, 2, 3]. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays – termed an amphiaster (“a star on both sides” [4]) – that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB) [5]. Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC re-assembled, and prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split/separate before NEB, and these entered mitosis with persistent monastral spindles. The chromatin-mediated RAN-GTP pathway could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but in its absence, the fidelity of bipolar spindle assembly is highly compromised. PMID:21439826
Transcriptional and posttranscriptional inhibition of HMGCR and PC biosynthesis by geraniol in 2 Hep-G2 cell proliferation linked pathways.

PubMed

Crespo, Rosana; Montero Villegas, Sandra; Abba, Martín C; de Bravo, Margarita G; Polo, Mónica P

2013-06-01

Geraniol, present in the essential oils of many aromatic plants, has in vitro and in vivo antitumor activity against several cell lines. We investigated the effects of geraniol on lipid metabolic pathways involved in Hep-G2 cell proliferation and found that geraniol inhibits the mevalonate pathway, phosphatidylcholine biosynthesis, cell growth, and cell cycle progression (with an arrest occurring at the G0/G1 interphase) and increases apoptosis. The expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting step in cholesterol synthesis, was inhibited at the transcriptional and posttranscriptional levels, as assessed by real-time RT-PCR, Western blots, and [(14)C]HMG-CoA-conversion radioactivity assays. That geraniol decreased cholesterogenesis but increased the incorporation of [(14)C]acetate into other nonsaponifiable metabolites indicated the existence of a second control point between squalene and cholesterol involved in redirecting the flow of cholesterol-derived carbon toward other metabolites of the mevalonate pathway. That exogenous mevalonate failed to restore growth in geraniol-inhibited cells suggests that, in addition to the inhibition of HMGCR, other dose-dependent actions exist through which geraniol can impact the mevalonate pathway and consequently inhibit cell proliferation. These results suggest that geraniol, a nontoxic compound found in many fruits and herbs, exhibits notable potential as a natural agent for combatting cancer and (or) cardiovascular diseases.
Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study.

PubMed

Ebner, Thomas; Montag, Markus; Montag, M; Van der Ven, K; Van der Ven, H; Ebner, T; Shebl, O; Oppelt, P; Hirchenhain, J; Krüssel, J; Maxrath, B; Gnoth, C; Friol, K; Tigges, J; Wünsch, E; Luckhaus, J; Beerkotte, A; Weiss, D; Grunwald, K; Struller, D; Etien, C

2015-04-01

Artificial oocyte activation has been proposed as a suitable means to overcome the problem of failed or impaired fertilization after intracytoplasmic sperm injection (ICSI). In a multicentre setting artificial oocyte activation was applied to 101 patients who were diagnosed with fertilization abnormalities (e.g. less than 50% fertilized oocytes) in a previous conventional ICSI cycle. Female gametes were activated for 15 min immediately after ICSI using a ready-to-use Ca(2+)-ionophore solution (A23187). Fertilization, pregnancy and live birth rates were compared with the preceding cycle without activation. The fertilization rate of 48% in the study cycles was significantly higher compared with the 25% in the control cycles (P < 0.001). Further splitting of the historical control group into failed (0%), low (1-30%) and moderate fertilization rate (31-50%) showed that all groups significantly benefitted (P < 0.001) in the ionophore cycle. Fewer patients had their embryo transfer cancelled compared with their previous treatments (1/101 versus 15/101). In total, 99% of the patients had an improved outcome with A23187 application resulting in a 28% live birth rate (35 babies). These data suggest that artificial oocyte activation using a ready-to-use compound is an efficient method. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Alteration of cell cycle progression by Sindbis virus infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yi, Ruirong; Saito, Kengo; Isegawa, Naohisa

We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Veromore » cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G{sub 1} phase preferred to proliferate during S/G{sub 2} phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G{sub 1} phase than in cells infected during S/G{sub 2} phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases.« less
Quantitative imaging with Fucci and mathematics to uncover temporal dynamics of cell cycle progression.

PubMed

Saitou, Takashi; Imamura, Takeshi

2016-01-01

Cell cycle progression is strictly coordinated to ensure proper tissue growth, development, and regeneration of multicellular organisms. Spatiotemporal visualization of cell cycle phases directly helps us to obtain a deeper understanding of controlled, multicellular, cell cycle progression. The fluorescent ubiquitination-based cell cycle indicator (Fucci) system allows us to monitor, in living cells, the G1 and the S/G2/M phases of the cell cycle in red and green fluorescent colors, respectively. Since the discovery of Fucci technology, it has found numerous applications in the characterization of the timing of cell cycle phase transitions under diverse conditions and various biological processes. However, due to the complexity of cell cycle dynamics, understanding of specific patterns of cell cycle progression is still far from complete. In order to tackle this issue, quantitative approaches combined with mathematical modeling seem to be essential. Here, we review several studies that attempted to integrate Fucci technology and mathematical models to obtain quantitative information regarding cell cycle regulatory patterns. Focusing on the technological development of utilizing mathematics to retrieve meaningful information from the Fucci producing data, we discuss how the combined methods advance a quantitative understanding of cell cycle regulation. © 2015 Japanese Society of Developmental Biologists.

Cell Cycle Control in the Early Embryonic Development of Aquatic Animal Species

PubMed Central

Siefert, Joseph C.; Clowdus, Emily A.; Sansam, Christopher L.

2016-01-01

The cell cycle is integrated with many aspects of embryonic development. Not only is proper control over the pace of cell proliferation important, but also the timing of cell cycle progression is coordinated with transcription, cell migration, and cell differentiation. Due to the ease with which the embryos of aquatic organisms can be observed and manipulated, they have been a popular choice for embryologists throughout history. In the cell cycle field, aquatic organisms have been extremely important because they have played a major role in the discovery and analysis of key regulators of the cell cycle. In particular, the frog Xenopus laevis has been instrumental for understanding how the basic embryonic cell cycle is regulated. More recently, the zebrafish has been used to understand how the cell cycle is remodeled during vertebrate development and how it is regulated during morphogenesis. This review describes how some of the unique strengths of aquatic species have been leveraged for cell cycle research and suggests how species such as Xenopus and zebrafish will continue to reveal the roles of the cell cycle in human biology and disease. PMID:26475527
Cell cycle arrest in the jewel wasp Nasonia vitripennis in larval diapause.

PubMed

Shimizu, Yuta; Mukai, Ayumu; Goto, Shin G

2018-04-01

Insects enter diapause to synchronise their life cycle with biotic and abiotic environmental conditions favourable for their development, reproduction, and survival. One of the most noticeable characteristics of diapause is the blockage of ontogeny. Although this blockage should occur with the cessation of cellular proliferation, i.e. cell cycle arrest, it was confirmed only in a few insect species and information on the molecular pathways involved in cell cycle arrest is limited. In the present study, we investigated developmental and cell cycle arrest in diapause larvae of the jewel wasp Nasonia vitripennis. Developmental and cell cycle arrest occur in the early fourth instar larval stage of N. vitripennis under short days. By entering diapause, the S fraction of the cell cycle disappears and approximately 80% and 20% of cells arrest their cell cycle in the G0/G1 and G2 phases, respectively. We further investigated expression of cell cycle regulatory genes and some housekeeping genes to dissect molecular mechanisms underlying the cell cycle arrest. Copyright © 2016 Elsevier Ltd. All rights reserved.
The oncolytic peptide LTX-315 triggers necrotic cell death

PubMed Central

Forveille, Sabrina; Zhou, Heng; Sauvat, Allan; Bezu, Lucillia; Müller, Kevin; Liu, Peng; Zitvogel, Laurence; Pierron, Gérard; Rekdal, Øystein; Kepp, Oliver; Kroemer, Guido

2015-01-01

The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects. PMID:26566869
Human embryonic stem cells fail to activate CHK1 and commit to apoptosis in response to DNA replication stress.

PubMed

Desmarais, Joëlle A; Hoffmann, Michele J; Bingham, Gregg; Gagou, Mary E; Meuth, Mark; Andrews, Peter W

2012-07-01

Pluripotent cells of the early embryo, to which embryonic stem cells (ESCs) correspond, give rise to all the somatic cells of the developing fetus. Any defects that occur in their genome or epigenome would have devastating consequences. Genetic and epigenetic change in human ESCs appear to be an inevitable consequence of long-term culture, driven by selection of variant cells that have a higher propensity for self-renewal rather than either differentiation or death. Mechanisms underlying the potentially separate events of mutation and subsequent selection of variants are poorly understood. Here, we show that human ESCs and their malignant counterpart, embryonal carcinoma (EC) cells, both fail to activate critical S-phase checkpoints when exposed to DNA replication inhibitors and commit to apoptosis instead. Human ESCs and EC cells also fail to form replication protein A, γH2AX, or RAD51 foci or load topoisomerase (DNA) II binding protein 1 onto chromatin in response to replication inhibitors. Furthermore, direct measurements of single-stranded DNA (ssDNA) show that these cells fail to generate the ssDNA regions in response to replication stress that are necessary for the activation of checkpoints and the initiation of homologous recombination repair to protect replication fork integrity and restart DNA replication. Taken together, our data suggest that pluripotent cells control genome integrity by the elimination of damaged cells through apoptosis rather than DNA repair, and therefore, mutations or epigenetic modifications resulting in an imbalance in cell death control could lead to genetic instability. Copyright © 2012 AlphaMed Press.
Modelling cell cycle synchronisation in networks of coupled radial glial cells.

PubMed

Barrack, Duncan S; Thul, Rüdiger; Owen, Markus R

2015-07-21

Radial glial cells play a crucial role in the embryonic mammalian brain. Their proliferation is thought to be controlled, in part, by ATP mediated calcium signals. It has been hypothesised that these signals act to locally synchronise cell cycles, so that clusters of cells proliferate together, shedding daughter cells in uniform sheets. In this paper we investigate this cell cycle synchronisation by taking an ordinary differential equation model that couples the dynamics of intracellular calcium and the cell cycle and extend it to populations of cells coupled via extracellular ATP signals. Through bifurcation analysis we show that although ATP mediated calcium release can lead to cell cycle synchronisation, a number of other asynchronous oscillatory solutions including torus solutions dominate the parameter space and cell cycle synchronisation is far from guaranteed. Despite this, numerical results indicate that the transient and not the asymptotic behaviour of the system is important in accounting for cell cycle synchronisation. In particular, quiescent cells can be entrained on to the cell cycle via ATP mediated calcium signals initiated by a driving cell and crucially will cycle in near synchrony with the driving cell for the duration of neurogenesis. This behaviour is highly sensitive to the timing of ATP release, with release at the G1/S phase transition of the cell cycle far more likely to lead to near synchrony than release during mid G1 phase. This result, which suggests that ATP release timing is critical to radial glia cell cycle synchronisation, may help us to understand normal and pathological brain development. Copyright © 2015 Elsevier Ltd. All rights reserved.
Unraveling Interfaces between Energy Metabolism and Cell Cycle in Plants.

PubMed

Siqueira, João Antonio; Hardoim, Pablo; Ferreira, Paulo C G; Nunes-Nesi, Adriano; Hemerly, Adriana S

2018-06-19

Oscillation in energy levels is widely variable in dividing and differentiated cells. To synchronize cell proliferation and energy fluctuations, cell cycle-related proteins have been implicated in the regulation of mitochondrial energy-generating pathways in yeasts and animals. Plants have chloroplasts and mitochondria, coordinating the cell energy flow. Recent findings suggest an integrated regulation of these organelles and the nuclear cell cycle. Furthermore, reports indicate a set of interactions between the cell cycle and energy metabolism, coordinating the turnover of proteins in plants. Here, we discuss how cell cycle-related proteins directly interact with energy metabolism-related proteins to modulate energy homeostasis and cell cycle progression. We provide interfaces between cell cycle and energy metabolism-related proteins that could be explored to maximize plant yield. Copyright © 2018 Elsevier Ltd. All rights reserved.
Parasites resistant to the antimalarial atovaquone fail to transmit by mosquitoes.

PubMed

Goodman, Christopher D; Siregar, Josephine E; Mollard, Vanessa; Vega-Rodríguez, Joel; Syafruddin, Din; Matsuoka, Hiroyuki; Matsuzaki, Motomichi; Toyama, Tomoko; Sturm, Angelika; Cozijnsen, Anton; Jacobs-Lorena, Marcelo; Kita, Kiyoshi; Marzuki, Sangkot; McFadden, Geoffrey I

2016-04-15

Drug resistance compromises control of malaria. Here, we show that resistance to a commonly used antimalarial medication, atovaquone, is apparently unable to spread. Atovaquone pressure selects parasites with mutations in cytochrome b, a respiratory protein with low but essential activity in the mammalian blood phase of the parasite life cycle. Resistance mutations rescue parasites from the drug but later prove lethal in the mosquito phase, where parasites require full respiration. Unable to respire efficiently, resistant parasites fail to complete mosquito development, arresting their life cycle. Because cytochrome b is encoded by the maternally inherited parasite mitochondrion, even outcrossing with wild-type strains cannot facilitate spread of resistance. Lack of transmission suggests that resistance will be unable to spread in the field, greatly enhancing the utility of atovaquone in malaria control. Copyright © 2016, American Association for the Advancement of Science.
A fully integrated new paradigm for lithium’s mode of action – lithium utilizes latent cellular fail-safe mechanisms

PubMed Central

van Woerkom, Arthur Ernst

2017-01-01

It is proposed that lithium’s therapeutic effects occur indirectly by augmenting a cascade of protective “fail-safe” pathways pre-configured to activate in response to a dangerous low cell [Mg++] situation, eg, posttraumatic brain injury, alongside relative cell adenosine triphosphate depletion. Lithium activates cell protection, as it neatly mimics a lowered intracellular [Mg++] level. PMID:28203080
Triggered intracellular calcium waves in dog and human left atrial myocytes from normal and failing hearts.

PubMed

Aistrup, Gary L; Arora, Rishi; Grubb, Søren; Yoo, Shin; Toren, Benjamin; Kumar, Manvinder; Kunamalla, Aaron; Marszalec, William; Motiwala, Tej; Tai, Shannon; Yamakawa, Sean; Yerrabolu, Satya; Alvarado, Francisco J; Valdivia, Hector H; Cordeiro, Jonathan M; Shiferaw, Yohannes; Wasserstrom, John Andrew

2017-11-01

Abnormal intracellular Ca2+ cycling contributes to triggered activity and arrhythmias in the heart. We investigated the properties and underlying mechanisms for systolic triggered Ca2+ waves in left atria from normal and failing dog hearts. Intracellular Ca2+ cycling was studied using confocal microscopy during rapid pacing of atrial myocytes (36 °C) isolated from normal and failing canine hearts (ventricular tachypacing model). In normal atrial myocytes (NAMs), Ca2+ waves developed during rapid pacing at rates ≥ 3.3 Hz and immediately disappeared upon cessation of pacing despite high sarcoplasmic reticulum (SR) load. In heart failure atrial myocytes (HFAMs), triggered Ca2+ waves (TCWs) developed at a higher incidence at slower rates. Because of their timing, TCW development relies upon action potential (AP)-evoked Ca2+ entry. The distribution of Ca2+ wave latencies indicated two populations of waves, with early events representing TCWs and late events representing conventional spontaneous Ca2+ waves. Latency analysis also demonstrated that TCWs arise after junctional Ca2+ release has occurred and spread to non-junctional (cell core) SR. TCWs also occurred in intact dog atrium and in myocytes from humans and pigs. β-adrenergic stimulation increased Ca2+ release and abolished TCWs in NAMs but was ineffective in HFAMs making this a potentially effective adaptive mechanism in normals but potentially arrhythmogenic in HF. Block of Ca-calmodulin kinase II also abolished TCWs, suggesting a role in TCW formation. Pharmacological manoeuvres that increased Ca2+ release suppressed TCWs as did interventions that decreased Ca2+ release but these also severely reduced excitation-contraction coupling. TCWs develop during the atrial AP and thus could affect AP duration, producing repolarization gradients and creating a substrate for reentry, particularly in HF where they develop at slower rates and a higher incidence. TCWs may represent a mechanism for the initiation of atrial fibrillation particularly in HF. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions please email: journals.permissions@oup.com.
Coordination of Myeloid Differentiation with Reduced Cell Cycle Progression by PU.1 Induction of MicroRNAs Targeting Cell Cycle Regulators and Lipid Anabolism.

PubMed

Solomon, Lauren A; Podder, Shreya; He, Jessica; Jackson-Chornenki, Nicholas L; Gibson, Kristen; Ziliotto, Rachel G; Rhee, Jess; DeKoter, Rodney P

2017-05-15

During macrophage development, myeloid progenitor cells undergo terminal differentiation coordinated with reduced cell cycle progression. Differentiation of macrophages from myeloid progenitors is accompanied by increased expression of the E26 transformation-specific transcription factor PU.1. Reduced PU.1 expression leads to increased proliferation and impaired differentiation of myeloid progenitor cells. It is not understood how PU.1 coordinates macrophage differentiation with reduced cell cycle progression. In this study, we utilized cultured PU.1-inducible myeloid cells to perform genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis coupled with gene expression analysis to determine targets of PU.1 that may be involved in regulating cell cycle progression. We found that genes encoding cell cycle regulators and enzymes involved in lipid anabolism were directly and inducibly bound by PU.1 although their steady-state mRNA transcript levels were reduced. Inhibition of lipid anabolism was sufficient to reduce cell cycle progression in these cells. Induction of PU.1 reduced expression of E2f1 , an important activator of genes involved in cell cycle and lipid anabolism, indirectly through microRNA 223. Next-generation sequencing identified microRNAs validated as targeting cell cycle and lipid anabolism for downregulation. These results suggest that PU.1 coordinates cell cycle progression with differentiation through induction of microRNAs targeting cell cycle regulators and lipid anabolism. Copyright © 2017 American Society for Microbiology.
Compliance with post-operative adjuvant chemotherapy in non-small cell lung cancer. An analysis of National Cancer Institute of Canada and intergroup trial JBR.10 and a review of the literature.

PubMed

Alam, Naveed; Shepherd, Frances A; Winton, Timothy; Graham, Barbara; Johnson, David; Livingston, Robert; Rigas, James; Whitehead, Marlo; Ding, Keyue; Seymour, Lesley

2005-03-01

Resected non-small cell lung cancer (NSCLC) has 5-years survival rates of 30-70%. The role of adjuvant chemotherapy remains unclear with poor compliance reported in most trials. The compliance with adjuvant chemotherapy (ACT) for stage IB and II NSCLC was analyzed using data from a North American multi-centre phase III study (accrual 1994-2001) that compared adjuvant chemotherapy to observation. Planned chemotherapy consisted of cisplatin (CIS) 50 mg/m2 days 1, 8 and vinorelbine (VIN) 25 mg/m2 days 1, 8, 15, 22 for four cycles; the VIN dose had been reduced from 30 mg/m2 after an initial cohort of patients experienced unacceptable toxicity. Four hundred and twenty-four patients were randomized after the amendment, 215 to the chemotherapy arm. Median age was 60 years, 64% were male and 84% had stage II disease. Thirty-seven patients completed one cycle, 14 completed two, 20 completed three and 108 patients completed all four cycles. Ten patients received no therapy. Multivariate analysis demonstrated statistically significant differences in compliance with extent of surgery, gender and age. Patients randomized in Canada were more likely to fail to complete chemotherapy due to refusal of therapy than their American counterparts. Patients who had pneumonectomies were more likely to discontinue therapy due to toxicity than those who had lesser resections. Extent of surgery may play a role in both the compliance and toxicity of ACT. Differences between nations in the perception of the risks and benefits of adjuvant chemotherapy regimens, both between physicians and patients, should be investigated further.
Testing of molded high temperature plastic actuator road seals for use in advanced aircraft hydraulic systems

NASA Technical Reports Server (NTRS)

Waterman, A. W.; Huxford, R. L.; Nelson, W. G.

1976-01-01

Molded high temperature plastic first and second stage rod seal elements were evaluated in seal assemblies to determine performance characteristics. These characteristics were compared with the performance of machined seal elements. The 6.35 cm second stage Chevron seal assembly was tested using molded Chevrons fabricated from five molding materials. Impulse screening tests conducted over a range of 311 K to 478 K revealed thermal setting deficiencies in the aromatic polyimide molding materials. Seal elements fabricated from aromatic copolyester materials structurally failed during impulse cycle calibration. Endurance testing of 3.85 million cycles at 450 K using MIL-H-83283 fluid showed poorer seal performance with the unfilled aromatic polyimide material than had been attained with seals machined from Vespel SP-21 material. The 6.35 cm first stage step-cut compression loaded seal ring fabricated from copolyester injection molding material failed structurally during impulse cycle calibration. Molding of complex shape rod seals was shown to be a potentially controllable technique, but additional molding material property testing is recommended.
Cell cycle-related metabolism and mitochondrial dynamics in a replication-competent pancreatic beta-cell line.

PubMed

Montemurro, Chiara; Vadrevu, Suryakiran; Gurlo, Tatyana; Butler, Alexandra E; Vongbunyong, Kenny E; Petcherski, Anton; Shirihai, Orian S; Satin, Leslie S; Braas, Daniel; Butler, Peter C; Tudzarova, Slavica

2017-01-01

Cell replication is a fundamental attribute of growth and repair in multicellular organisms. Pancreatic beta-cells in adults rarely enter cell cycle, hindering the capacity for regeneration in diabetes. Efforts to drive beta-cells into cell cycle have so far largely focused on regulatory molecules such as cyclins and cyclin-dependent kinases (CDKs). Investigations in cancer biology have uncovered that adaptive changes in metabolism, the mitochondrial network, and cellular Ca 2+ are critical for permitting cells to progress through the cell cycle. Here, we investigated these parameters in the replication-competent beta-cell line INS 832/13. Cell cycle synchronization of this line permitted evaluation of cell metabolism, mitochondrial network, and cellular Ca 2+ compartmentalization at key cell cycle stages. The mitochondrial network is interconnected and filamentous at G1/S but fragments during the S and G2/M phases, presumably to permit sorting to daughter cells. Pyruvate anaplerosis peaks at G1/S, consistent with generation of biomass for daughter cells, whereas mitochondrial Ca 2+ and respiration increase during S and G2/M, consistent with increased energy requirements for DNA and lipid synthesis. This synchronization approach may be of value to investigators performing live cell imaging of Ca 2+ or mitochondrial dynamics commonly undertaken in INS cell lines because without synchrony widely disparate data from cell to cell would be expected depending on position within cell cycle. Our findings also offer insight into why replicating beta-cells are relatively nonfunctional secreting insulin in response to glucose. They also provide guidance on metabolic requirements of beta-cells for the transition through the cell cycle that may complement the efforts currently restricted to manipulating cell cycle to drive beta-cells through cell cycle.
A Short-Term Advantage for Syngamy in the Origin of Eukaryotic Sex: Effects of Cell Fusion on Cell Cycle Duration and Other Effects Related to the Duration of the Cell Cycle-Relationship between Cell Growth Curve and the Optimal Size of the Species, and Circadian Cell Cycle in Photosynthetic Unicellular Organisms.

PubMed

Mancebo Quintana, J M; Mancebo Quintana, S

2012-01-01

The origin of sex is becoming a vexatious issue for Evolutionary Biology. Numerous hypotheses have been proposed, based on the genetic effects of sex, on trophic effects or on the formation of cysts and syncytia. Our approach addresses the change in cell cycle duration which would cause cell fusion. Several results are obtained through graphical and mathematical analysis and computer simulations. (1) In poor environments, cell fusion would be an advantageous strategy, as fusion between cells of different size shortens the cycle of the smaller cell (relative to the asexual cycle), and the majority of mergers would occur between cells of different sizes. (2) The easiest-to-evolve regulation of cell proliferation (sexual/asexual) would be by modifying the checkpoints of the cell cycle. (3) A regulation of this kind would have required the existence of the G2 phase, and sex could thus be the cause of the appearance of this phase. Regarding cell cycle, (4) the exponential curve is the only cell growth curve that has no effect on the optimal cell size in unicellular species; (5) the existence of a plateau with no growth at the end of the cell cycle explains the circadian cell cycle observed in unicellular algae.
A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells

PubMed Central

Jarrin, Miguel; Pandit, Tanushree; Gunhaga, Lena

2012-01-01

In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals. PMID:22718906
Transcription Factor Binding Profiles Reveal Cyclic Expression of Human Protein-coding Genes and Non-coding RNAs

PubMed Central

Cheng, Chao; Ung, Matthew; Grant, Gavin D.; Whitfield, Michael L.

2013-01-01

Cell cycle is a complex and highly supervised process that must proceed with regulatory precision to achieve successful cellular division. Despite the wide application, microarray time course experiments have several limitations in identifying cell cycle genes. We thus propose a computational model to predict human cell cycle genes based on transcription factor (TF) binding and regulatory motif information in their promoters. We utilize ENCODE ChIP-seq data and motif information as predictors to discriminate cell cycle against non-cell cycle genes. Our results show that both the trans- TF features and the cis- motif features are predictive of cell cycle genes, and a combination of the two types of features can further improve prediction accuracy. We apply our model to a complete list of GENCODE promoters to predict novel cell cycle driving promoters for both protein-coding genes and non-coding RNAs such as lincRNAs. We find that a similar percentage of lincRNAs are cell cycle regulated as protein-coding genes, suggesting the importance of non-coding RNAs in cell cycle division. The model we propose here provides not only a practical tool for identifying novel cell cycle genes with high accuracy, but also new insights on cell cycle regulation by TFs and cis-regulatory elements. PMID:23874175
Cell Cycle Control by PTEN.

PubMed

Brandmaier, Andrew; Hou, Sheng-Qi; Shen, Wen H

2017-07-21

Continuous and error-free chromosome inheritance through the cell cycle is essential for genomic stability and tumor suppression. However, accumulation of aberrant genetic materials often causes the cell cycle to go awry, leading to malignant transformation. In response to genotoxic stress, cells employ diverse adaptive mechanisms to halt or exit the cell cycle temporarily or permanently. The intrinsic machinery of cycling, resting, and exiting shapes the cellular response to extrinsic stimuli, whereas prevalent disruption of the cell cycle machinery in tumor cells often confers resistance to anticancer therapy. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and a guardian of the genome that is frequently mutated or deleted in human cancer. Moreover, it is increasingly evident that PTEN deficiency disrupts the fundamental processes of genetic transmission. Cells lacking PTEN exhibit cell cycle deregulation and cell fate reprogramming. Here, we review the role of PTEN in regulating the key processes in and out of cell cycle to optimize genomic integrity. Copyright © 2017 Elsevier Ltd. All rights reserved.
Pharmacological inhibition of myostatin/TGF-β receptor/pSmad3 signaling rescues muscle regenerative responses in mouse model of type 1 diabetes.

PubMed

Jeong, Jaemin; Conboy, Michael J; Conboy, Irina M

2013-08-01

To study the influence of acute experimental diabetes on the regenerative potential of muscle stem (satellite) cells in mice. Male C57BL/6 young mice were injected with a single dose of streptozotocin (STZ, 180 mg/kg, ip) to induce diabetes. The diabetic mice were treated with insulin (0.75 U/kg, ip), follistatin (12 μg/kg, im) or Alk5 inhibitor (5 μmol/L per kg, sc) once a day. On the first day when high glucose levels were found, cardiotoxin (CTX) was focally injected into tibialis anterior and gastronemius muscles of the mice. The muscles were harvested 3 d and 5 d after CTX injection, and myofibers and satellite cells were isolated. Quantitative ex-vivo and in-vivo assays of myogenic potential were used to evaluate the muscle regenerative responses. The satellite cells from the diabetic mice 3 d after CTX injection fail to activate, and the repair of muscle deteriorates, resembling that observed in old control mice. Furthermore, the satellite cells have excessive levels of myostatin, TGF-β receptor 1, pSmad3 and the cell cycle inhibitor p15, while the level of TGF-β1 remain unchanged. Treatment of the diabetic mice with insulin rescued muscle regenerative responses, and restored the expression levels of myostatin, TGF-β receptor 1, pSmad3, and p15 to those similar of healthy controls. Treatment of the diabetic mice with the myostatin antagonist follistatin, or with the Alk5 inhibitor of TGF-β receptor 1 (which did not diminish the blood glucose levels) rescued muscle regenerative responses and attenuated the myostatin/TGFβ receptor/pSmad3 signaling. The muscle regenerative responses are incapacitated and repair of the tissue fails within hours after the initiation of hyperglycemia in a mouse model of type 1 diabetes, but stem cell function is rescued by insulin, as well as follistatin or an Alk5 inhibitor that blocks TGF-β receptor signaling.
Benefits of utilizing CellProfiler as a characterization tool for U–10Mo nuclear fuel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collette, R.; Douglas, J.; Patterson, L.

2015-07-15

Automated image processing techniques have the potential to aid in the performance evaluation of nuclear fuels by eliminating judgment calls that may vary from person-to-person or sample-to-sample. Analysis of in-core fuel performance is required for design and safety evaluations related to almost every aspect of the nuclear fuel cycle. This study presents a methodology for assessing the quality of uranium–molybdenum fuel images and describes image analysis routines designed for the characterization of several important microstructural properties. The analyses are performed in CellProfiler, an open-source program designed to enable biologists without training in computer vision or programming to automatically extract cellularmore » measurements from large image sets. The quality metric scores an image based on three parameters: the illumination gradient across the image, the overall focus of the image, and the fraction of the image that contains scratches. The metric presents the user with the ability to ‘pass’ or ‘fail’ an image based on a reproducible quality score. Passable images may then be characterized through a separate CellProfiler pipeline, which enlists a variety of common image analysis techniques. The results demonstrate the ability to reliably pass or fail images based on the illumination, focus, and scratch fraction of the image, followed by automatic extraction of morphological data with respect to fission gas voids, interaction layers, and grain boundaries. - Graphical abstract: Display Omitted - Highlights: • A technique is developed to score U–10Mo FIB-SEM image quality using CellProfiler. • The pass/fail metric is based on image illumination, focus, and area scratched. • Automated image analysis is performed in pipeline fashion to characterize images. • Fission gas void, interaction layer, and grain boundary coverage data is extracted. • Preliminary characterization results demonstrate consistency of the algorithm.« less
Agile Walking Robot

NASA Technical Reports Server (NTRS)

Larimer, Stanley J.; Lisec, Thomas R.; Spiessbach, Andrew J.; Waldron, Kenneth J.

1990-01-01

Proposed agile walking robot operates over rocky, sandy, and sloping terrain. Offers stability and climbing ability superior to other conceptual mobile robots. Equipped with six articulated legs like those of insect, continually feels ground under leg before applying weight to it. If leg sensed unexpected object or failed to make contact with ground at expected point, seeks alternative position within radius of 20 cm. Failing that, robot halts, examines area around foot in detail with laser ranging imager, and replans entire cycle of steps for all legs before proceeding.

Hair Follicle Bulge Stem Cells Appear Dispensable for the Acute Phase of Wound Re-epithelialization.

PubMed

Garcin, Clare L; Ansell, David M; Headon, Denis J; Paus, Ralf; Hardman, Matthew J

2016-05-01

The cutaneous healing response has evolved to occur rapidly, in order to minimize infection and to re-establish epithelial homeostasis. Rapid healing is achieved through complex coordination of multiple cell types, which importantly includes specific cell populations within the hair follicle (HF). Under physiological conditions, the epithelial compartments of HF and interfollicular epidermis remain discrete, with K15(+ve) bulge stem cells contributing progeny for HF reconstruction during the hair cycle and as a basis for hair shaft production during anagen. Only upon wounding do HF cells migrate from the follicle to contribute to the neo-epidermis. However, the identity of the first-responding cells, and in particular whether this process involves a direct contribution of K15(+ve) bulge cells to the early stage of epidermal wound repair remains unclear. Here we demonstrate that epidermal injury in murine skin does not induce bulge activation during early epidermal wound repair. Specifically, bulge cells of uninjured HFs neither proliferate nor appear to migrate out of the bulge niche upon epidermal wounding. In support of these observations, Diphtheria toxin-mediated partial ablation of K15(+ve) bulge cells fails to delay wound healing. Our data suggest that bulge cells only respond to epidermal wounding during later stages of repair. We discuss that this response may have evolved as a protective safeguarding mechanism against bulge stem cell exhaust and tumorigenesis. Stem Cells 2016;34:1377-1385. © 2016 The Authors. Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.
The therapeutic potential of cell cycle targeting in multiple myeloma.

PubMed

Maes, Anke; Menu, Eline; Veirman, Kim De; Maes, Ken; Vand Erkerken, Karin; De Bruyne, Elke

2017-10-27

Proper cell cycle progression through the interphase and mitosis is regulated by coordinated activation of important cell cycle proteins (including cyclin-dependent kinases and mitotic kinases) and several checkpoint pathways. Aberrant activity of these cell cycle proteins and checkpoint pathways results in deregulation of cell cycle progression, which is one of the key hallmarks of cancer. Consequently, intensive research on targeting these cell cycle regulatory proteins identified several candidate small molecule inhibitors that are able to induce cell cycle arrest and even apoptosis in cancer cells. Importantly, several of these cell cycle regulatory proteins have also been proposed as therapeutic targets in the plasma cell malignancy multiple myeloma (MM). Despite the enormous progress in the treatment of MM the past 5 years, MM still remains most often incurable due to the development of drug resistance. Deregulated expression of the cyclins D is observed in virtually all myeloma patients, emphasizing the potential therapeutic interest of cyclin-dependent kinase inhibitors in MM. Furthermore, other targets have also been identified in MM, such as microtubules, kinesin motor proteins, aurora kinases, polo-like kinases and the anaphase promoting complex/cyclosome. This review will provide an overview of the cell cycle proteins and checkpoint pathways deregulated in MM and discuss the therapeutic potential of targeting proteins or protein complexes involved in cell cycle control in MM.
Do patients with a failed metal-on-metal hip implant with a pseudotumor present differences in their peripheral blood lymphocyte subpopulations?

PubMed

Catelas, Isabelle; Lehoux, Eric A; Hurda, Ian; Baskey, Stephen J; Gala, Luca; Foster, Ryan; Kim, Paul R; Beaulé, Paul E

2015-12-01

Early adverse tissue reactions around metal-on-metal (MoM) hip replacements, especially pseudotumors, are a major concern. Because the causes and pathomechanisms of these pseudotumors remain largely unknown, clinical monitoring of patients with MoM bearings is challenging. The purpose of this study was to compare the lymphocyte subpopulations in peripheral blood from patients with a failed MoM hip implant with and without a pseudotumor and patients with a well-functioning MoM hip implant without a pseudotumor. Potential differences in the systemic immune response are expected to reflect local differences in the periprosthetic tissues. Consenting patients who underwent a revision of a failed MoM hip implant at The Ottawa Hospital (TOH) from 2011 to 2014, or presented with a well-functioning MoM hip implant for a postoperative clinical followup at TOH from 2012 to 2013, were recruited for this study, unless they met any of the exclusion criteria (including diagnosed conditions that can affect peripheral blood lymphocyte subpopulations). Patients with a failed implant were divided into two groups: those with a pseudotumor (two hip resurfacings and five total hip arthroplasties [THAs]) and those without a pseudotumor (10 hip resurfacings and two THAs). Patients with a well-functioning MoM hip implant (nine resurfacings and three THAs) at 5 or more years postimplantation and who did not have a pseudotumor as demonstrated sonographically served as the control group. Peripheral blood subpopulations of T cells (specifically T helper [Th] and cytotoxic T [Tc]), B cells, natural killer (NK) cells, memory T and B cells as well as type 1 (expressing interferon-γ) and type 2 (expressing interleukin-4) Th and Tc cells were analyzed by flow cytometry after immunostaining. Serum concentrations of cobalt and chromium were measured by inductively coupled plasma-mass spectrometry. The mean percentages of total memory T cells and, specifically, memory Th and memory Tc cells were lower in patients with a failed MoM hip implant with a pseudotumor than in both patients with a failed implant without a pseudotumor and patients with a well-functioning implant without a pseudotumor (memory Th cells: 29% ± 5% [means ± SD] versus 55% ± 17%, d = 1.8, 95% confidence interval [CI] [1.2, 2.5] and versus 48% ± 14%, d = 1.6, 95% CI [1.0, 2.2], respectively; memory Tc cells: 18% ± 5% versus 45% ± 14%, d = 2.3, 95% CI [1.5, 3.1] and versus 41% ± 12%, d = 2.3, 95% CI [1.5, 3.1], respectively; p < 0.001 in all cases). The mean percentage of memory B cells was also lower in patients with a failed MoM hip implant with a pseudotumor than in patients with a well-functioning implant without a pseudotumor (12% ± 8% versus 29% ± 16%, d = 1.3, 95% CI [0.7, 1.8], p = 0.025). In addition, patients with a failed MoM hip implant with a pseudotumor had overall lower percentages of type 1 Th cells than both patients with a failed implant without a pseudotumor and patients with a well-functioning implant without a pseudotumor (5.5% [4.9%-5.8%] [median with interquartile range] versus 8.7% [6.5%-10.2%], d = 1.4, 95% CI [0.8, 2.0] and versus 9.6% [6.4%-11.1%], d = 1.6, 95% CI [1.0, 2.2], respectively; p ≤ 0.010 in both cases). Finally, serum cobalt concentrations in patients with a failed MoM hip implant with a pseudotumor were overall higher than those in patients with a well-functioning implant without a pseudotumor (5.8 µg/L [2.9-17.0 µg/L] versus 0.9 µg/L [0.6-1.3 µg/L], d = 2.2, 95% CI [1.4, 2.9], p < 0.001). Overall, results suggest the presence of a type IV hypersensitivity reaction, with a predominance of type 1 Th cells, in patients with a failed MoM hip implant with a pseudotumor. The lower percentages of memory T cells (specifically Th and Tc) as well as type 1 Th cells in peripheral blood of patients with a failed MoM hip implant with a pseudotumor could potentially become diagnostic biomarkers for the detection of pseudotumors. Although implant design (hip resurfacing or THA) did not seem to affect the results, as suggested by the scatter of the data with respect to this parameter, future studies with additional patients could include the analysis of implant design in addition to correlations with histological analyses of specific Th subsets in periprosthetic tissues.
Effect of cycling on the lithium/electrolyte interface in organic electrolytes

NASA Technical Reports Server (NTRS)

Surampudi, S.; Shen, D. H.; Huang, C.-K.; Narayanan, S. R.; Attia, A.; Halpert, G.; Peled, E.

1993-01-01

Nondestructive methods such as ac impedance spectroscopy and microcalorimetry are used to study the effect of cell cycling on the lithium/electrolyte interface. The reactivity of both uncycled and cycled lithium towards various electrolytes is examined by measuring the heat evolved from the cells under open-circuit conditions at 25 C by microcalorimetry. Cycled cells at the end of charge/discharge exhibited considerably higher heat output compared with the uncycled cells. After 30 d of storage, the heat output of the cycled cells is similar to that of the uncycled cells. The cell internal resistance increases with cycling, and this is attributed to the degradation of the electrolyte with cycling.
Temporal fluxomics reveals oscillations in TCA cycle flux throughout the mammalian cell cycle.

PubMed

Ahn, Eunyong; Kumar, Praveen; Mukha, Dzmitry; Tzur, Amit; Shlomi, Tomer

2017-11-06

Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal-fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse-chase LC-MS-based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine-derived fluxes: Oxidation of glucose-derived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.
Playing with the cell cycle to build the spinal cord.

PubMed

Molina, Angie; Pituello, Fabienne

2017-12-01

A fundamental issue in nervous system development and homeostasis is to understand the mechanisms governing the balance between the maintenance of proliferating progenitors versus their differentiation into post-mitotic neurons. Accumulating data suggest that the cell cycle and core regulators of the cell cycle machinery play a major role in regulating this fine balance. Here, we focus on the interplay between the cell cycle and cellular and molecular events governing spinal cord development. We describe the existing links between the cell cycle and interkinetic nuclear migration (INM). We show how the different morphogens patterning the neural tube also regulate the cell cycle machinery to coordinate proliferation and patterning. We give examples of how cell cycle core regulators regulate transcriptionally, or post-transcriptionally, genes involved in controlling the maintenance versus the differentiation of neural progenitors. Finally, we describe the changes in cell cycle kinetics occurring during neural tube patterning and at the time of neuronal differentiation, and we discuss future research directions to better understand the role of the cell cycle in cell fate decisions. Copyright © 2017 Elsevier Inc. All rights reserved.
Cell cycle proteins as promising targets in cancer therapy.

PubMed

Otto, Tobias; Sicinski, Piotr

2017-01-27

Cancer is characterized by uncontrolled tumour cell proliferation resulting from aberrant activity of various cell cycle proteins. Therefore, cell cycle regulators are considered attractive targets in cancer therapy. Intriguingly, animal models demonstrate that some of these proteins are not essential for proliferation of non-transformed cells and development of most tissues. By contrast, many cancers are uniquely dependent on these proteins and hence are selectively sensitive to their inhibition. After decades of research on the physiological functions of cell cycle proteins and their relevance for cancer, this knowledge recently translated into the first approved cancer therapeutic targeting of a direct regulator of the cell cycle. In this Review, we focus on proteins that directly regulate cell cycle progression (such as cyclin-dependent kinases (CDKs)), as well as checkpoint kinases, Aurora kinases and Polo-like kinases (PLKs). We discuss the role of cell cycle proteins in cancer, the rationale for targeting them in cancer treatment and results of clinical trials, as well as the future therapeutic potential of various cell cycle inhibitors.
Cell cycle nucleic acids, polypeptides and uses thereof

DOEpatents

Gordon-Kamm, William J [Urbandale, IA; Lowe, Keith S [Johnston, IA; Larkins, Brian A [Tucson, AZ; Dilkes, Brian R [Tucson, AZ; Sun, Yuejin [Westfield, IN

2007-08-14

The invention provides isolated nucleic acids and their encoded proteins that are involved in cell cycle regulation. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering cell cycle protein content, cell cycle progression, cell number and/or composition of plants.
Quantitative Cell Cycle Analysis Based on an Endogenous All-in-One Reporter for Cell Tracking and Classification.

PubMed

Zerjatke, Thomas; Gak, Igor A; Kirova, Dilyana; Fuhrmann, Markus; Daniel, Katrin; Gonciarz, Magdalena; Müller, Doris; Glauche, Ingmar; Mansfeld, Jörg

2017-05-30

Cell cycle kinetics are crucial to cell fate decisions. Although live imaging has provided extensive insights into this relationship at the single-cell level, the limited number of fluorescent markers that can be used in a single experiment has hindered efforts to link the dynamics of individual proteins responsible for decision making directly to cell cycle progression. Here, we present fluorescently tagged endogenous proliferating cell nuclear antigen (PCNA) as an all-in-one cell cycle reporter that allows simultaneous analysis of cell cycle progression, including the transition into quiescence, and the dynamics of individual fate determinants. We also provide an image analysis pipeline for automated segmentation, tracking, and classification of all cell cycle phases. Combining the all-in-one reporter with labeled endogenous cyclin D1 and p21 as prime examples of cell-cycle-regulated fate determinants, we show how cell cycle and quantitative protein dynamics can be simultaneously extracted to gain insights into G1 phase regulation and responses to perturbations. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
Interplay between cancer cell cycle and metabolism: Challenges, targets and therapeutic opportunities.

PubMed

Roy, Debmalya; Sheng, Gao Ying; Herve, Semukunzi; Carvalho, Evandro; Mahanty, Arpan; Yuan, Shengtao; Sun, Li

2017-05-01

A growing interest has emerged in the field of studying the cross-talk between cancer cell cycle and metabolism. In this review, we aimed to present how metabolism and cell cycle are correlated and how cancer cells get energy to drive cell cycle. Cell proliferation and cell death largely depend on the metabolic activity of the cell. Cell cycle proteins, e.g. cyclin D, cyclin dependent kinase (CDK), some pro-apoptotic and anti-apoptotic proteins, and P53 have been shown to be regulated by metabolic crosstalk. Dysregulation of this cross-talk between metabolism and cell cycle leads to degenerative disorder(s) and cancer. It is not fully understood the actual reason of aberration between metabolism and cell cycle, but it is a hallmark of cancer research. Herein, we discussed the role of some regulatory molecules relative of cell cycle and metabolism and highlight how they control the function of each other. We also pointed out, current therapeutic opportunities and some additional crucial therapeutic targets on these fields that could be a breakthrough in cancer research. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight cells. An update

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1991-01-01

An update of validation test results confirming the breakthrough in LEO cycle life of nickel-hydrogen cells containing 26 percent potassium hydroxide (KOH) electrolyte is presented. A breakthrough in the LEO cycle life of individual pressure vessel nickel-hydrogen cells is reported. The cycle life of boiler plate cells containing 26 percent KOH electrolyte was about 40,000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH.
Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight cells - An update

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1991-01-01

An update of validation test results confirming the breakthrough in LEO cycle life of nickel-hydrogen cells containing 26 percent potassium hydroxide (KOH) electrolyte is presented. A breakthrough in the LEO cycle life of individual pressure vessel nickel-hydrogen cells is reported. The cycle life of boiler plate cells containing 26 percent KOH electrolyte was about 40,000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH.
Effect of LEO cycling on 125 Ah advanced design IPV nickel-hydrogen flight cells - An update

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1991-01-01

An update of validation test results confirming the breakthrough in LEO cycle life of nickel-hydrogen cells containing 26 percent potassium hydroxide (KOH) electrolyte is presented. A breakthrough in the LEO cycle life of individual pressure vessel nickel-hydrogen cells is reported. The cycle life of boiler plate cells containing 26 percent KOH electrolyte was about 40,000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH.
Exploring the Underlying Mechanisms of the Xenopus laevis Embryonic Cell Cycle.

PubMed

Zhang, Kun; Wang, Jin

2018-05-31

The cell cycle is an indispensable process in proliferation and development. Despite significant efforts, global quantification and physical understanding are still challenging. In this study, we explored the mechanisms of the Xenopus laevis embryonic cell cycle by quantifying the underlying landscape and flux. We uncovered the Mexican hat landscape of the Xenopus laevis embryonic cell cycle with several local basins and barriers on the oscillation path. The local basins characterize the different phases of the Xenopus laevis embryonic cell cycle, and the local barriers represent the checkpoints. The checkpoint mechanism of the cell cycle is revealed by the landscape basins and barriers. While landscape shape determines the stabilities of the states on the oscillation path, the curl flux force determines the stability of the cell cycle flow. Replication is fundamental for biology of living cells. We quantify the input energy (through the entropy production) as the thermodynamic requirement for initiation and sustainability of single cell life (cell cycle). Furthermore, we also quantify curl flux originated from the input energy as the dynamical requirement for the emergence of a new stable phase (cell cycle). This can provide a new quantitative insight for the origin of single cell life. In fact, the curl flux originated from the energy input or nutrition supply determines the speed and guarantees the progression of the cell cycle. The speed of the cell cycle is a hallmark of cancer. We characterized the quality of the cell cycle by the coherence time and found it is supported by the flux and energy cost. We are also able to quantify the degree of time irreversibility by the cross correlation function forward and backward in time from the stochastic traces in the simulation or experiments, providing a way for the quantification of the time irreversibility and the flux. Through global sensitivity analysis upon landscape and flux, we can identify the key elements for controlling the cell cycle speed. This can help to design an effective strategy for drug discovery against cancer.
Cell-cycle control in the face of damage--a matter of life or death.

PubMed

Clarke, Paul R; Allan, Lindsey A

2009-03-01

Cells respond to DNA damage or defects in the mitotic spindle by activating checkpoints that arrest the cell cycle. Alternatively, damaged cells can undergo cell death by the process of apoptosis. The correct balance between these pathways is important for the maintenance of genomic integrity while preventing unnecessary cell death. Although the molecular mechanisms of the cell cycle and apoptosis have been elucidated, the links between them have not been clear. Recent work, however, indicates that common components directly link the regulation of apoptosis with cell-cycle checkpoints operating during interphase, whereas in mitosis, the control of apoptosis is directly coupled to the cell-cycle machinery. These findings shed new light on how the balance between cell-cycle progression and cell death is controlled.
The cell cycle of early mammalian embryos: lessons from genetic mouse models.

PubMed

Artus, Jérôme; Babinet, Charles; Cohen-Tannoudji, Michel

2006-03-01

Genes coding for cell cycle components predicted to be essential for its regulation have been shown to be dispensable in mice, at the whole organism level. Such studies have highlighted the extraordinary plasticity of the embryonic cell cycle and suggest that many aspects of in vivo cell cycle regulation remain to be discovered. Here, we discuss the particularities of the mouse early embryonic cell cycle and review the mutations that result in cell cycle defects during mouse early embryogenesis, including deficiencies for genes of the cyclin family (cyclin A2 and B1), genes involved in cell cycle checkpoints (Mad2, Bub3, Chk1, Atr), genes involved in ubiquitin and ubiquitin-like pathways (Uba3, Ubc9, Cul1, Cul3, Apc2, Apc10, Csn2) as well as genes the function of which had not been previously ascribed to cell cycle regulation (Cdc2P1, E4F and Omcg1).
Model-Based Analysis of Cell Cycle Responses to Dynamically Changing Environments

PubMed Central

Seaton, Daniel D; Krishnan, J

2016-01-01

Cell cycle progression is carefully coordinated with a cell’s intra- and extracellular environment. While some pathways have been identified that communicate information from the environment to the cell cycle, a systematic understanding of how this information is dynamically processed is lacking. We address this by performing dynamic sensitivity analysis of three mathematical models of the cell cycle in Saccharomyces cerevisiae. We demonstrate that these models make broadly consistent qualitative predictions about cell cycle progression under dynamically changing conditions. For example, it is shown that the models predict anticorrelated changes in cell size and cell cycle duration under different environments independently of the growth rate. This prediction is validated by comparison to available literature data. Other consistent patterns emerge, such as widespread nonmonotonic changes in cell size down generations in response to parameter changes. We extend our analysis by investigating glucose signalling to the cell cycle, showing that known regulation of Cln3 translation and Cln1,2 transcription by glucose is sufficient to explain the experimentally observed changes in cell cycle dynamics at different glucose concentrations. Together, these results provide a framework for understanding the complex responses the cell cycle is capable of producing in response to dynamic environments. PMID:26741131
A dual-color marker system for in vivo visualization of cell cycle progression in Arabidopsis.

PubMed

Yin, Ke; Ueda, Minako; Takagi, Hitomi; Kajihara, Takehiro; Sugamata Aki, Shiori; Nobusawa, Takashi; Umeda-Hara, Chikage; Umeda, Masaaki

2014-11-01

Visualization of the spatiotemporal pattern of cell division is crucial to understand how multicellular organisms develop and how they modify their growth in response to varying environmental conditions. The mitotic cell cycle consists of four phases: S (DNA replication), M (mitosis and cytokinesis), and the intervening G1 and G2 phases; however, only G2/M-specific markers are currently available in plants, making it difficult to measure cell cycle duration and to analyze changes in cell cycle progression in living tissues. Here, we developed another cell cycle marker that labels S-phase cells by manipulating Arabidopsis CDT1a, which functions in DNA replication origin licensing. Truncations of the CDT1a coding sequence revealed that its carboxy-terminal region is responsible for proteasome-mediated degradation at late G2 or in early mitosis. We therefore expressed this region as a red fluorescent protein fusion protein under the S-specific promoter of a histone 3.1-type gene, HISTONE THREE RELATED2 (HTR2), to generate an S/G2 marker. Combining this marker with the G2/M-specific CYCB1-GFP marker enabled us to visualize both S to G2 and G2 to M cell cycle stages, and thus yielded an essential tool for time-lapse imaging of cell cycle progression. The resultant dual-color marker system, Cell Cycle Tracking in Plant Cells (Cytrap), also allowed us to identify root cells in the last mitotic cell cycle before they entered the endocycle. Our results demonstrate that Cytrap is a powerful tool for in vivo monitoring of the plant cell cycle, and thus for deepening our understanding of cell cycle regulation in particular cell types during organ development. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.
A Short-Term Advantage for Syngamy in the Origin of Eukaryotic Sex: Effects of Cell Fusion on Cell Cycle Duration and Other Effects Related to the Duration of the Cell Cycle—Relationship between Cell Growth Curve and the Optimal Size of the Species, and Circadian Cell Cycle in Photosynthetic Unicellular Organisms

PubMed Central

Mancebo Quintana, J. M.; Mancebo Quintana, S.

2012-01-01

The origin of sex is becoming a vexatious issue for Evolutionary Biology. Numerous hypotheses have been proposed, based on the genetic effects of sex, on trophic effects or on the formation of cysts and syncytia. Our approach addresses the change in cell cycle duration which would cause cell fusion. Several results are obtained through graphical and mathematical analysis and computer simulations. (1) In poor environments, cell fusion would be an advantageous strategy, as fusion between cells of different size shortens the cycle of the smaller cell (relative to the asexual cycle), and the majority of mergers would occur between cells of different sizes. (2) The easiest-to-evolve regulation of cell proliferation (sexual/asexual) would be by modifying the checkpoints of the cell cycle. (3) A regulation of this kind would have required the existence of the G2 phase, and sex could thus be the cause of the appearance of this phase. Regarding cell cycle, (4) the exponential curve is the only cell growth curve that has no effect on the optimal cell size in unicellular species; (5) the existence of a plateau with no growth at the end of the cell cycle explains the circadian cell cycle observed in unicellular algae. PMID:22666626
Cell Cycle Regulation of Stem Cells by MicroRNAs.

PubMed

Mens, Michelle M J; Ghanbari, Mohsen

2018-06-01

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules involved in the regulation of gene expression. They are involved in the fine-tuning of fundamental biological processes such as proliferation, differentiation, survival and apoptosis in many cell types. Emerging evidence suggests that miRNAs regulate critical pathways involved in stem cell function. Several miRNAs have been suggested to target transcripts that directly or indirectly coordinate the cell cycle progression of stem cells. Moreover, previous studies have shown that altered expression levels of miRNAs can contribute to pathological conditions, such as cancer, due to the loss of cell cycle regulation. However, the precise mechanism underlying miRNA-mediated regulation of cell cycle in stem cells is still incompletely understood. In this review, we discuss current knowledge of miRNAs regulatory role in cell cycle progression of stem cells. We describe how specific miRNAs may control cell cycle associated molecules and checkpoints in embryonic, somatic and cancer stem cells. We further outline how these miRNAs could be regulated to influence cell cycle progression in stem cells as a potential clinical application.

Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster.

PubMed

Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura

2016-07-01

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed D: rosophila, R: bf, E: 2F A: nd M: yb/ M: ulti-vulva class B: (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. Copyright © 2016 by the Genetics Society of America.
Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster

PubMed Central

Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura

2016-01-01

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo. However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed Drosophila, Rbf, E2F and Myb/Multi-vulva class B (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. PMID:27184390
Scratch2 prevents cell cycle re-entry by repressing miR-25 in postmitotic primary neurons.

PubMed

Rodríguez-Aznar, Eva; Barrallo-Gimeno, Alejandro; Nieto, M Angela

2013-03-20

During the development of the nervous system the regulation of cell cycle, differentiation, and survival is tightly interlinked. Newly generated neurons must keep cell cycle components under strict control, as cell cycle re-entry leads to neuronal degeneration and death. However, despite their relevance, the mechanisms controlling this process remain largely unexplored. Here we show that Scratch2 is involved in the control of the cell cycle in neurons in the developing spinal cord of the zebrafish embryo. scratch2 knockdown induces postmitotic neurons to re-enter mitosis. Scratch2 prevents cell cycle re-entry by maintaining high levels of the cycle inhibitor p57 through the downregulation of miR-25. Thus, Scratch2 appears to safeguard the homeostasis of postmitotic primary neurons by preventing cell cycle re-entry.
An extensive program of periodic alternative splicing linked to cell cycle progression

PubMed Central

Dominguez, Daniel; Tsai, Yi-Hsuan; Weatheritt, Robert; Wang, Yang; Blencowe, Benjamin J; Wang, Zefeng

2016-01-01

Progression through the mitotic cell cycle requires periodic regulation of gene function at the levels of transcription, translation, protein-protein interactions, post-translational modification and degradation. However, the role of alternative splicing (AS) in the temporal control of cell cycle is not well understood. By sequencing the human transcriptome through two continuous cell cycles, we identify ~1300 genes with cell cycle-dependent AS changes. These genes are significantly enriched in functions linked to cell cycle control, yet they do not significantly overlap genes subject to periodic changes in steady-state transcript levels. Many of the periodically spliced genes are controlled by the SR protein kinase CLK1, whose level undergoes cell cycle-dependent fluctuations via an auto-inhibitory circuit. Disruption of CLK1 causes pleiotropic cell cycle defects and loss of proliferation, whereas CLK1 over-expression is associated with various cancers. These results thus reveal a large program of CLK1-regulated periodic AS intimately associated with cell cycle control. DOI: http://dx.doi.org/10.7554/eLife.10288.001 PMID:27015110
Analysis of growth of tetraploid nuclei in roots of Vicia faba.

PubMed

Bansal, J; Davidson, D

1978-03-01

Growth of nuclei of a marked population of cells was determined from G1 to prophase in roots of Vicia faba. The cells were marked by inducing them to become tetraploid by treatment with 0.002% colchicine for 1 hr. Variation in nuclear volume is large; it is established in early G1 and maintained through interphase and into prophase. One consequence of this variation is that there is considerable overlap between volumes of nuclei of different ages in the cell cycle; nuclear volume, we suggest, cannot be used as an accurate indicator of the age of the cell in its growth cycle. Nuclei exhibit considerable variation in their growth rate through the cell cycle. Of the marked population of cells, about 65% had completed a cell cycle 14--15 hr after they were formed. These tetraploid nuclei have a cell cycle duration similar to that of fast cycling diploid cells of the same roots. Since they do complete a cell cycle, at least 65% of the nuclei studied must come from rapidly proliferating cells, showing that variability in nuclear volumes must be present in growing cells and cannot be attributed solely to the presence, in our samples, of non-cycling cells.
SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

PubMed Central

Siu, Fai Y.; Spanggord, Richard J.; Doudna, Jennifer A.

2007-01-01

The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRP-receptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo. Using both biochemical and genetic experiments, we show here that SRP RNA enhances GTPase activity of the SRP–receptor complex above a critical threshold required for cell viability. Furthermore, this stimulation is a property of the SRP RNA tetraloop. SRP RNA tetraloop mutants that confer defective growth phenotypes can assemble into SRP–receptor complexes, but fail to stimulate GTP hydrolysis in these complexes in vitro. Tethered hydroxyl radical probing data reveal that specific positioning of the RNA tetraloop within the SRP–receptor complex is required to stimulate GTPase activity to a level sufficient to support cell growth. These results explain why no external GAP is needed and why the phylogenetically conserved SRP RNA tetraloop is required in vivo. PMID:17164479
Polo-like kinase 1 is essential for early embryonic development and tumor suppression.

PubMed

Lu, Lin-Yu; Wood, Jamie L; Minter-Dykhouse, Katherine; Ye, Lin; Saunders, Thomas L; Yu, Xiaochun; Chen, Junjie

2008-11-01

Polo-like kinases (Plks) are serine/threonine kinases that are highly conserved in organisms from yeasts to humans. Previous reports have shown that Plk1 is critical for all stages of mitosis and may play a role in DNA replication during S phase. While much work has focused on Plk1, little is known about the physiological function of Plk1 in vivo. To address this question, we generated Plk1 knockout mice. Plk1 homozygous null mice were embryonic lethal, and early Plk1(-/-) embryos failed to survive after the eight-cell stage. Immunocytochemistry studies revealed that Plk1-null embryos were arrested outside the mitotic phase, suggesting that Plk1 is important for proper cell cycle progression. It has been postulated that Plk1 is a potential oncogene, due to its overexpression in a variety of tumors and tumor cell lines. While the Plk1 heterozygotes were healthy at birth, the incidence of tumors in these animals was threefold greater than that in their wild-type counterparts, demonstrating that the loss of one Plk1 allele accelerates tumor formation. Collectively, our data support that Plk1 is important for early embryonic development and may function as a haploinsufficient tumor suppressor.
Control of complex networks requires both structure and dynamics

NASA Astrophysics Data System (ADS)

Gates, Alexander J.; Rocha, Luis M.

2016-04-01

The study of network structure has uncovered signatures of the organization of complex systems. However, there is also a need to understand how to control them; for example, identifying strategies to revert a diseased cell to a healthy state, or a mature cell to a pluripotent state. Two recent methodologies suggest that the controllability of complex systems can be predicted solely from the graph of interactions between variables, without considering their dynamics: structural controllability and minimum dominating sets. We demonstrate that such structure-only methods fail to characterize controllability when dynamics are introduced. We study Boolean network ensembles of network motifs as well as three models of biochemical regulation: the segment polarity network in Drosophila melanogaster, the cell cycle of budding yeast Saccharomyces cerevisiae, and the floral organ arrangement in Arabidopsis thaliana. We demonstrate that structure-only methods both undershoot and overshoot the number and which sets of critical variables best control the dynamics of these models, highlighting the importance of the actual system dynamics in determining control. Our analysis further shows that the logic of automata transition functions, namely how canalizing they are, plays an important role in the extent to which structure predicts dynamics.
Synthetic activation of caspases: Artificial death switches

PubMed Central

MacCorkle, Rebecca A.; Freeman, Kevin W.; Spencer, David M.

1998-01-01

The development of safe vectors for gene therapy requires fail-safe mechanisms to terminate therapy or remove genetically altered cells. The ideal “suicide switch” would be nonimmunogenic and nontoxic when uninduced and able to trigger cell death independent of tissue type or cell cycle stage. By using chemically induced dimerization, we have developed powerful death switches based on the cysteine proteases, caspase-1 ICE (interleukin-1β converting enzyme) and caspase-3 YAMA. In both cases, aggregation of the target protein is achieved by a nontoxic lipid-permeable dimeric FK506 analog that binds to the attached FK506-binding proteins, FKBPs. We find that intracellular cross-linking of caspase-1 or caspase-3 is sufficient to trigger rapid apoptosis in a Bcl-xL-independent manner, suggesting that these conditional proapoptotic molecules can bypass intracellular checkpoint genes, such as Bcl-xL, that limit apoptosis. Because these chimeric molecules are derived from autologous proteins, they should be nonimmunogenic and thus ideal for long-lived gene therapy vectors. These properties should also make chemically induced apoptosis useful for developmental studies, for treating hyperproliferative disorders, and for developing animal models to a wide variety of diseases. PMID:9520421
Flow cytometry analysis of cell cycle and specific cell synchronization with butyrate

USDA-ARS?s Scientific Manuscript database

Synchronized cells have been invaluable in many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in MDBK cells. The possibility of using butyrate-blocked cells to obtain synchronized cells was explored and the properties of butyrate-induced cell ...
Cryogenic Absorption Cells Operating Inside a Bruker IFS-125HR: First Results for 13CH4 at 7 Micrometers

NASA Technical Reports Server (NTRS)

Sung, K.; Mantz, A. W.; Smith, M. A. H.; Brown, L. R.; Crawford, T. J.; Devi, V. M.; Benner, D. C.

2010-01-01

New absorption cells designed specifically to achieve stable temperatures down to 66 K inside the sample compartment of an evacuated Bruker IFS-125HR Fourier transform spectrometer (FTS) were developed at Connecticut College and tested at the Jet Propulsion Laboratory (JPL). The temperature stabilized cryogenic cells with path lengths of 24.29 and 20.38 cm were constructed of oxygen free high conductivity (OFHC) copper and fitted with wedged ZnSe windows using vacuum tight indium seals. In operation, the temperature-controlled cooling by a closed-cycle helium refrigerator achieved stability of 0.01 K. The unwanted absorption features arising from cryodeposits on the cell windows at low temperatures were eliminated by building an internal vacuum shroud box around the cell which significantly minimized the growth of cryodeposits. The effects of vibrations from the closed-cycle helium refrigerator on the FTS spectra were characterized. Using this set up, several high-resolution spectra of methane isotopologues broadened with nitrogen were recorded in the 1200-1800 per centimeter spectral region at various sample temperatures between 79.5 and 296 K. Such data are needed to characterize the temperature dependence of spectral line shapes at low temperatures for remote sensing of outer planets and their moons. Initial analysis of a limited number of spectra in the region of the R(2) manifold of the v4 fundamental band of 13CH4 indicated that an empirical power law used for the temperature dependence of the N2-broadened line widths would fail to fit the observed data in the entire temperature range from 80 to 296 K; instead, it follows a temperature-dependence similar to that reported by Mondelain et al. [17,18]. The initial test was very successful proving that a high precision Fourier transform spectrometer with a completely evacuated optical path can be configured for spectroscopic studies at low temperatures relevant to the planetary atmospheres.
Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight battery cells

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1990-01-01

A breakthrough in low earth orbit (LEO) cycle life of individual pressure vessel (IPV) nickel hydrogen battery cells was reported. The cycle life of boiler plate cells containing 26 percent potassium hydroxide (KOH) electrolyte was about 40,000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH. The effect of KOH concentration on cycle life was studied. The cycle regime was a stressful accelerated LEO, which consisted of a 27.5 min charge followed by a 17.5 min charge (2 x normal rate). The depth of discharge (DOD) was 80 percent. The cell temperature was maintained at 23 C. The next step is to validate these results using flight hardware and a real time LEO test. NASA Lewis has a contract with the Naval Weapons Support Center (NWSC), Crane, Indiana, to validate the boiler plate test results. Six 48 A-hr Hughes recirculation design IPV nickel-hydrogen flight battery cells are being evaluated. Three of the cells contain 26 percent KOH (test cells) and three contain 31 percent KOH (control cells). They are undergoing real time LEO cycle life testing. The cycle regime is a 90-min LEO orbit consisting of a 54-min charge followed by a 36-min discharge. The depth-of-discharge is 80 percent. The cell temperature is maintained at 10 C. The cells were cycled for over 8000 cycles in the continuing test. There were no failures for the cells containing 26 percent KOH. There was two failures, however, for the cells containing 31 percent KOH.
Effect of KOH concentration on LEO cycle life of IPV nickel-hydrogen flight battery cells

NASA Technical Reports Server (NTRS)

Smithrick, John J.; Hall, Stephen W.

1990-01-01

A breakthrough in the low-earth-orbit (LEO) cycle life of individual pressure vessel (IPV) nickel hydrogen battery cells is reported. The cycle life of boiler plate cells containing 26 percent potassium hydroxide (KOH) electrolyte was about 40,000 LEO cycles compared to 3500 cycles for cells containing 31 percent KOH. The effect of KOH concentration on cycle life was studied. The cycle regime was a stressful accelerated LEO, which consisted of a 27.5 min charge followed by a 17.5 min charge (2 x normal rate). The depth of discharge (DOD) was 80 percent. The cell temperature was maintained at 23 C. The next step is to validate these results using flight hardware and real time LEO test. NASA Lewis has a contract with the Naval Weapons Support Center (NWSC), Crane, Indiana to validate the boiler plate test results. Six 48 A-hr Hughes recirculation design IPV nickel-hydrogen flight battery cells are being evaluated. Three of the cells contain 26 percent KOH (test cells) and three contain 31 percent KOH (control cells). They are undergoing real time LEO cycle life testing. The cycle regime is a 90-min LEO orbit consisting of a 54-min charge followed by a 36-min discharge. The depth-of-discharge is 80 percent. The cell temperature is maintained at 10 C. The cells were cycled for over 8000 cycles in the continuing test. There were no failures for the cells containing 26 percent KOH. There were two failures, however, for the cells containing 31 percent KOH.
Perivascular epithelial cell tumor (PEComa) of the pancreas

PubMed Central

Zhang, Shuisheng; Chen, Fang; Huang, Xiaozhun; Jiang, Qinglong; Zhao, Yajie; Chen, Yingtai; Zhang, Jianwei; Ma, Jie; Yuan, Wei; Xu, Quan; Zhao, Jiuda; Wang, Chengfeng

2017-01-01

Abstract Rationale: Perivascular epithelial cell tumors (PEComas) of the pancreas are rare mesenchymal tumors and, to our knowledge, only 20 cases have been reported to date. Patient concerns: We report a 43-year-old female who presented with upper abdominal pain for 1 year. She underwent an exploratory laparotomy at a local hospital, which failed to resect the tumor. Five months later, she came to the Chinese National Cancer Center for surgery. Preoperative imaging revealed an 11.5-cm-sized mass located in the head of the pancreas. At the microscopic level, the tumor was composed of epithelioid and spindle cells possessing clear to focally granular eosinophilic cytoplasm, which grew in a nested and alveolar pattern around blood vessels. The tumor cells showed immunoreactivity for human melanoma black 45 (HMB-45), but did not express epithelial or endocrine markers. Diagnoses: Pancreatic PEComa. Interventions: Pancreaticoduodenectomy, partial hepatectomy, and vascular replacement were performed. After the surgery, the patient received 4 cycles of chemotherapy. Outcomes: The patient is free of recurrence and metastasis 1.5 years after surgical resection. Lessons: PEComa should be recognized as a preoperative differential diagnosis of pancreatic tumors. For treatment, removal of the tumor should be attempted, and in the case of tumors with malignant tendencies, the addition of chemotherapy should be considered. PMID:28562565
tRNAs promote nuclear import of HIV-1 intracellular reverse transcription complexes.

PubMed

Zaitseva, Lyubov; Myers, Richard; Fassati, Ariberto

2006-10-01

Infection of non-dividing cells is a biological property of HIV-1 crucial for virus transmission and AIDS pathogenesis. This property depends on nuclear import of the intracellular reverse transcription and pre-integration complexes (RTCs/PICs). To identify cellular factors involved in nuclear import of HIV-1 RTCs, cytosolic extracts were fractionated by chromatography and import activity examined by the nuclear import assay. A near-homogeneous fraction was obtained, which was active in inducing nuclear import of purified and labeled RTCs. The active fraction contained tRNAs, mostly with defective 3' CCA ends. Such tRNAs promoted HIV-1 RTC nuclear import when synthesized in vitro. Active tRNAs were incorporated into and recovered from virus particles. Mutational analyses indicated that the anticodon loop mediated binding to the viral complex whereas the T-arm may interact with cellular factors involved in nuclear import. These tRNA species efficiently accumulated into the nucleus on their own in a energy- and temperature-dependent way. An HIV-1 mutant containing MLV gag did not incorporate tRNA species capable of inducing HIV-1 RTC nuclear import and failed to infect cell cycle-arrested cells. Here we provide evidence that at least some tRNA species can be imported into the nucleus of human cells and promote HIV-1 nuclear import.
Perivascular epithelial cell tumor (PEComa) of the pancreas: A case report and review of literature.

PubMed

Zhang, Shuisheng; Chen, Fang; Huang, Xiaozhun; Jiang, Qinglong; Zhao, Yajie; Chen, Yingtai; Zhang, Jianwei; Ma, Jie; Yuan, Wei; Xu, Quan; Zhao, Jiuda; Wang, Chengfeng

2017-06-01

Perivascular epithelial cell tumors (PEComas) of the pancreas are rare mesenchymal tumors and, to our knowledge, only 20 cases have been reported to date. We report a 43-year-old female who presented with upper abdominal pain for 1 year. She underwent an exploratory laparotomy at a local hospital, which failed to resect the tumor. Five months later, she came to the Chinese National Cancer Center for surgery. Preoperative imaging revealed an 11.5-cm-sized mass located in the head of the pancreas. At the microscopic level, the tumor was composed of epithelioid and spindle cells possessing clear to focally granular eosinophilic cytoplasm, which grew in a nested and alveolar pattern around blood vessels. The tumor cells showed immunoreactivity for human melanoma black 45 (HMB-45), but did not express epithelial or endocrine markers. Pancreatic PEComa. Pancreaticoduodenectomy, partial hepatectomy, and vascular replacement were performed. After the surgery, the patient received 4 cycles of chemotherapy. The patient is free of recurrence and metastasis 1.5 years after surgical resection. PEComa should be recognized as a preoperative differential diagnosis of pancreatic tumors. For treatment, removal of the tumor should be attempted, and in the case of tumors with malignant tendencies, the addition of chemotherapy should be considered.
The E7 oncoprotein associates with Mi2 and histone deacetylase activity to promote cell growth.

PubMed Central

Brehm, A; Nielsen, S J; Miska, E A; McCance, D J; Reid, J L; Bannister, A J; Kouzarides, T

1999-01-01

E7 is the main transforming protein of human papilloma virus type 16 (HPV16) which is implicated in the formation of cervical cancer. The transforming activity of E7 has been attributed to its interaction with the retinoblastoma (Rb) tumour suppressor. However, Rb binding is not sufficient for transformation by E7. Mutations within a zinc finger domain, which is dispensable for Rb binding, also abolish E7 transformation functions. Here we show that HPV16 E7 associates with histone deacetylase in vitro and in vivo, via its zinc finger domain. Using a genetic screen, we identify Mi2beta, a component of the recently identified NURD histone deacetylase complex, as a protein that binds directly to the E7 zinc finger. A zinc finger point mutant which is unable to bind Mi2beta and histone deacetylase but is still able to bind Rb fails to overcome cell cycle arrest in osteosarcoma cells. Our results suggest that the binding to a histone deacetylase complex is an important parameter for the growthpromoting activity of the human papilloma virus E7 protein. This provides the first indication that viral oncoproteins control cell proliferation by targeting deacetylation pathways. PMID:10228159
Dynamic changes to survivin subcellular localization are initiated by DNA damage

PubMed Central

Asumen, Maritess Gay; Ifeacho, Tochukwu V; Cockerham, Luke; Pfandl, Christina; Wall, Nathan R

2010-01-01

Subcellular distribution of the apoptosis inhibitor survivin and its ability to relocalize as a result of cell cycle phase or therapeutic insult has led to the hypothesis that these subcellular pools may coincide with different survivin functions. The PIK kinases (ATM, ATR and DNA-PK) phosphorylate a variety of effector substrates that propagate DNA damage signals, resulting in various biological outputs. Here we demonstrate that subcellular repartitioning of survivin in MCF-7 cells as a result of UV light-mediated DNA damage is dependent upon DNA damage-sensing proteins as treatment with the pan PIK kinase inhibitor wortmannin repartitioned survivin in the mitochondria and diminished it from the cytosol and nucleus. Mitochondrial redistribution of survivin, such as was recorded after wortmannin treatment, occurred in cells lacking any one of the three DNA damage sensing protein kinases: DNA-PK, ATM or ATR. However, failed survivin redistribution from the mitochondria in response to low-dose UV occurred only in the cells lacking ATM, implying that ATM may be the primary kinase involved in this process. Taken together, this data implicates survivian’s subcellular distribution is a dynamic physiological process that appears responsive to UV light-initiated DNA damage and that its distribution may be responsible for its multifunctionality. PMID:20856848
Requirement for the Murine Zinc Finger Protein ZFR in Perigastrulation Growth and Survival

PubMed Central

Meagher, Madeleine J.; Braun, Robert E.

2001-01-01

The transition from preimplantation to postimplantation development leads to the initiation of complex cellular differentiation and morphogenetic movements, a dramatic decrease in cell cycle length, and a commensurate increase in the size of the embryo. Accompanying these changes is the need for the transfer of nutrients from the mother to the embryo and the elaboration of sophisticated genetic networks that monitor genomic integrity and the homeostatic control of cellular growth, differentiation, and programmed cell death. To determine the function of the murine zinc finger protein ZFR in these events, we generated mice carrying a null mutation in the gene encoding it. Homozygous mutant embryos form normal-appearing blastocysts that implant and initiate the process of gastrulation. Mutant embryos form mesoderm but they are delayed in their development and fail to form normal anterior embryonic structures. Loss of ZFR function leads to both an increase in programmed cell death and a decrease in mitotic index, especially in the region of the distal tip of the embryonic ectoderm. Mutant embryos also have an apparent reduction in apical vacuoles in the columnar visceral endoderm cells in the extraembryonic region. Together, these cellular phenotypes lead to a dramatic development delay and embryonic death by 8 to 9 days of gestation, which are independent of p53 function. PMID:11283266
Addition of a suture anchor for coracoclavicular fixation to a superior locking plate improves stability of type IIB distal clavicle fractures.

PubMed

Madsen, Wes; Yaseen, Zaneb; LaFrance, Russell; Chen, Tony; Awad, Hani; Maloney, Michael; Voloshin, Ilya

2013-06-01

The purpose of this study was to determine the effect of coracoclavicular (CC) fixation on biomechanical stability in type IIB distal clavicle fractures fixed with plate and screws. Twelve fresh-frozen matched cadaveric specimens were used to create type IIB distal clavicle fractures. Dual-energy x-ray absorptiometry (DEXA) scans ensured similar bone quality. Group 1 (6 specimens) was stabilized with a superior precontoured distal clavicle locking plate and supplemental suture anchor CC fixation. Group 2 (6 specimens) followed the same construct without CC fixation. Each specimen was cyclically loaded in the coronal plane at 40 to 80 N for 17,500 cycles. Load-to-failure testing was performed on the specimens that did not fail cyclic loading. Outcome measures included mode of failure and the number of cycles or load required to create 10 mm of displacement in the construct. All specimens (12 of 12) completed cyclic testing without failure and underwent load-to-failure testing. Group 1 specimens failed at a mean of 808.5 N (range, 635.4 to 952.3 N), whereas group 2 specimens failed at a mean of 401.3 N (range, 283.6 to 656.0 N) (P = .005). Group 1 specimens failed by anchor pullout without coracoid fracture (4 of 6) and distal clavicle fracture fragment fragmentation (1 of 6); one specimen did not fail at the maximal load the materials testing machine was capable of exerting (1,000 N). Group 2 specimens failed by distal clavicle fracture fragment fragmentation (3 of 6) and acromioclavicular (AC) joint displacement (1 of 6); 2 specimens did not fail at the maximal load of the materials testing machine. During cyclic loading, type IIB distal clavicle fractures with and without CC fixation remain stable. CC fixation adds stability to type IIB distal clavicle fractures fixed with plate and screws when loaded to failure. CC fixation for distal clavicle fractures is a useful adjunct to plate-and-screw fixation to augment stability of the fracture. Copyright © 2013 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

PubMed

Bonazza, Camila; Andrade, Sheila Siqueira; Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J; Girão, Manoel J B C; Oliva, Maria Luiza V; Castro, Rodrigo Aquino

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity.
Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids

PubMed Central

Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J.; Girão, Manoel J. B. C.; Oliva, Maria Luiza V.

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity. PMID:27391384
Follow up of infertile patients after failed ART cycles: a preliminary report from Iran and Turkey.

PubMed

Khalili, Mohammad Ali; Kahraman, Semra; Ugur, Mete Gurol; Agha-Rahimi, Azam; Tabibnejad, Nasim

2012-03-01

Assisted reproductive technology (ART) has become an established and increasingly successful form of treatment for infertility. However, significant numbers of cycles fail after embryo transfer (ET) and it becomes necessary to follow up the infertile couples after failed ART treatments. The main goal was to follow up the infertile patients after failed IVF/ICSI+ET treatments in Iran and Turkey. 198 infertile couples from Iran and 355 infertile couples from Turkey were followed up after IVF/ICSI failures. The patients' demographic data, the couples' decisions about continuation of treatment and the spontaneous pregnancy rates were compared in the two countries. The drop-out rate was higher in Iran (28.3%) than in Turkey (23.4%). The reasons for treatment discontinuation in Iran and Turkey were: financial problem (33.9% vs. 41%), hopeless (10.7% vs. 22.9%), fear of drug side-effects (7.1% vs. 12%), achieving pregnancy (37.5% vs. 19.6%), child adoption (5.4% vs. 2.4%), lack of spouse cooperation (5.4% vs. 2.4%), and divorce (0% vs. 2.4%). Spontaneous pregnancy was significantly higher in Iran (10.1%) than in Turkey (3.9%). There was correlation between duration of infertility and female factor infertility with spontaneous pregnancy. Since the majority of couples that discontinued treatment had financial problems, it is essential for health professionals to support infertile couples during their childlessness crisis. Copyright Â© 2011 Elsevier Ireland Ltd. All rights reserved.
Cell cycle in egg cell and its progression during zygotic development in rice.

PubMed

Sukawa, Yumiko; Okamoto, Takashi

2018-03-01

Rice egg is arrested at G1 phase probably by OsKRP2. After fusion with sperm, karyogamy, OsWEE1-mediated parental DNA integrity in zygote nucleus, zygote progresses cell cycle to produce two-celled embryo. In angiosperms, female and male gametes exist in gametophytes after the complementation of meiosis and the progression of nuclear/cell division of the haploid cell. Within the embryo sac, the egg cell is specially differentiated for fertilization and subsequent embryogenesis, and cellular programs for embryonic development, such as restarting the cell cycle and de novo gene expression, are halted. There is only limited knowledge about how the cell cycle in egg cells restarts toward zygotic division, although the conversion of the cell cycle from a quiescent and arrested state to an active state is the most evident transition of cell status from egg cell to zygote. This is partly due to the difficulty in direct access and analysis of egg cells, zygotes and early embryos, which are deeply embedded in ovaries. In this study, precise relative DNA amounts in the nuclei of egg cells, developing zygotes and cells of early embryos were measured, and the cell cycle of a rice egg cell was estimated as the G1 phase with a 1C DNA level. In addition, increases in DNA content in zygote nuclei via karyogamy and DNA replication were also detectable according to progression of the cell cycle. In addition, expression profiles for cell cycle-related genes in egg cells and zygotes were also addressed, and it was suggested that OsKRP2 and OsWEE1 function in the inhibition of cell cycle progression in egg cells and in checkpoint of parental DNA integrity in zygote nucleus, respectively.
Toll-like receptor 4 is involved in the cell cycle modulation and required for effective human cytomegalovirus infection in THP-1 macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arcangeletti, Maria-Cristina, E-mail: mariacristina.arcangeletti@unipr.it; Germini, Diego; Rodighiero, Isabella

2013-05-25

Suitable host cell metabolic conditions are fundamental for the effective development of the human cytomegalovirus (HCMV) lytic cycle. Indeed, several studies have demonstrated the ability of this virus to interfere with cell cycle regulation, mainly by blocking proliferating cells in G1 or G1/S. In the present study, we demonstrate that HCMV deregulates the cell cycle of THP-1 macrophages (a cell line irreversibly arrested in G0) by pushing them into S and G2 phases. Moreover, we show that HCMV infection of THP-1 macrophages leads to Toll-like receptor 4 (TLR4) activation. Since various studies have indicated TLR4 to be involved in promotingmore » cell proliferation, here we investigate the possible role of TLR4 in the observed HCMV-induced cell cycle perturbation. Our data strongly support TLR4 as a mediator of HCMV-triggered cell cycle activation in THP-1 macrophages favouring, in turn, the development of an efficient viral lytic cycle. - Highlights: ► We studied HCMV infection impact on THP-1 macrophage cell cycle. ► We analysed the role played by Toll-like receptor (TLR) 4 upon HCMV infection. ► HCMV pushes THP-1 macrophages (i.e. resting cells) to re-enter the cell cycle. ► TLR4 pathway inhibition strongly affects the effectiveness of HCMV replication. ► TLR4 pathway inhibition significantly decreases HCMV-induced cell cycle re-entry.« less
Restrictions in Cell Cycle Progression of Adult Vestibular Supporting Cells in Response to Ectopic Cyclin D1 Expression

PubMed Central

Loponen, Heidi; Ylikoski, Jukka; Albrecht, Jeffrey H.; Pirvola, Ulla

2011-01-01

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27Kip1 and p21Cip1 expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells. PMID:22073316
Restrictions in cell cycle progression of adult vestibular supporting cells in response to ectopic cyclin D1 expression.

PubMed

Loponen, Heidi; Ylikoski, Jukka; Albrecht, Jeffrey H; Pirvola, Ulla

2011-01-01

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27(Kip1) and p21(Cip1) expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells.
Slow-cycling stem cells in hydra contribute to head regeneration

PubMed Central

Govindasamy, Niraimathi; Murthy, Supriya; Ghanekar, Yashoda

2014-01-01

ABSTRACT Adult stem cells face the challenge of maintaining tissue homeostasis by self-renewal while maintaining their proliferation potential over the lifetime of an organism. Continuous proliferation can cause genotoxic/metabolic stress that can compromise the genomic integrity of stem cells. To prevent stem cell exhaustion, highly proliferative adult tissues maintain a pool of quiescent stem cells that divide only in response to injury and thus remain protected from genotoxic stress. Hydra is a remarkable organism with highly proliferative stem cells and ability to regenerate at whole animal level. Intriguingly, hydra does not display consequences of high proliferation, such as senescence or tumour formation. In this study, we investigate if hydra harbours a pool of slow-cycling stem cells that could help prevent undesirable consequences of continuous proliferation. Hydra were pulsed with the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) and then chased in the absence of EdU to monitor the presence of EdU-retaining cells. A significant number of undifferentiated cells of all three lineages in hydra retained EdU for about 8–10 cell cycles, indicating that these cells did not enter cell cycle. These label-retaining cells were resistant to hydroxyurea treatment and were predominantly in the G2 phase of cell cycle. Most significantly, similar to mammalian quiescent stem cells, these cells rapidly entered cell division during head regeneration. This study shows for the first time that, contrary to current beliefs, cells in hydra display heterogeneity in their cell cycle potential and the slow-cycling cells in this population enter cell cycle during head regeneration. These results suggest an early evolution of slow-cycling stem cells in multicellular animals. PMID:25432513
Investigating Conservation of the Cell-Cycle-Regulated Transcriptional Program in the Fungal Pathogen, Cryptococcus neoformans

PubMed Central

Sierra, Crystal S.; Haase, Steven B.

2016-01-01

The pathogenic yeast Cryptococcus neoformans causes fungal meningitis in immune-compromised patients. Cell proliferation in the budding yeast form is required for C. neoformans to infect human hosts, and virulence factors such as capsule formation and melanin production are affected by cell-cycle perturbation. Thus, understanding cell-cycle regulation is critical for a full understanding of virulence factors for disease. Our group and others have demonstrated that a large fraction of genes in Saccharomyces cerevisiae is expressed periodically during the cell cycle, and that proper regulation of this transcriptional program is important for proper cell division. Despite the evolutionary divergence of the two budding yeasts, we found that a similar percentage of all genes (~20%) is periodically expressed during the cell cycle in both yeasts. However, the temporal ordering of periodic expression has diverged for some orthologous cell-cycle genes, especially those related to bud emergence and bud growth. Genes regulating DNA replication and mitosis exhibited a conserved ordering in both yeasts, suggesting that essential cell-cycle processes are conserved in periodicity and in timing of expression (i.e. duplication before division). In S. cerevisiae cells, we have proposed that an interconnected network of periodic transcription factors (TFs) controls the bulk of the cell-cycle transcriptional program. We found that temporal ordering of orthologous network TFs was not always maintained; however, the TF network topology at cell-cycle commitment appears to be conserved in C. neoformans. During the C. neoformans cell cycle, DNA replication genes, mitosis genes, and 40 genes involved in virulence are periodically expressed. Future work toward understanding the gene regulatory network that controls cell-cycle genes is critical for developing novel antifungals to inhibit pathogen proliferation. PMID:27918582
AS160 controls eukaryotic cell cycle and proliferation by regulating the CDK inhibitor p21.

PubMed

Gongpan, Pianchou; Lu, Yanting; Wang, Fang; Xu, Yuhui; Xiong, Wenyong

2016-07-02

AS160 (TBC1D4) has been implicated in multiple biological processes. However, the role and the mechanism of action of AS160 in the regulation of cell proliferation remain unclear. In this study, we demonstrated that AS160 knockdown led to blunted cell proliferation in multiple cell types, including fibroblasts and cancer cells. The results of cell cycle analysis showed that these cells were arrested in the G1 phase. Intriguingly, this inhibition of cell proliferation and the cell cycle arrest caused by AS160 depletion were glucose independent. Moreover, AS160 silencing led to a marked upregulation of the expression of the cyclin-dependent kinase inhibitor p21. Furthermore, whereas AS160 overexpression resulted in p21 downregulation and rescued the arrested cell cycle in AS160-depeleted cells, p21 silencing rescued the inhibited cell cycle and proliferation in the cells. Thus, our results demonstrated that AS160 regulates glucose-independent eukaryotic cell proliferation through p21-dependent control of the cell cycle, and thereby revealed a molecular mechanism of AS160 modulation of cell cycle and proliferation that is of general physiological significance.
Mps1 kinase regulates tumor cell viability via its novel role in mitochondria

PubMed Central

Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

2016-01-01

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells. PMID:27383047
Mps1 kinase regulates tumor cell viability via its novel role in mitochondria.

PubMed

Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

2016-07-07

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells.
A dual transcriptional reporter and CDK-activity sensor marks cell cycle entry and progression in C. elegans

PubMed Central

van Rijnberk, Lotte M.; van der Horst, Suzanne E. M.; van den Heuvel, Sander; Ruijtenberg, Suzan

2017-01-01

Development, tissue homeostasis and tumor suppression depend critically on the correct regulation of cell division. Central in the cell division process is the decision whether to enter the next cell cycle and commit to going through the S and M phases, or to remain temporarily or permanently arrested. Cell cycle studies in genetic model systems could greatly benefit from visualizing cell cycle commitment in individual cells without the need of fixation. Here, we report the development and characterization of a reporter to monitor cell cycle entry in the nematode C. elegans. This reporter combines the mcm-4 promoter, to reveal Rb/E2F-mediated transcriptional control, and a live-cell sensor for CDK-activity. The CDK sensor was recently developed for use in human cells and consists of a DNA Helicase fragment fused to eGFP. Upon phosphorylation by CDKs, this fusion protein changes in localization from the nucleus to the cytoplasm. The combined regulation of transcription and subcellular localization enabled us to visualize the moment of cell cycle entry in dividing seam cells during C. elegans larval development. This reporter is the first to reflect cell cycle commitment in C. elegans and will help further genetic studies of the mechanisms that underlie cell cycle entry and exit. PMID:28158315
Non-24-Hour Sleep-Wake Disorder Revisited - A Case Study.

PubMed

Garbazza, Corrado; Bromundt, Vivien; Eckert, Anne; Brunner, Daniel P; Meier, Fides; Hackethal, Sandra; Cajochen, Christian

2016-01-01

The human sleep-wake cycle is governed by two major factors: a homeostatic hourglass process (process S), which rises linearly during the day, and a circadian process C, which determines the timing of sleep in a ~24-h rhythm in accordance to the external light-dark (LD) cycle. While both individual processes are fairly well characterized, the exact nature of their interaction remains unclear. The circadian rhythm is generated by the suprachiasmatic nucleus ("master clock") of the anterior hypothalamus, through cell-autonomous feedback loops of DNA transcription and translation. While the phase length (tau) of the cycle is relatively stable and genetically determined, the phase of the clock is reset by external stimuli ("zeitgebers"), the most important being the LD cycle. Misalignments of the internal rhythm with the LD cycle can lead to various somatic complaints and to the development of circadian rhythm sleep disorders (CRSD). Non-24-hour sleep-wake disorders (N24HSWD) is a CRSD affecting up to 50% of totally blind patients and characterized by the inability to maintain a stable entrainment of the typically long circadian rhythm (tau > 24.5 h) to the LD cycle. The disease is rare in sighted individuals and the pathophysiology less well understood. Here, we present the case of a 40-year-old sighted male, who developed a misalignment of the internal clock with the external LD cycle following the treatment for Hodgkin's lymphoma (ABVD regimen, four cycles and AVD regimen, four cycles). A thorough clinical assessment, including actigraphy, melatonin profiles and polysomnography led to the diagnosis of non-24-hour sleep-wake disorders (N24HSWD) with a free-running rhythm of tau = 25.27 h. A therapeutic intervention with bright light therapy (30 min, 10,000 lux) in the morning and melatonin administration (0.5-0.75 mg) in the evening failed to entrain the free-running rhythm, although a longer treatment duration and more intense therapy might have been successful. The sudden onset and close timely connection led us to hypothesize that the chemotherapy might have caused a mutation of the molecular clock components leading to the observed elongation of the circadian period.
The Adder Phenomenon Emerges from Independent Control of Pre- and Post-Start Phases of the Budding Yeast Cell Cycle.

PubMed

Chandler-Brown, Devon; Schmoller, Kurt M; Winetraub, Yonatan; Skotheim, Jan M

2017-09-25

Although it has long been clear that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained poorly understood. In budding yeast, cell size primarily modulates the duration of the cell-division cycle by controlling the G1/S transition known as Start. We have recently shown that the rate of progression through Start increases with cell size, because cell growth dilutes the cell-cycle inhibitor Whi5 in G1. Recent phenomenological studies in yeast and bacteria have shown that these cells add an approximately constant volume during each complete cell cycle, independent of their size at birth. These results seem to be in conflict, as the phenomenological studies suggest that cells measure the amount they grow, rather than their size, and that size control acts over the whole cell cycle, rather than specifically in G1. Here, we propose an integrated model that unifies the adder phenomenology with the molecular mechanism of G1/S cell-size control. We use single-cell microscopy to parameterize a full cell-cycle model based on independent control of pre- and post-Start cell-cycle periods. We find that our model predicts the size-independent amount of cell growth during the full cell cycle. This suggests that the adder phenomenon is an emergent property of the independent regulation of pre- and post-Start cell-cycle periods rather than the consequence of an underlying molecular mechanism measuring a fixed amount of growth. Copyright © 2017 Elsevier Ltd. All rights reserved.
77 FR 4650 - Airworthiness Directives; General Electric Company Turbofan Engines

Federal Register 2010, 2011, 2012, 2013, 2014

2012-01-31

... blade borescope inspection (BSI) or a failed engine core vibration survey, establishes a new lower life... LPT rotor stage 3 disk removal after a failed HPT blade BSI or a failed engine core vibration survey... engine test cell as part of an engine manual performance run fulfill the vibration survey requirements of...
TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Li; College of Life Sciences, Hainan Normal University, Haikou, Hainan 571158; Huang, Yong

2014-03-07

Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressedmore » cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.« less
A single cyclin–CDK complex is sufficient for both mitotic and meiotic progression in fission yeast

PubMed Central

Gutiérrez-Escribano, Pilar; Nurse, Paul

2015-01-01

The dominant model for eukaryotic cell cycle control proposes that cell cycle progression is driven by a succession of CDK complexes with different substrate specificities. However, in fission yeast it has been shown that a single CDK complex generated by the fusion of the Cdc13 cyclin with the CDK protein Cdc2 can drive the mitotic cell cycle. Meiosis is a modified cell cycle programme in which a single S-phase is followed by two consecutive rounds of chromosome segregation. Here we systematically analyse the requirements of the different fission yeast cyclins for meiotic cell cycle progression. We also show that a single Cdc13–Cdc2 complex, in the absence of the other cyclins, can drive the meiotic cell cycle. We propose that qualitatively different CDK complexes are not absolutely required for cell cycle progression either during mitosis or meiosis, and that a single CDK complex can drive both cell cycle programmes. PMID:25891897
Immunocytochemistry of cell surface heparan sulfate proteoglycan in mouse tissues. A light and electron microscopic study.

PubMed

Hayashi, K; Hayashi, M; Jalkanen, M; Firestone, J H; Trelstad, R L; Bernfield, M

1987-10-01

The core protein of the proteoglycan at the cell surface of NMuMG mouse mammary epithelial cells bears both heparan and chondroitin sulfate chains and is recognized by the monoclonal antibody 281-2. Using this antibody and the peroxidase-antiperoxidase staining technique in adult mouse tissues, we found that the antibody recognizes the antigen in a highly restricted distribution, staining a variety of epithelial cells but no cells derived from embryonic mesoderm or neural crest. The antibody fails to stain any stromal (mesenchymal) or neuronal cells, with the exception of plasma cells and Leydig cells. Squamous and transitional epithelia stain intensely over their entire surfaces, whereas cuboidal and columnar epithelia stain moderately and only at the lateral surface of the basal cells. Within squamous and transitional epithelial tissues that undergo physiological regeneration (e.g., epidermis), the most superficial and differentiated cell types fail to stain. Within glandular and branched epithelia (e.g., pancreas), the secretory alveolar cells fail to stain. When evaluated by electron microscopy, granular deposits of stain are seen on the plasma membrane, especially on lateral surfaces, but none are noted within the cells or the basement membrane. These results indicate that in adult tissues the core protein of this heparan sulfate-rich proteoglycan is expressed almost exclusively at epithelial cell surfaces. Expression appears to be lost as the cells become either mature or highly differentiated.
Soybean agglutinin binding to corneal endothelial cell surfaces disrupts in situ monolayer integrity and actin organization and interferes with wound repair.

PubMed

Gordon, Sheldon R; Wood, Meredith

2009-03-01

Rat corneal endothelium demonstrates cell-surface soybean agglutinin (SBA) binding during organ-culture or injury. When organ-cultured in medium containing SBA, the endothelial monolayer is disrupted because of cell-cell and cell-matrix alterations. SBA binding disorganizes the circumferential microfilament bundles (CMBs), an effect that is partially prevented by phallacidin preincubation. This disruption is reversible if tissues are returned to standard culture medium. Serum heightens SBA binding, whereas puromycin prevents it. Neither actinomycin D nor alpha-amanitin inhibits SBA binding, suggesting that SBA-binding protein(s) may be post-transcriptionally regulated. During injury-induced cell migration in the presence of SBA, cellular processes are blunted and fail to extend significantly outward. By 72 h post-injury, cells of SBA-treated tissues repopulate the wound but demonstrate little association with neighboring cells. Cells migrating in the presence of N-acetylgalactosamine appear normal but also fail to reassociate with other cells in the jury zone. Immunofluorescent staining for ZO-1 reveals punctuate patterns in cells of control tissues, whereas neither SBA- nor N-acetylgalactosamine-treated tissues exhibit ZO-1 staining. Terminal N-acetylgalactosamine removal fails to affect cell morphology, actin organization, or migration but prevents lectin binding. Our results suggest that SBA binding reflects the synthesis of a stress-induced protein(s) that may play a role in reestablishing cell-cell relationships during monolayer reorganization following injury.

Hypoxia induces p53 accumulation in the S-phase and accumulation of hypophosphorylated retinoblastoma protein in all cell cycle phases of human melanoma cells.

PubMed Central

Danielsen, T.; Hvidsten, M.; Stokke, T.; Solberg, K.; Rofstad, E. K.

1998-01-01

Hypoxia has been shown to induce accumulation of p53 and of hypophosphorylated retinoblastoma protein (pRb) in tumour cells. In this study, the cell cycle dependence of p53 accumulation and pRb hypophosphorylation in four human melanoma cell lines that are wild type for p53 was investigated using two-parameter flow cytometry measurements of p53 or pRb protein content and DNA content. The hypoxia-induced increase in p53 protein was higher in S-phase than in G1 and G2 phases in all cell lines. The accumulation of p53 in S-phase during hypoxia was not related to hypoxia-induced apoptosis or substantial cell cycle specific cell inactivation during the first 24 h of reoxygenation. pRb was hypophosphorylated in all cell cycle phases by hypoxia treatment. The results did not support a direct link between p53 and pRb during hypoxia because p53 was induced in a cell cycle-specific manner, whereas no cell cycle-dependent differences in pRb hypophosphorylation were detected. Only a fraction of the cell populations (0.60+/-0.10) showed hypophosphorylated pRb. Thus, pRb is probably not the only mediator of the hypoxia-induced cell cycle block seen in all cells and all cell cycle phases. Moreover, the cell cycle-dependent induction of p53 by hypoxia suggests that the primary function of p53 accumulation during hypoxia is other than to arrest the cells. Images Figure 4 Figure 7 PMID:9862563
Nuclear receptor TLX regulates cell cycle progression in neural stem cells of the developing brain.

PubMed

Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

2008-01-01

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain.
Nuclear Receptor TLX Regulates Cell Cycle Progression in Neural Stem Cells of the Developing Brain

PubMed Central

Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

2008-01-01

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain. PMID:17901127
Reversible Block of Mouse Neural Stem Cell Differentiation in the Absence of Dicer and MicroRNAs

PubMed Central

Sansom, Stephen N.; Alsiö, Jessica M.; Kaneda, Masahiro; Smith, James; O'Carroll, Donal; Tarakhovsky, Alexander; Livesey, Frederick J.

2010-01-01

Background To investigate the functions of Dicer and microRNAs in neural stem (NS) cell self-renewal and neurogenesis, we established neural stem cell lines from the embryonic mouse Dicer-null cerebral cortex, producing neural stem cell lines that lacked all microRNAs. Principal Findings Dicer-null NS cells underwent normal self-renewal and could be maintained in vitro indefinitely, but had subtly altered cell cycle kinetics and abnormal heterochromatin organisation. In the absence of all microRNAs, Dicer-null NS cells were incapable of generating either glial or neuronal progeny and exhibited a marked dependency on exogenous EGF for survival. Dicer-null NS cells assumed complex differences in mRNA and protein expression under self-renewing conditions, upregulating transcripts indicative of self-renewing NS cells and expressing genes characteristic of differentiating neurons and glia. Underlining the growth-factor dependency of Dicer-null NS cells, many regulators of apoptosis were enriched in expression in these cells. Dicer-null NS cells initiate some of the same gene expression changes as wild-type cells under astrocyte differentiating conditions, but also show aberrant expression of large sets of genes and ultimately fail to complete the differentiation programme. Acute replacement of Dicer restored their ability to differentiate to both neurons and glia. Conclusions The block in differentiation due to loss of Dicer and microRNAs is reversible and the significantly altered phenotype of Dicer-null NS cells does not constitute a permanent transformation. We conclude that Dicer and microRNAs function in this system to maintain the neural stem cell phenotype and to facilitate the completion of differentiation. PMID:20976144
Gemcitabine treatment causes resistance and malignancy of pancreatic cancer stem-like cells via induction of lncRNA HOTAIR

PubMed Central

Wang, Li; Dong, Ping; Wang, Weiguo; Huang, Mingquan; Tian, Bole

2017-01-01

Gemcitabine is the first-line chemotherapeutic agent for advanced adenocarcinoma of the pancreas, despite the high risk of chemoresistance as a major disadvantage. In the past few years, significant advances have been made in the field of pancreatic cancer stem-like cells (CSCs) and their critical roles in drug resistance, invasion and metastasis, which are tightly regulated by long non-coding RNAs (lncRNAs). The present study demonstrated that HOX antisense intergenic RNA (HOTAIR) is not different between the pancreatic cancer cell line PANC-1 and its enriched CSC sub-population. However, after gemcitabine treatment, the expression levels of HOTAIR in CSCs were induced, but not in PANC-1 cells. HOTAIR induced by gemcitabine failed to cause chemoresistance, but promoted the clonogenicity, proliferation and migration of the cells. By introducing HOTAIR using lentivirus, chemoresistance was induced and the self-renewal capacity, proliferation and migration were significantly promoted. By contrast, HOTAIR knockdown in PANC-1 CSCs treated with or without gemcitabine decreased the cell proliferation, altered the cell cycle progression and induced apoptosis, demonstrating its critical roles in regulating the malignant character of PANC-1 CSCs. In conclusion, the present study demonstrated that HOTAIR may be induced by gemcitabine and acts as a tumor promoter by inhibiting the chemosensitivity, and promoting the self-renewal capacity, proliferation and migration of PANC-1 CSCs, which supports its potential application as a novel therapeutic approach for pancreatic cancer. PMID:29201179
Genetic podocyte lineage reveals progressive podocytopenia with parietal cell hyperplasia in a murine model of cellular/collapsing focal segmental glomerulosclerosis.

PubMed

Suzuki, Taisei; Matsusaka, Taiji; Nakayama, Makiko; Asano, Takako; Watanabe, Teruo; Ichikawa, Iekuni; Nagata, Michio

2009-05-01

Focal segmental glomerulosclerosis (FSGS) is a progressive renal disease, and the glomerular visceral cell hyperplasia typically observed in cellular/collapsing FSGS is an important pathological factor in disease progression. However, the cellular features that promote FSGS currently remain obscure. To determine both the origin and phenotypic alterations in hyperplastic cells in cellular/collapsing FSGS, the present study used a previously described FSGS model in p21-deficient mice with visceral cell hyperplasia and identified the podocyte lineage by genetic tagging. The p21-deficient mice with nephropathy showed significantly higher urinary protein levels, extracapillary hyperplastic indices on day 5, and glomerular sclerosis indices on day 14 than wild-type controls. X-gal staining and immunohistochemistry for podocyte and parietal epithelial cell (PEC) markers revealed progressive podocytopenia with capillary collapse accompanied by PEC hyperplasia leading to FSGS. In our investigation, non-tagged cells expressed neither WT1 nor nestin. Ki-67, a proliferation marker, was rarely associated with podocytes but was expressed at high levels in PECs. Both terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy failed to show evidence of significant podocyte apoptosis on days 5 and 14. These findings suggest that extensive podocyte loss and simultaneous PEC hyperplasia is an actual pathology that may contribute to the progression of cellular/collapsing FSGS in this mouse model. Additionally, this is the first study to demonstrate the regulatory role of p21 in the PEC cell cycle.
Cell cycle gene expression under clinorotation

NASA Astrophysics Data System (ADS)

Artemenko, Olga

2016-07-01

Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.
The alpha-fetoprotein (AFP) third domain: a search for AFP interaction sites of cell cycle proteins.

PubMed

Mizejewski, G J

2016-09-01

The carboxy-terminal third domain of alpha-fetoprotein (AFP-3D) is known to harbor binding and/or interaction sites for hydrophobic ligands, receptors, and binding proteins. Such reports have established that AFP-3D consists of amino acid (AA) sequence stretches on the AFP polypeptide that engages in protein-to-protein interactions with various ligands and receptors. Using a computer software program specifically designed for such interactions, the present report identified AA sequence fragments on AFP-3D that could potentially interact with a variety of cell cycle proteins. The cell cycle proteins identified were (1) cyclins, (2) cyclin-dependent kinases, (3) cell cycle-associated proteins (inhibitors, checkpoints, initiators), and (4) ubiquitin ligases. Following detection of the AFP-3D to cell cycle protein interaction sites, the computer-derived AFP localization AA sequences were compared and aligned with previously reported hydrophobic ligand and receptor interaction sites on AFP-3D. A literature survey of the association of cell cycle proteins with AFP showed both positive relationships and correlations. Previous reports of experimental AFP-derived peptides effects on various cell cycle proteins served to confirm and verify the present computer cell cycle protein identifications. Cell cycle protein interactions with AFP-CD peptides have been reported in cultured MCF-7 breast cancer cells subjected to mRNA microarray analysis. After 7 days in culture with MCF-7 cells, the AFP-derived peptides were shown to downregulate cyclin E, SKP2, checkpoint suppressors, cyclin-dependent kinases, and ubiquitin ligases that modulate cyclin E/CdK2 transition from the G1 to the S-phase of the cell cycle. Thus, the experimental data on AFP-CD interaction with cell cycle proteins were consistent with the "in silico" findings.
Analyzing the dynamics of cell cycle processes from fixed samples through ergodic principles

PubMed Central

Wheeler, Richard John

2015-01-01

Tools to analyze cyclical cellular processes, particularly the cell cycle, are of broad value for cell biology. Cell cycle synchronization and live-cell time-lapse observation are widely used to analyze these processes but are not available for many systems. Simple mathematical methods built on the ergodic principle are a well-established, widely applicable, and powerful alternative analysis approach, although they are less widely used. These methods extract data about the dynamics of a cyclical process from a single time-point “snapshot” of a population of cells progressing through the cycle asynchronously. Here, I demonstrate application of these simple mathematical methods to analysis of basic cyclical processes—cycles including a division event, cell populations undergoing unicellular aging, and cell cycles with multiple fission (schizogony)—as well as recent advances that allow detailed mapping of the cell cycle from continuously changing properties of the cell such as size and DNA content. This includes examples using existing data from mammalian, yeast, and unicellular eukaryotic parasite cell biology. Through the ongoing advances in high-throughput cell analysis by light microscopy, electron microscopy, and flow cytometry, these mathematical methods are becoming ever more important and are a powerful complementary method to traditional synchronization and time-lapse cell cycle analysis methods. PMID:26543196
Angular-dependent light scattering from cancer cells in different phases of the cell cycle.

PubMed

Lin, Xiaogang; Wan, Nan; Weng, Lingdong; Zhou, Yong

2017-10-10

Cancer cells in different phases of the cell cycle result in significant differences in light scattering properties. In order to harvest cancer cells in particular phases of the cell cycle, we cultured cancer cells through the process of synchronization. Flow cytometric analysis was applied to check the results of cell synchronization and prepare for light scattering measurements. Angular-dependent light scattering measurements of cancer cells arrested in the G1, S, and G2 phases have been performed. Based on integral calculations for scattering intensities from 5° to 10° and from 110° to 150°, conclusions have been reached. Clearly, the sizes of the cancer cells in different phases of the cell cycle dominated the forward scatter. Accompanying the increase of cell size with the progression of the cell cycle, the forward scattering intensity also increased. Meanwhile, the DNA content of cancer cells in every phase of the cell cycle is responsible for light scattering at large scatter angles. The higher the DNA content of cancer cells was, the greater the positive effect on the high-scattering intensity. As expected, understanding the relationships between the light scattering from cancer cells and cell cycles will aid in the development of cancer diagnoses. Also, it may assist in the guidance of antineoplastic drugs clinically.
Pregnancy derived from human zygote pronuclear transfer in a patient who had arrested embryos after IVF.

PubMed

Zhang, John; Zhuang, Guanglun; Zeng, Yong; Grifo, Jamie; Acosta, Carlo; Shu, Yimin; Liu, Hui

2016-10-01

Nuclear transfer of an oocyte into the cytoplasm of another enucleated oocyte has shown that embryogenesis and implantation are influenced by cytoplasmic factors. We report a case of a 30-year-old nulligravida woman who had two failed IVF cycles characterized by all her embryos arresting at the two-cell stage and ultimately had pronuclear transfer using donor oocytes. After her third IVF cycle, eight out of 12 patient oocytes and 12 out of 15 donor oocytes were fertilized. The patient's pronuclei were transferred subzonally into an enucleated donor cytoplasm resulting in seven reconstructed zygotes. Five viable reconstructed embryos were transferred into the patient's uterus resulting in a triplet pregnancy with fetal heartbeats, normal karyotypes and nuclear genetic fingerprinting matching the mother's genetic fingerprinting. Fetal mitochondrial DNA profiles were identical to those from donor cytoplasm with no detection of patient's mitochondrial DNA. This report suggests that a potentially viable pregnancy with normal karyotype can be achieved through pronuclear transfer. Ongoing work to establish the efficacy and safety of pronuclear transfer will result in its use as an aid for human reproduction. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant1[C][W

PubMed Central

Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

2014-01-01

The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants. PMID:25037213
Chromium-picolinate therapy in diabetes care: individual outcomes require new guidelines and navigation by predictive diagnostics

PubMed Central

Yeghiazaryan, Kristina; Schild, Hans H.; Golubnitschaja, Olga

2013-01-01

Aims Nephropathy is the leading secondary complication of metabolic syndrome. Nutritional supplement by chromium-picolinate is assumed to have renoprotective effects. However, potential toxic effects reported increase concerns about safety of chromium-picolinate. The experimental design aimed at determining, whether the treatment with clinically relevant doses of chromium-picolinate can harm individual oucomes through DNA damage and extensive alterations in central detoxification / cell-cycle regulating pathways in treatment of diabetes. Methods The study was performed in a double-blind manner. Well-acknowledged animal model of db/db-mice and clinically relevant doses of chromium-picolinate were used. As an index of DNA-damage, measurement of DNA-breaks was performed using “Comet Assay”-analysis. Individual and group-specific expression patterns of SOD-1 and P53 were evaluated to get insights into central detoxification and cell-cycle regulating pathways under treatment conditions. Results Experimental data revealed highly individual reaction under treatment conditions. Highest variability of DNA-damage was monitored under prolonged treatment with high dosage of CrPic. Expression patterns demonstrated a correlation with subcellular imaging and dosage-dependent suppression under chromium-picolinate treatment. Interpretation and recommendations Population at-risk for diabetes is huge and increasing in pandemic scale. One of the reasons might be the failed attempt to prevent the disease by application of artificial supplements and drugs with hardly recognised individual risks. Consequently, a multimodal approach of integrative medicine by predictive diagnostics, targeted prevention and individually created treatment algorithms is highly desirable. PMID:23017160
Disruption of clathrin-mediated trafficking causes centrosome overduplication and senescence.

PubMed

Olszewski, Maciej B; Chandris, Panagiotis; Park, Bum-Chan; Eisenberg, Evan; Greene, Lois E

2014-01-01

The Hsc70 cochaperone, G cyclin-associated kinase (GAK), has been shown to be essential for the chaperoning of clathrin by Hsc70 in the cell. In this study, we used conditional GAK knockout mouse embryonic fibroblasts (MEFs) to determine the effect of completely inhibiting clathrin-dependent trafficking on the cell cycle. After GAK was knocked out, the cells developed the unusual phenotype of having multiple centrosomes, but at the same time failed to divide and ultimately became senescent. To explain this phenotype, we examined the signaling profile and found that mitogenic stimulation of the GAK KO cells and the control cells were similar except for increased phosphorylation of Akt. In addition, the disruption of intracellular trafficking caused by knocking out GAK destabilized the lysosomal membranes, resulting in DNA damage due to iron leakage. Knocking down clathrin heavy chain or inhibiting dynamin largely reproduced the GAK KO phenotype, but inhibiting only clathrin-mediated endocytosis by knocking down adaptor protein (AP2) caused growth arrest and centrosome overduplication, but no DNA damage or senescence. We conclude that disruption of clathrin-dependent trafficking induces senescence accompanied by centrosome overduplication because of a combination of DNA damage and changes in mitogenic signaling that uncouples centrosomal duplication from DNA replication. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Gabrielson, Marike; Reizer, Edwin; Stål, Olle

An increasing body of evidence is pointing towards mitochondrial regulation of the cell cycle. In a previous study of HER2-positive tumours we could demonstrate a common loss in the gene encoding for the mitochondrial transporter SLC25A43 and also a significant relation between SLC25A43 protein expression and S-phase fraction. Here, we investigated the consequence of suppressed SLC25A43 expression on cell cycle progression and proliferation in breast epithelial cells. In the present study, we suppressed SLC25A43 using siRNA in immortalised non-cancerous breast epithelial MCF10A cells and HER2-positive breast cancer cells BT-474. Viability, apoptosis, cell proliferation rate, cell cycle phase distribution, and nuclearmore » Ki-67 and p21, were assessed by flow cytometry. Cell cycle related gene expressions were analysed using real-time PCR. We found that SLC25A43 knockdown in MCF10A cells significantly inhibited cell cycle progression during G{sub 1}-to-S transition, thus significantly reducing the proliferation rate and fraction of Ki-67 positive MCF10A cells. In contrast, suppressed SLC25A43 expression in BT-474 cells resulted in a significantly increased proliferation rate together with an enhanced G{sub 1}-to-S transition. This was reflected by an increased fraction of Ki-67 positive cells and reduced level of nuclear p21. In line with our previous results, we show a role for SLC25A43 as a regulator of cell cycle progression and proliferation through a putative mitochondrial checkpoint. These novel data further strengthen the connection between mitochondrial function and the cell cycle, both in non-malignant and in cancer cells. - Highlights: • Proposed cell cycle regulation through the mitochondrial transporter SLC25A43. • SLC25A43 alters cell proliferation rate and cell cycle progression. • Suppressed SLC25A43 influences transcription of cell cycle regulatory genes.« less
Deregulated expression of Cdc6 as BCR/ABL-dependent survival factor in chronic myeloid leukemia cells.

PubMed

Zhang, Jia-Hua; He, Yan-Li; Zhu, Rui; Du, Wen; Xiao, Jun-Hua

2017-06-01

Chronic myeloid leukemia is characterized by the presence of the reciprocal translocation t(9;22) and the BCR/ABL oncogene. The BCR/ABL oncogene activates multiple signaling pathways and involves the dysregulation of oncogenes during the progression of chronic myeloid leukemia. The cell division cycle protein 6, an essential regulator of DNA replication, is elevated in some human cancer cells. However, the expression of cell division cycle protein 6 in chronic myeloid leukemia and the underlying regulatory mechanism remain to be elucidated. In this study, our data showed that cell division cycle protein 6 expression was significantly upregulated in primary chronic myeloid leukemia cells and the chronic myeloid leukemia cell line K562 cells, as compared to the normal bone marrow mononuclear cells. BCR/ABL kinase inhibitor STI571 or BCR/ABL small interfering RNA could significantly downregulate cell division cycle protein 6 messenger RNA expression in K562 cells. Moreover, phosphoinositide 3-kinase/AKT pathway inhibitor LY294002 and Janus kinase/signal transducer and activator of transcription pathway inhibitor AG490 could downregulate cell division cycle protein 6 expression in K562 cells, but not RAS/mitogen-activated protein kinase pathway inhibitor PD98059 had such effect. Cell division cycle protein 6 gene silencing by small interfering RNA effectively resulted in decrease of proliferation, increase of apoptosis, and arrest of cell cycle in K562 cells. These findings have demonstrated that cell division cycle protein 6 overexpression may contribute to the high proliferation and low apoptosis in chronic myeloid leukemia cells and can be regulated by BCR/ABL signal transduction through downstream phosphoinositide 3-kinase/Akt and Janus kinase/signal transducer and activator of transcription pathways, suggesting cell division cycle protein 6 as a potential therapeutic target in chronic myeloid leukemia.
Modeling Bi-modality Improves Characterization of Cell Cycle on Gene Expression in Single Cells

PubMed Central

Danaher, Patrick; Finak, Greg; Krouse, Michael; Wang, Alice; Webster, Philippa; Beechem, Joseph; Gottardo, Raphael

2014-01-01

Advances in high-throughput, single cell gene expression are allowing interrogation of cell heterogeneity. However, there is concern that the cell cycle phase of a cell might bias characterizations of gene expression at the single-cell level. We assess the effect of cell cycle phase on gene expression in single cells by measuring 333 genes in 930 cells across three phases and three cell lines. We determine each cell's phase non-invasively without chemical arrest and use it as a covariate in tests of differential expression. We observe bi-modal gene expression, a previously-described phenomenon, wherein the expression of otherwise abundant genes is either strongly positive, or undetectable within individual cells. This bi-modality is likely both biologically and technically driven. Irrespective of its source, we show that it should be modeled to draw accurate inferences from single cell expression experiments. To this end, we propose a semi-continuous modeling framework based on the generalized linear model, and use it to characterize genes with consistent cell cycle effects across three cell lines. Our new computational framework improves the detection of previously characterized cell-cycle genes compared to approaches that do not account for the bi-modality of single-cell data. We use our semi-continuous modelling framework to estimate single cell gene co-expression networks. These networks suggest that in addition to having phase-dependent shifts in expression (when averaged over many cells), some, but not all, canonical cell cycle genes tend to be co-expressed in groups in single cells. We estimate the amount of single cell expression variability attributable to the cell cycle. We find that the cell cycle explains only 5%–17% of expression variability, suggesting that the cell cycle will not tend to be a large nuisance factor in analysis of the single cell transcriptome. PMID:25032992
Equilibrium between cell division and apoptosis in immortal cells as an alternative to the G1 restriction mechanism in mammalian cells.

PubMed

Dedov, Vadim N; Dedova, Irina V; Nicholson, Garth A

2004-04-01

Starvation arrests cultured mammalian cells in the G(1) restriction point of the cell cycle, whereas cancer cells generally lose the regulatory control of the cell cycle. Human lymphocytes, infected with Epstein-Barr virus (EBV), also lose their cell cycle control and produce immortal lymphoblastoid cell lines. We show that during starvation, EBV-lymphoblasts override the cell cycle arrest in the G(1) restriction point and continue cell division. Simultaneously, starvation activates apoptosis in an approximately half of the daughter cells in each cell generation. Continuos cell division and partial removal of cells by apoptosis results in stabilization of viable cell numbers, where a majority of viable cells are in the G(1) phase of the cell cycle. In contrast to starvation, anticancer drug etoposide activates apoptosis indiscriminately in all EBV-lymphoblasts and convertes all the viable cells into apoptotic. We conclude that the removal of surplus cells by apoptosis may represent a survival mechanism of transformed (i.e., cancer) cell population in nutrient restricted conditions, whereas nontransformed mammalian cells are arrested in the G(1) restriction point of the cell cycle.
[Effects of methyl tertiary butyl ether on cell cycle and cell apoptosis].

PubMed

Zhou, W; Huang, G; Zhang, H; Ye, S

2000-07-01

To explore the effects of the new gasoline additive, methyl tertiary butyl ether (MTBE) on cell cycle and cell apoptosis. Flow cytometry was used to evaluate the effect of MTBE (1, 2, 4 microl/ml, 24 h) on NIH/3T3 cell cycles; and the effect of MTBE on Hela cell apoptosis was evaluated by detecting cell survival using crystal violet staining. Flow cytometry showed that MTBE could change NIH/3T3 cell cycles, decrease the number of cells in S stage, and arrest cells at G(2) + M stage. The results suggested that MTBE could affect NIH/3T3 cell cycles and induce cell proliferation. This situation existed 48 hours after the treatment, and cell cycles came back normal 96 hours after the treatment. By detecting cell survival using crystal violet staining, we found that MTBE could inhibit the apoptosis of Hela cells which was induced by tumor necrosis factor (TNF)alpha and cycloheximide. MTBE's carcinogenicity to animals may relate to induction of cell proliferation and inhibition of cell apoptosis.
KOH concentration effect on the cycle life of nickel-hydrogen cells. 4: Results of failure analyse

NASA Technical Reports Server (NTRS)

Lim, H. S.; Verzwyvelt, S. A.

1989-01-01

Effects of KOH concentrations on failure modes and mechanisms of nickel-hydrogen cells were studied using long cycled boiler plate cells containing electrolytes of various KOH concentrations ranging 21 to 36 percent. Life of these cells were up to 40,000 cycles in an accelerated low earth orbit (LEO) cycle regime at 80 percent depth of discharge. An interim life test results were reported earlier in J. Power Sources, 22, 213-220, 1988. The results of final life test, end-of-life cell performance, and teardown analyses are discussed. These teardown analyses included visual observations, measurements of nickel electrode capacity in an electrolyte-flooded cell, dimensional changes of cell components, SEM studies on cell cross section, BET surface area and pore volume distribution in cycled nickel electrodes, and chemical analyses. Cycle life of a nickel-hydrogen cell was improved tremendously as KOH concentration was decreased from 36 to 31 percent and from 31 to 26 percent while effect of further concentration decrease was complicated as described in our earlier report. Failure mode of high concentration (31 to 36 percent) cells was gradual capacity decrease, while that of low concentration (21 to 26 percent) cells was mainly formation of a soft short. Long cycled (25,000 to 40,000 cycles) nickel electrodes were expanded more than 50 percent of the initial value, but no correlation was found between this expansion and measured capacity. All electrodes cycled in low concentration (21 to 26 percent) cells had higher capacity than those cycled in high concentration (31 to 36 percent) cells.

The cell-cycle interactome: a source of growth regulators?

PubMed

Blomme, Jonas; Inzé, Dirk; Gonzalez, Nathalie

2014-06-01

When plants develop, cell proliferation and cell expansion are tightly controlled in order to generate organs with a determinate final size such as leaves. Several studies have demonstrated the importance of the cell proliferation phase for leaf growth, illustrating that cell-cycle regulation is crucial for correct leaf development. A large and complex set of interacting proteins that constitute the cell-cycle interactome controls the transition from one cell-cycle phase to another. Here, we review the current knowledge on cell-cycle regulators from this interactome affecting final leaf size when their expression is altered, mainly in Arabidopsis. In addition to the description of mutants of CYCLIN-DEPENDENT KINASES (CDKs), CYCLINS (CYCs), and their transcriptional and post-translational regulators, a phenotypic analysis of gain- and loss-of-function mutants for 27 genes encoding proteins that interact with cell-cycle proteins is presented. This compilation of information shows that when cell-cycle-related genes are mis-expressed, leaf growth is often altered and that, seemingly, three main trends appear to be crucial in the regulation of final organ size by cell-cycle-related genes: (i) cellular compensation; (ii) gene dosage; and (iii) correct transition through the G2/M phase by ANAPHASE PROMOTING COMPLEX/CYCLOSOME (APC/C) activation. In conclusion, this meta-analysis shows that the cell-cycle interactome is enriched in leaf growth regulators, and illustrates the potential to identify new leaf growth regulators among putative new cell-cycle regulators. © The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Performance of Li-Ion Cells Under Battery Voltage Charge Control

NASA Technical Reports Server (NTRS)

Rao, Gopalakrishna M.; Vaidyanathan, Hari; Day, John H. (Technical Monitor)

2001-01-01

A study consisting of electrochemical characterization and Low-Earth-Orbit (LEO) cycling of Li-Ion cells from three vendors was initiated in 1999 to determine the cycling performance and to infuse the new technology in the future NASA missions. The 8-cell batteries included in this evaluation are prismatic cells manufactured by Mine Safety Appliances Company (MSA), cylindrical cells manufactured by SAFT and prismatic cells manufactured by Yardney Technical Products, Inc. (YTP). The three batteries were cycle tested in the LEO regime at 40% depth of discharge, and under a charge control technique that consists of battery voltage clamp with a current taper. The initial testing was conducted at 20 C; however, the batteries were cycled also intermittently at low temperatures. YTP 20 Ah cells consisted of mixed-oxide (Co and Ni) positive, graphitic carbon negative, LIPF6 salt mixed with organic carbonate solvents. The battery voltage clamp was 32 V. The low temperature cycling tests started after 4575 cycles at 20 C. The cells were not capable of cycling. at low temperature since the charge acceptance at battery level was poor. There was a cell in the battery that showed too high an end-of-charge (EOC) voltage thereby limiting the ability to charge the rest of the cells in the battery. The battery has completed 6714 cycles. SAFT 12 Ah cells consisted of mixed-oxide (Co and NO positive, graphitic carbon negative, LiPF6 salt mixed with organic carbonate solvents. The battery voltage clamp was for 30.8 V. The low temperature cycling tests started after 4594 cycles at 20 C. A cell that showed low end of discharge (EOD) and EOC voltages and three other cells that showed higher EOC voltages limited the charge acceptance at the selected voltage limit during charge. The cells were capable of cycling at 10 C and 0 C but the charge voltage limit had to be increased to 34.3 V (4.3 V per cell). The low temperature cycling may have induced poor chargeability since the voltage had to be increased to achieve the required charge input. The battery has completed 6226 cycles. MSA 10 Ah cells consisted of Co oxide positive, graphitic carbon negative, LiPF6 salt mixed with organic carbonate solvents. The battery voltage clamp was 30.8 V. The low temperature cycling tests were started after 2182 cycles at 20 C. The cells were capable of cycling at 10 C and 0 C. Like SAFT, the voltage limit on charge had to be increased to 36 V (4.5 V per cell). There was a cell (cell S/N 13) in the battery that showed poor performance features such as low EOD voltage and high EOC voltage. The battery has completed 3441 cycles. A reconditioning procedure that consisted of C15 charge to a taper current of C/100 and C/20 discharge improved the voltage behavior of SAFT and MSA cells with no significant effect on YTP cells. We have demonstrated that the charge operation with VT clamp at battery rather than at cell level is feasible for onboard Li-Ion battery operation.
Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis.

PubMed

Loor, Gabriel; Kondapalli, Jyothisri; Schriewer, Jacqueline M; Chandel, Navdeep S; Vanden Hoek, Terry L; Schumacker, Paul T

2010-12-15

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X(L) protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them. Copyright © 2010 Elsevier Inc. All rights reserved.
Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis

PubMed Central

Loor, Gabriel; Kondapalli, Jyothisri; Schriewer, Jacqueline M.; Chandel, Navdeep S.; Vanden Hoek, Terry L.; Schumacker, Paul T.

2010-01-01

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, while other studies implicate activation of the mitochondrial permeability transition poreas the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, while it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetyl cysteine and exogenous glutathione (GSH), or by over-expression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells over-expressing Cu, Zn-SOD or MnSOD. Over-expression of antiapoptotic Bcl-XLprotected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochromec, Bax/Bak, caspase-9 and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them. PMID:20937380
Misexpression of cyclin D1 in embryonic germ cells promotes testicular teratoma initiation

PubMed Central

Lanza, Denise G.; Dawson, Emily P.; Rao, Priya; Heaney, Jason D.

2016-01-01

ABSTRACT Testicular teratomas result from anomalies in embryonic germ cell development. In the 129 family of inbred mouse strains, teratomas arise during the same developmental period that male germ cells normally enter G1/G0 mitotic arrest and female germ cells initiate meiosis (the mitotic:meiotic switch). Dysregulation of this switch associates with teratoma susceptibility and involves three germ cell developmental abnormalities seemingly critical for tumor initiation: delayed G1/G0 mitotic arrest, retention of pluripotency, and misexpression of genes normally restricted to embryonic female and adult male germ cells. One misexpressed gene, cyclin D1 (Ccnd1), is a known regulator of cell cycle progression and an oncogene in many tissues. Here, we investigated whether Ccnd1 misexpression in embryonic germ cells is a determinant of teratoma susceptibility in mice. We found that CCND1 localizes to teratoma-susceptible germ cells that fail to enter G1/G0 arrest during the mitotic:meiotic switch and is the only D-type cyclin misexpressed during this critical developmental time frame. We discovered that Ccnd1 deficiency in teratoma-susceptible mice significantly reduced teratoma incidence and suppressed the germ cell proliferation and pluripotency abnormalities associated with tumor initiation. Importantly, Ccnd1 expression was dispensable for somatic cell development and male germ cell specification and maturation in tumor-susceptible mice, implying that the mechanisms by which Ccnd1 deficiency reduced teratoma incidence were germ cell autonomous and specific to tumorigenesis. We conclude that misexpression of Ccnd1 in male germ cells is a key component of a larger pro-proliferative program that disrupts the mitotic:meiotic switch and predisposes 129 inbred mice to testicular teratocarcinogenesis. PMID:26901436
Zebrafish cdc6 hypomorphic mutation causes Meier-Gorlin syndrome-like phenotype.

PubMed

Yao, Likun; Chen, Jing; Wu, Xiaotong; Jia, Shunji; Meng, Anming

2017-11-01

Cell Division Cycle 6 (Cdc6) is a component of pre-replicative complex (preRC) forming on DNA replication origins in eukaryotes. Recessive mutations in ORC1, ORC4, ORC6, CDT1 or CDC6 of the preRC in human cause Meier-Gorlin syndrome (MGS) that is characterized by impaired post-natal growth, short stature and microcephaly. However, vertebrate models of MGS have not been reported. Through N-ethyl-N-nitrosourea mutagenesis and Cas9 knockout, we generate several cdc6 mutant lines in zebrafish. Loss-of-function mutations of cdc6, as manifested by cdc6tsu4305 and cdc6tsu7cd mutants, lead to embryonic lethality due to cell cycle arrest at the S phase and extensive apoptosis. Embryos homozygous for a cdc6 hypomorphic mutation, cdc6tsu21cd, develop normally during embryogenesis. Later on, compared with their wild-type (WT) siblings, cdc6tsu21cd mutant fish show growth retardation, and their body weight and length in adulthood are greatly reduced, which resemble human MGS. Surprisingly, cdc6tsu21cd mutant fish become males with a short life and fail to mate with WT females, suggesting defective reproduction. Overexpression of Cdc6 mutant forms, which mimic human CDC6(T323R) mutation found in a MGS patient, in zebrafish cdc6tsu4305 mutant embryos partially represses cell death phenotype, suggesting that the human CDC6(T323R) mutation is a hypomorph. cdc6tsu21cd mutant fish will be useful to detect more tissue defects and develop medical treatment strategies for MGS patients. © The Author 2017. Published by Oxford University Press.
Does calcium influx regulate melatonin production through the circadian pacemaker in chick pineal cells? Effects of nitrendipine, Bay K 8644, Co2+, Mn2+, and low external Ca2+.

PubMed

Zatz, M; Mullen, D A

1988-11-01

We have recently described a system, using dispersed chick pineal cells in static culture, which displays a persistent, photosensitive, circadian rhythm of melatonin production and release. Here, we describe the effects of nitrendipine (NTR) (a dihydropyridine 'antagonist' of L-type calcium channels), Bay K 8644 (BK) (a dihydropyridine calcium channel 'agonist'), cobalt and manganese ions (both inorganic calcium channel blockers), and low external calcium concentrations, on the melatonin rhythm. NTR inhibited and BK stimulated melatonin output; they were potent and effective. Co2+, Mn2+, and low external Ca2+ markedly inhibited melatonin output. These results support a role for calcium influx through voltage-dependent calcium channels (L-type) in the regulation of melatonin production. Four or 8 h pulses of white light or darkness, in otherwise constant red light, cause, in addition to acute effects, phase-dependent phase shifts of the melatonin rhythm in subsequent cycles. Such phase shifts indicate an effect on (proximal to) the pacemaker generating the rhythm. Four or 8 h pulses of NTR, BK, Co2+, or low Ca2+, however, did not appreciably alter the phase of subsequent melatonin cycles. Neither did BK interfere with phase shifts induced by light pulses. Mn2+ pulses did induce phase-dependent phase shifts, but, unlike those evoked by light or dark pulses, these were all delays. Such effects of Mn2+ in other systems have been attributed to, and are characteristic of, 'metabolic inhibitors'. On balance, the results fail to support a prominent role for calcium influx in regulating the pacemaker underlying the circadian rhythm in chick pineal cells. Rather, calcium influx appears to regulate melatonin production primarily by acting on the melatonin-synthesizing apparatus, distal to the pacemaker.
Zebrafish cdc6 hypomorphic mutation causes Meier-Gorlin syndrome-like phenotype

PubMed Central

Yao, Likun; Chen, Jing; Wu, Xiaotong; Jia, Shunji; Meng, Anming

2017-01-01

Abstract Cell Division Cycle 6 (Cdc6) is a component of pre-replicative complex (preRC) forming on DNA replication origins in eukaryotes. Recessive mutations in ORC1, ORC4, ORC6, CDT1 or CDC6 of the preRC in human cause Meier-Gorlin syndrome (MGS) that is characterized by impaired post-natal growth, short stature and microcephaly. However, vertebrate models of MGS have not been reported. Through N-ethyl-N-nitrosourea mutagenesis and Cas9 knockout, we generate several cdc6 mutant lines in zebrafish. Loss-of-function mutations of cdc6, as manifested by cdc6tsu4305 and cdc6tsu7cd mutants, lead to embryonic lethality due to cell cycle arrest at the S phase and extensive apoptosis. Embryos homozygous for a cdc6 hypomorphic mutation, cdc6tsu21cd, develop normally during embryogenesis. Later on, compared with their wild-type (WT) siblings, cdc6tsu21cd mutant fish show growth retardation, and their body weight and length in adulthood are greatly reduced, which resemble human MGS. Surprisingly, cdc6tsu21cd mutant fish become males with a short life and fail to mate with WT females, suggesting defective reproduction. Overexpression of Cdc6 mutant forms, which mimic human CDC6(T323R) mutation found in a MGS patient, in zebrafish cdc6tsu4305 mutant embryos partially represses cell death phenotype, suggesting that the human CDC6(T323R) mutation is a hypomorph. cdc6tsu21cd mutant fish will be useful to detect more tissue defects and develop medical treatment strategies for MGS patients. PMID:28985365
Mitigating Thermal Runaway Risk in Lithium Ion Batteries

NASA Technical Reports Server (NTRS)

Darcy, Eric; Jeevarajan, Judy; Russell, Samuel

2014-01-01

The JSC/NESC team has successfully demonstrated Thermal Runaway (TR) risk reduction in a lithium ion battery for human space flight by developing and implementing verifiable design features which interrupt energy transfer between adjacent electrochemical cells. Conventional lithium ion (li-Ion) batteries can fail catastrophically as a result of a single cell going into thermal runaway. Thermal runaway results when an internal component fails to separate electrode materials leading to localized heating and complete combustion of the lithium ion cell. Previously, the greatest control to minimize the probability of cell failure was individual cell screening. Combining thermal runaway propagation mitigation design features with a comprehensive screening program reduces both the probability, and the severity, of a single cell failure.
Inhibitor effects during the cell cycle in Chlamydomonas reinhardtii. Determination of transition points in asynchronous cultures

PubMed Central

1975-01-01

A wide variety of inhibitors (drugs, antibiotics, and antimetabolites) will block cell division within an ongoing cell cycle in autotrophic cultures of Chlamydomonas reinhardtii. To determine when during the cell cycle a given inhibitor is effective in preventing cell division, a technique is described which does not rely on the use of synchronous cultures. The technique permits the measurement of transition points, the cell cycle stage at which the subsequent cell division becomes insensitive to the effects of an inhibitor. A map of transition points in the cell cycle reveals that they are grouped into two broad periods, the second and fourth quarters. In general, inhibitors which block organellar DNA, RNA, and protein synthesis have second-quarter transition points, while those which inhibit nuclear cytoplasmic macromolecular synthesis have fourth-quarter transition points. The specific grouping of these transition points into two periods suggests that the synthesis of organellar components is completed midway through the cell cycle and that the synthesis of nonorganellar components required for cell division is not completed until late in the cell cycle. PMID:1176526
Identification of Primary Transcriptional Regulation of Cell Cycle-Regulated Genes upon DNA Damage

PubMed Central

Zhou, Tong; Chou, Jeff; Mullen, Thomas E.; Elkon, Rani; Zhou, Yingchun; Simpson, Dennis A.; Bushel, Pierre R.; Paules, Richard S.; Lobenhofer, Edward K.; Hurban, Patrick; Kaufmann, William K.

2007-01-01

The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation (IR)-treated normal human fibroblasts identified 150 of 406 cycle-regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization. PMID:17404513
The Yeast Cyclin-Dependent Kinase Routes Carbon Fluxes to Fuel Cell Cycle Progression.

PubMed

Ewald, Jennifer C; Kuehne, Andreas; Zamboni, Nicola; Skotheim, Jan M

2016-05-19

Cell division entails a sequence of processes whose specific demands for biosynthetic precursors and energy place dynamic requirements on metabolism. However, little is known about how metabolic fluxes are coordinated with the cell division cycle. Here, we examine budding yeast to show that more than half of all measured metabolites change significantly through the cell division cycle. Cell cycle-dependent changes in central carbon metabolism are controlled by the cyclin-dependent kinase (Cdk1), a major cell cycle regulator, and the metabolic regulator protein kinase A. At the G1/S transition, Cdk1 phosphorylates and activates the enzyme Nth1, which funnels the storage carbohydrate trehalose into central carbon metabolism. Trehalose utilization fuels anabolic processes required to reliably complete cell division. Thus, the cell cycle entrains carbon metabolism to fuel biosynthesis. Because the oscillation of Cdk activity is a conserved feature of the eukaryotic cell cycle, we anticipate its frequent use in dynamically regulating metabolism for efficient proliferation. Copyright © 2016 Elsevier Inc. All rights reserved.
Towards Predicting the Response of a Solid Tumour to Chemotherapy and Radiotherapy Treatments: Clinical Insights from a Computational Model

PubMed Central

Powathil, Gibin G.; Adamson, Douglas J. A.; Chaplain, Mark A. J.

2013-01-01

In this paper we use a hybrid multiscale mathematical model that incorporates both individual cell behaviour through the cell-cycle and the effects of the changing microenvironment through oxygen dynamics to study the multiple effects of radiation therapy. The oxygenation status of the cells is considered as one of the important prognostic markers for determining radiation therapy, as hypoxic cells are less radiosensitive. Another factor that critically affects radiation sensitivity is cell-cycle regulation. The effects of radiation therapy are included in the model using a modified linear quadratic model for the radiation damage, incorporating the effects of hypoxia and cell-cycle in determining the cell-cycle phase-specific radiosensitivity. Furthermore, after irradiation, an individual cell's cell-cycle dynamics are intrinsically modified through the activation of pathways responsible for repair mechanisms, often resulting in a delay/arrest in the cell-cycle. The model is then used to study various combinations of multiple doses of cell-cycle dependent chemotherapies and radiation therapy, as radiation may work better by the partial synchronisation of cells in the most radiosensitive phase of the cell-cycle. Moreover, using this multi-scale model, we investigate the optimum sequencing and scheduling of these multi-modality treatments, and the impact of internal and external heterogeneity on the spatio-temporal patterning of the distribution of tumour cells and their response to different treatment schedules. PMID:23874170
A Multiplexed High-Content Screening Approach Using the Chromobody Technology to Identify Cell Cycle Modulators in Living Cells.

PubMed

Schorpp, Kenji; Rothenaigner, Ina; Maier, Julia; Traenkle, Bjoern; Rothbauer, Ulrich; Hadian, Kamyar

2016-10-01

Many screening hits show relatively poor quality regarding later efficacy and safety. Therefore, small-molecule screening efforts shift toward high-content analysis providing more detailed information. Here, we describe a novel screening approach to identify cell cycle modulators with low toxicity by combining the Cell Cycle Chromobody (CCC) technology with the CytoTox-Glo (CTG) cytotoxicity assay. The CCC technology employs intracellularly functional single-domain antibodies coupled to a fluorescent protein (chromobodies) to visualize the cell cycle-dependent redistribution of the proliferating cell nuclear antigen (PCNA) in living cells. This image-based cell cycle analysis was combined with determination of dead-cell protease activity in cell culture supernatants by the CTG assay. We adopted this multiplex approach to high-throughput format and screened 960 Food and Drug Administration (FDA)-approved drugs. By this, we identified nontoxic compounds, which modulate different cell cycle stages, and validated selected hits in diverse cell lines stably expressing CCC. Additionally, we independently validated these hits by flow cytometry as the current state-of-the-art format for cell cycle analysis. This study demonstrates that CCC imaging is a versatile high-content screening approach to identify cell cycle modulators, which can be multiplexed with cytotoxicity assays for early elimination of toxic compounds during screening. © 2016 Society for Laboratory Automation and Screening.
Cell cycle activation in p21 dependent pathway: An alternative mechanism of organophosphate induced dopaminergic neurodegeneration.

PubMed

Wani, Willayat Yousuf; Kandimalla, Ramesh J L; Sharma, Deep Raj; Kaushal, Alka; Ruban, Anand; Sunkaria, Aditya; Vallamkondu, Jayalakshmi; Chiarugi, Alberto; Reddy, P Hemachandra; Gill, Kiran Dip

2017-07-01

In the previous study, we demonstrated that dichlorvos induces oxidative stress in dopaminergic neuronal cells and subsequent caspase activation mediates apoptosis. In the present study, we evaluated the effect and mechanism of dichlorvos induced oxidative stress on cell cycle activation in NGF-differentiated PC12 cells. Dichlorvos exposure resulted in oxidative DNA damage along with activation of cell cycle machinery in differentiated PC12 cells. Dichlorvos exposed cells exhibited an increased expression of p53, cyclin-D1, pRb and decreased expression of p21suggesting a re-entry of differentiated cells into the cell cycle. Cell cycle analysis of dichlorvos exposed cells revealed a reduction of cells in the G 0 /G 1 phase of the cell cycle (25%), and a concomitant increase of cells in S phase (30%) and G2/M phase (43.3%) compared to control PC12 cells. Further, immunoblotting of cytochrome c, Bax, Bcl-2 and cleaved caspase-3 revealed that dichlorvos induces a caspase-dependent cell death in PC12 cells. These results suggest that Dichlorvos exposure has the potential to generate oxidative stress which evokes activation of cell cycle machinery leading to apoptotic cell death via cytochrome c release from mitochondria and subsequent caspase-3 activation in differentiated PC12 cells. Copyright © 2016 Elsevier B.V. All rights reserved.
Cryosurgery of genital warts in cases in which podophyllin treatment failed or was contraindicated.

PubMed Central

Ghosh, A K

1977-01-01

A total of 56 cases with warts in which podophyllin treatment was either contraindicated or a failure, were treated with nitrous oxide using a Keymed 500 cryosurgical appliance. Forty-three patients who completed treatment were followed-up for three months. Thirty-seven of these had received applications of podophyllin twice weekly for an average period of three months before cryosurgery. The remaining cases in whom podophyllin was considered to be contraindicated included five with vulval warts (two were pregnant, two were bronchial asthmatics taking oral prednisolone, and one was taking oral clomiphene citrate), and a diabetic patients with penile warts. Thirty-two were cured and 11 relapsed. The latter were re-treated at the end of the follow-up period with two freezing cycles of 45 seconds at an interval of 30 minutes. Seven were cured and the remaining four men who failed to respond belonged to the podophyllin-resistant group and included three with metal warts and one homosexual with anogenital warts. Cryosurgery gave a cumulative success rate of 91%. A single freezing cycle was free from complications but a double freezing cycle was often followed by severe local reaction. Images PMID:843897
Decoupling of Nuclear Division Cycles and Cell Size during the Coenocytic Growth of the Ichthyosporean Sphaeroforma arctica.

PubMed

Ondracka, Andrej; Dudin, Omaya; Ruiz-Trillo, Iñaki

2018-06-18

Coordination of the cell division cycle with the growth of the cell is critical to achieve cell size homeostasis [1]. Mechanisms coupling the cell division cycle with cell growth have been described across diverse eukaryotic taxa [2-4], but little is known about how these processes are coordinated in organisms that undergo more complex life cycles, such as coenocytic growth. Coenocytes (multinucleate cells formed by sequential nuclear divisions without cytokinesis) are commonly found across the eukaryotic kingdom, including in animal and plant tissues and several lineages of unicellular eukaryotes [5]. Among the organisms that form coenocytes are ichthyosporeans, a lineage of unicellular holozoans that are of significant interest due to their phylogenetic placement as one of the closest relatives of animals [6]. Here, we characterize the coenocytic cell division cycle in the ichthyosporean Sphaeroforma arctica. We observe that, in laboratory conditions, S. arctica cells undergo a uniform and easily synchronizable coenocytic cell cycle, reaching up to 128 nuclei per cell before cellularization and release of daughter cells. Cycles of nuclear division occur synchronously within the coenocyte and in regular time intervals (11-12 hr). We find that the growth of cell volume is dependent on concentration of nutrients in the media; in contrast, the rate of nuclear division cycles is constant over a range of nutrient concentrations. Together, the results suggest that nuclear division cycles in the coenocytic growth of S. arctica are driven by a timer, which ensures periodic and synchronous nuclear cycles independent of the cell size and growth. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.
Real-time tracking of cell cycle progression during CD8+ effector and memory T-cell differentiation

PubMed Central

Kinjyo, Ichiko; Qin, Jim; Tan, Sioh-Yang; Wellard, Cameron J.; Mrass, Paulus; Ritchie, William; Doi, Atsushi; Cavanagh, Lois L.; Tomura, Michio; Sakaue-Sawano, Asako; Kanagawa, Osami; Miyawaki, Atsushi; Hodgkin, Philip D.; Weninger, Wolfgang

2015-01-01

The precise pathways of memory T-cell differentiation are incompletely understood. Here we exploit transgenic mice expressing fluorescent cell cycle indicators to longitudinally track the division dynamics of individual CD8+ T cells. During influenza virus infection in vivo, naive T cells enter a CD62Lintermediate state of fast proliferation, which continues for at least nine generations. At the peak of the anti-viral immune response, a subpopulation of these cells markedly reduces their cycling speed and acquires a CD62Lhi central memory cell phenotype. Construction of T-cell family division trees in vitro reveals two patterns of proliferation dynamics. While cells initially divide rapidly with moderate stochastic variations of cycling times after each generation, a slow-cycling subpopulation displaying a CD62Lhi memory phenotype appears after eight divisions. Phenotype and cell cycle duration are inherited by the progeny of slow cyclers. We propose that memory precursors cell-intrinsically modulate their proliferative activity to diversify differentiation pathways. PMID:25709008
Real-time tracking of cell cycle progression during CD8+ effector and memory T-cell differentiation.

PubMed

Kinjyo, Ichiko; Qin, Jim; Tan, Sioh-Yang; Wellard, Cameron J; Mrass, Paulus; Ritchie, William; Doi, Atsushi; Cavanagh, Lois L; Tomura, Michio; Sakaue-Sawano, Asako; Kanagawa, Osami; Miyawaki, Atsushi; Hodgkin, Philip D; Weninger, Wolfgang

2015-02-24

The precise pathways of memory T-cell differentiation are incompletely understood. Here we exploit transgenic mice expressing fluorescent cell cycle indicators to longitudinally track the division dynamics of individual CD8(+) T cells. During influenza virus infection in vivo, naive T cells enter a CD62L(intermediate) state of fast proliferation, which continues for at least nine generations. At the peak of the anti-viral immune response, a subpopulation of these cells markedly reduces their cycling speed and acquires a CD62L(hi) central memory cell phenotype. Construction of T-cell family division trees in vitro reveals two patterns of proliferation dynamics. While cells initially divide rapidly with moderate stochastic variations of cycling times after each generation, a slow-cycling subpopulation displaying a CD62L(hi) memory phenotype appears after eight divisions. Phenotype and cell cycle duration are inherited by the progeny of slow cyclers. We propose that memory precursors cell-intrinsically modulate their proliferative activity to diversify differentiation pathways.
Identification of Cell Cycle-regulated Genes in Fission YeastD⃞

PubMed Central

Peng, Xu; Karuturi, R. Krishna Murthy; Miller, Lance D.; Lin, Kui; Jia, Yonghui; Kondu, Pinar; Wang, Long; Wong, Lim-Soon; Liu, Edison T.; Balasubramanian, Mohan K.; Liu, Jianhua

2005-01-01

Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements. Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found ∼140 genes that are cell cycle regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Our complete data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/CDC. PMID:15616197

Altered cell cycle-related gene expression in brain and lymphocytes from a transgenic mouse model of Alzheimer's disease [amyloid precursor protein/presenilin 1 (PS1)].

PubMed

Esteras, Noemí; Bartolomé, Fernando; Alquézar, Carolina; Antequera, Desireé; Muñoz, Úrsula; Carro, Eva; Martín-Requero, Ángeles

2012-09-01

Cumulative evidence indicates that aberrant re-expression of many cell cycle-related proteins and inappropriate neuronal cell cycle control are critical events in Alzheimer's disease (AD) pathogenesis. Evidence of cell cycle activation in post-mitotic neurons has also been observed in murine models of AD, despite the fact that most of these mice do not show massive loss of neuronal bodies. Dysfunction of the cell cycle appears to affect cells other than neurons, as peripheral cells, such as lymphocytes and fibroblasts from patients with AD, show an altered response to mitogenic stimulation. We sought to determine whether cell cycle disturbances are present simultaneously in both brain and peripheral cells from the amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD, in order to validate the use of peripheral cells from patients not only to study cell cycle abnormalities as a pathogenic feature of AD, but also as a means to test novel therapeutic approaches. By using cell cycle pathway-specific RT(2)Profiler™ PCR Arrays, we detected changes in a number of cell cycle-related genes in brain as well as in lymphocytes from APP/PS1 mice. Moreover, we found enhanced 5'-bromo-2'-deoxyuridine incorporation into DNA in lymphocytes from APP/PS1 mice, and increased expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA), and the cyclin-dependent kinase (CDK) inhibitor Cdkn2a, as detected by immunohistochemistry in cortical neurons of the APP/PS1 mice. Taken together, the cell cycle-related changes in brain and blood cells reported here support the mitosis failure hypothesis in AD and validate the use of peripheral cells as surrogate tissue to study the molecular basis of AD pathogenesis. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.
Pseudolaric Acid B Induced Cell Cycle Arrest, Autophagy and Senescence in Murine Fibrosarcoma L929 Cell

PubMed Central

hua Yu, Jing; yu Liu, Chun; bin Zheng, Gui; Zhang, Li Ying; hui Yan, Ming; yan Zhang, Wen; ying Meng, Xian; fang Yu, Xiao

2013-01-01

Objective: PAB induced various cancer cell apoptosis, cell cycle arrest and senescence. But in cell line murine fibrosarcoma L929, PAB did not induce apoptosis, but autophagy, therefore it was thought by us as a good model to research the relationship of cell cycle arrest, autophagy and senescence bypass apoptosis. Methods: Inhibitory ratio was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Phase contrast microscopy visualized cell morphology. Hoechst 33258 staining for nuclear change, propidium iodode (PI) staining for cell cycle, monodansylcadaverine (MDC) staining for autophagy, and rodanmine 123 staining for mitochondrial membrane potential (MMP) were measured by fluorescence microscopy or flowcytometry. Apoptosis was determined by DNA ladder test. Protein kinase C (PKC) activity was detected by PKC assay kit. SA-β-galactosidase assay was used to detect senescence. Protein expression was examined by western blot. Results: PAB inhibited L929 cell growth in time-and dose-dependent manner. At 12 h, 80 μmol/L PAB induced obvious mitotic arrest; at 24 h, PAB began to induce autophagy; at 36 h, cell-treated with PAB slip into G1 cell cycle; and 3 d PAB induced senescence. In time sequence PAB induced firstly cell cycle arrest, then autophagy, then slippage into G1 phase, lastly senescence. Senescent cells had high level of autophagy, inhibiting autophagy led to apoptosis, and no senescence. PAB activated PKC activity to induce cell cycle arrest, autophagy and senescence, inhibiting PKC activity suppressed cell cycle arrest, autophagy and senescence. Conclusion: PAB induced cell cycle arrest, autophagy and senescence in murine fibrosarcoma L929 cell through PKC. PMID:23630435
ATRX Plays a Key Role in Maintaining Silencing at Interstitial Heterochromatic Loci and Imprinted Genes

PubMed Central

Voon, Hsiao P.J.; Hughes, Jim R.; Rode, Christina; De La Rosa-Velázquez, Inti A.; Jenuwein, Thomas; Feil, Robert; Higgs, Douglas R.; Gibbons, Richard J.

2015-01-01

Summary Histone H3.3 is a replication-independent histone variant, which replaces histones that are turned over throughout the entire cell cycle. H3.3 deposition at euchromatin is dependent on HIRA, whereas ATRX/Daxx deposits H3.3 at pericentric heterochromatin and telomeres. The role of H3.3 at heterochromatic regions is unknown, but mutations in the ATRX/Daxx/H3.3 pathway are linked to aberrant telomere lengthening in certain cancers. In this study, we show that ATRX-dependent deposition of H3.3 is not limited to pericentric heterochromatin and telomeres but also occurs at heterochromatic sites throughout the genome. Notably, ATRX/H3.3 specifically localizes to silenced imprinted alleles in mouse ESCs. ATRX KO cells failed to deposit H3.3 at these sites, leading to loss of the H3K9me3 heterochromatin modification, loss of repression, and aberrant allelic expression. We propose a model whereby ATRX-dependent deposition of H3.3 into heterochromatin is normally required to maintain the memory of silencing at imprinted loci. PMID:25865896
The spindle protein CHICA mediates localization of the chromokinesin Kid to the mitotic spindle.

PubMed

Santamaria, Anna; Nagel, Susanna; Sillje, Herman H W; Nigg, Erich A

2008-05-20

Microtubule-based motor proteins provide essential forces for bipolar organization of spindle microtubules and chromosome movement, prerequisites of chromosome segregation during the cell cycle. Here, we describe the functional characterization of a novel spindle protein, termed "CHICA," that was originally identified in a proteomic survey of the human spindle apparatus [1]. We show that CHICA localizes to the mitotic spindle and is both upregulated and phosphorylated during mitosis. CHICA-depleted cells form shorter spindles and fail to organize a proper metaphase plate, highly reminiscent of the phenotype observed upon depletion of the chromokinesin Kid, a key mediator of polar ejection forces [2-6]. We further show that CHICA coimmunoprecipitates with Kid and is required for the spindle localization of Kid without affecting its chromosome association. Moreover, upon depletion of either CHICA or Kid (or both proteins simultaneously), chromosomes collapse onto the poles of monastrol-induced monopolar spindles. We conclude that CHICA represents a novel interaction partner of the chromokinesin Kid that is required for the generation of polar ejection forces and chromosome congression.
SAMHD1 controls cell cycle status, apoptosis and HIV-1 infection in monocytic THP-1 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bonifati, Serena; Daly, Michele B.; St Gelais, Corine

SAMHD1 limits HIV-1 infection in non-dividing myeloid cells by decreasing intracellular dNTP pools. HIV-1 restriction by SAMHD1 in these cells likely prevents activation of antiviral immune responses and modulates viral pathogenesis, thus highlighting a critical role of SAMHD1 in HIV-1 physiopathology. Here, we explored the function of SAMHD1 in regulating cell proliferation, cell cycle progression and apoptosis in monocytic THP-1 cells. Using the CRISPR/Cas9 technology, we generated THP-1 cells with stable SAMHD1 knockout. We found that silencing of SAMHD1 in cycling cells stimulates cell proliferation, redistributes cell cycle population in the G{sub 1}/G{sub 0} phase and reduces apoptosis. These alterationsmore » correlated with increased dNTP levels and more efficient HIV-1 infection in dividing SAMHD1 knockout cells relative to control. Our results suggest that SAMHD1, through its dNTPase activity, affects cell proliferation, cell cycle distribution and apoptosis, and emphasize a key role of SAMHD1 in the interplay between cell cycle regulation and HIV-1 infection.« less
Potential antitumor agent from the endophytic fungus Pestalotiopsis photiniae induces apoptosis via the mitochondrial pathway in HeLa cells.

PubMed

Chen, Chuan; Hu, Shu-Yuan; Luo, Du-Qiang; Zhu, Si-Yu; Zhou, Chuan-Qi

2013-10-01

4-(3',3'-Dimethylallyloxy)-5-methyl-6-methoxy-phthalide (DMMP) has previously been isolated from the endophytic fungus Pestalotiopsis photiniae. Although the cytotoxic activities of DMMP have been reported, little is known concerning the molecular mechanism of its cytotoxic effect. In the present study, we investigated the effect of DMMP on the growth of several types of cancer cell lines and investigated the mechanism of its antiproliferative effect. DMMP caused the growth inhibition of human cancer lines HeLa, MCF7 and MDA-MB-231, but had little antiproliferative effect on MRC5 normal lung cells. DMMP also significantly caused cell cycle arrest in the G1 phase and upregulated the cyclin-dependent kinase inhibitor p27KIPI protein in the HeLa cells. Moreover DMMP was able to induce marked nuclear apoptotic morphology in HeLa cells. DMMP induced apoptosis and loss of mitochondrial membrane potential (ΔΨm) in the HeLa cells. Although the activated forms of caspase-9 and -3 in HeLa cells were detected, pretreatment with caspase inhibitors (Ac-DEVD-CHO and Z-VAD-FMK) failed to attenuate DMMP-induced cell death. In addition, protein levels of the p53 family members, p53 and p73, were upregulated, and DMMP significantly increased the mRNA expression of pro-apoptotic Bcl-2 family genes (PUMA, NOXA, Bax, Bad and Bim). HPV E6-E7 mRNA levels were reduced. In conclusion, DMMP demonstrates potential for use in the treatment of cervical cancer.
Heterokaryon analysis of muscle differentiation: regulation of the postmitotic state.

PubMed

Clegg, C H; Hauschka, S D

1987-08-01

MM14 mouse myoblasts withdraw irreversibly from the cell cycle and become postmitotic within a few hours of being deprived of fibroblast growth factor (Clegg, C. H., T. A. Linkhart, B. B. Olwin, and S. D. Hauschka, 1987, J. Cell Biol., 105:949-956). To examine the mechanisms that may regulate this developmental state of skeletal muscle, we tested the mitogen responsiveness of various cell types after their polyethylene glycol-mediated fusion with post-mitotic myocytes. Heterokaryons containing myocytes and quiescent nonmyogenic cells such as 3T3, L cell, and a differentiation-defective myoblast line (DD-1) responded to mitogen-rich medium by initiating DNA synthesis. Myonuclei replicated DNA and reexpressed thymidine kinase. In contrast, (myocyte x G1 myoblast) heterokaryons failed to replicate DNA in mitogen-rich medium and became postmitotic. This included cells with a nuclear ratio of three myoblasts to one myocyte. Proliferation dominance in (myocyte x 3T3 cell) and (myocyte x DD-1) heterokaryons was conditionally regulated by the timing of mitogen treatment; such cells became postmitotic when mitogen exposure was delayed for as little as 6 h after cell fusion. In addition, (myocyte x DD-1) heterokaryons expressed a muscle-specific trait and lost epidermal growth factor receptors when they became postmitotic. These results demonstrate that DNA synthesis is not irreversibly blocked in skeletal muscle; myonuclei readily express proliferation-related functions when provided with a mitogenic signal. Rather, myocyte-specific repression of DNA synthesis in heterokaryons argues that the postmitotic state of skeletal muscle is regulated by diffusible factors that inhibit processes of cellular mitogenesis.
Rethinking cell-cycle-dependent gene expression in Schizosaccharomyces pombe.

PubMed

Cooper, Stephen

2017-11-01

Three studies of gene expression during the division cycle of Schizosaccharomyces pombe led to the proposal that a large number of genes are expressed at particular times during the S. pombe cell cycle. Yet only a small fraction of genes proposed to be expressed in a cell-cycle-dependent manner are reproducible in all three published studies. In addition to reproducibility problems, questions about expression amplitudes, cell-cycle timing of expression, synchronization artifacts, and the problem with methods for synchronizing cells must be considered. These problems and complications prompt the idea that caution should be used before accepting the conclusion that there are a large number of genes expressed in a cell-cycle-dependent manner in S. pombe.
Molecular machinery of signal transduction and cell cycle regulation in Plasmodium.

PubMed

Koyama, Fernanda C; Chakrabarti, Debopam; Garcia, Célia R S

2009-05-01

The regulation of the Plasmodium cell cycle is not understood. Although the Plasmodium falciparum genome is completely sequenced, about 60% of the predicted proteins share little or no sequence similarity with other eukaryotes. This feature impairs the identification of important proteins participating in the regulation of the cell cycle. There are several open questions that concern cell cycle progression in malaria parasites, including the mechanism by which multiple nuclear divisions is controlled and how the cell cycle is managed in all phases of their complex life cycle. Cell cycle synchrony of the parasite population within the host, as well as the circadian rhythm of proliferation, are striking features of some Plasmodium species, the molecular basis of which remains to be elucidated. In this review we discuss the role of indole-related molecules as signals that modulate the cell cycle in Plasmodium and other eukaryotes, and we also consider the possible role of kinases in the signal transduction and in the responses it triggers.
The Rts1 Regulatory Subunit of Protein Phosphatase 2A Is Required for Control of G1 Cyclin Transcription and Nutrient Modulation of Cell Size

PubMed Central

Artiles, Karen; Anastasia, Stephanie; McCusker, Derek; Kellogg, Douglas R.

2009-01-01

The key molecular event that marks entry into the cell cycle is transcription of G1 cyclins, which bind and activate cyclin-dependent kinases. In yeast cells, initiation of G1 cyclin transcription is linked to achievement of a critical cell size, which contributes to cell-size homeostasis. The critical cell size is modulated by nutrients, such that cells growing in poor nutrients are smaller than cells growing in rich nutrients. Nutrient modulation of cell size does not work through known critical regulators of G1 cyclin transcription and is therefore thought to work through a distinct pathway. Here, we report that Rts1, a highly conserved regulatory subunit of protein phosphatase 2A (PP2A), is required for normal control of G1 cyclin transcription. Loss of Rts1 caused delayed initiation of bud growth and delayed and reduced accumulation of G1 cyclins. Expression of the G1 cyclin CLN2 from an inducible promoter rescued the delayed bud growth in rts1Δ cells, indicating that Rts1 acts at the level of transcription. Moreover, loss of Rts1 caused altered regulation of Swi6, a key component of the SBF transcription factor that controls G1 cyclin transcription. Epistasis analysis revealed that Rts1 does not work solely through several known critical upstream regulators of G1 cyclin transcription. Cells lacking Rts1 failed to undergo nutrient modulation of cell size. Together, these observations demonstrate that Rts1 is a key player in pathways that link nutrient availability, cell size, and G1 cyclin transcription. Since Rts1 is highly conserved, it may function in similar pathways in vertebrates. PMID:19911052
Dermal Aged and Fetal Fibroblasts Realign in Response to Mechanical Strain

NASA Technical Reports Server (NTRS)

Sawyer, Christine; Grymes, Rose; Alvarez, Teresa (Technical Monitor)

1994-01-01

Integrins specifically recognize and bind extracellular matrix components, providing physical anchor points and functional setpoints. Focal adhesion complexes, containing integrin and cytoskeletal proteins, are potential mechanoreceptors, poised to distribute applied forces through the cytoskeleton. Pursuing the hypothesis that cells both perceive and respond to external force, we applied a stretch/relaxation regimen to normal human fetal and aged dermal fibroblast monolayers cultured on flexible membranes. The frequency and magnitude of the applied force is precisely controlled by the Flexercell Unit(Trademark). A protocol of stretch (20% elongation of the monolayer) at a frequency of 6 cycles/min caused a progressive change from a randomly distributed pattern of cells to a symmetric, radial distribution with cells aligned parallel to the applied force. We have coined the term 'orienteering' as the process of active alignment of cells in response to applied force. Cytochalasin D was added in graded doses to investigate the role of the actin cytoskeleton in force perception and transmission. A clear dose response was found; at high concentrations orienteering was abolished; and the drug's impact was reversible. The two cell strains used were similar in their alignment behavior and in their responses to cytochalasin D. Orienteering was influenced by cell density, and the cell strains studied differed in this respect. Fetal cells, unlike their aged counterparts, failed to orient at high cell density. In both cell strains, mid-density cultures aligned rapidly and sparse cultures lagged. These results indicate that both cell-cell adhesion and cytoskeleton integrity are critical in mediating the orienteering response. Differences between these two cell strains may relate to their expression of extracellular matrix molecules (fibronectin, collagen type 1) integrins and their relative binding affinities.
In vitro synergistic antitumor efficacy of sequentially combined chemotherapy/icotinib in non‑small cell lung cancer cell lines.

PubMed

Wang, Min-Cong; Liang, Xuan; Liu, Zhi-Yan; Cui, Jie; Liu, Ying; Jing, Li; Jiang, Li-Li; Ma, Jie-Qun; Han, Li-Li; Guo, Qian-Qian; Yang, Cheng-Cheng; Wang, Jing; Wu, Tao; Nan, Ke-Jun; Yao, Yu

2015-01-01

The concurrent administration of chemotherapy and epidermal growth factor receptor‑tyrosine kinase inhibitors (EGFR‑TKIs) has previously produced a negative interaction and failed to confer a survival benefit to non‑small cell lung cancer (NSCLC) patients compared with first‑line cytotoxic chemotherapy. The present study aimed to investigate the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib in NSCLC cell lines and clarify the underlying mechanisms. HCC827, H1975, H1299 and A549 human NSCLC cell lines with wild‑type and mutant EGFR genes were used as in vitro models to define the differential effects of various schedules of cisplatin/paclitaxel with icotinib treatments on cell growth, proliferation, cell cycle distribution, apoptosis, and EGFR signaling pathway. Sequence‑dependent antiproliferative effects differed among the four NSCLC cell lines, and were not associated with EGFR mutation, constitutive expression levels of EGFR or downstream signaling molecules. The antiproliferative effect of cisplatin plus paclitaxel followed by icotinib was superior to that of cisplatin or paclitaxel followed by icotinib in the HCC827, H1975, H1299 and A549 cell lines, and induced more cell apoptosis and G0/G1 phase arrest. Cisplatin and paclitaxel significantly increased the expression of EGFR phosphorylation in the HCC827 cell line. However, only paclitaxel increased the expression of EGFR phosphorylation in the H1975 cell line. Cisplatin/paclitaxel followed by icotinib influenced the expression of p‑EGFR and p‑AKT, although the expression of p‑ERK1/2 remained unchanged. The results suggest that the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib differed among the NSCLC cell lines. The results also provide molecular evidence to support clinical treatment strategies for NSCLC patients.
The Abbreviated Pluripotent Cell Cycle

PubMed Central

Kapinas, Kristina; Grandy, Rodrigo; Ghule, Prachi; Medina, Ricardo; Becker, Klaus; Pardee, Arthur; Zaidi, Sayyed K.; Lian, Jane; Stein, Janet; van Wijnen, Andre; Stein, Gary

2013-01-01

Human embryonic stem cells and induced pluripotent stem cells proliferate rapidly and divide symmetrically producing equivalent progeny cells. In contrast, lineage committed cells acquire an extended symmetrical cell cycle. Self-renewal of tissue-specific stem cells is sustained by asymmetric cell division where one progeny cell remains a progenitor while the partner progeny cell exits the cell cycle and differentiates. There are three principal contexts for considering the operation and regulation of the pluripotent cell cycle: temporal, regulatory andstructural. The primary temporal context that the pluripotent self-renewal cell cycle of human embryonic stem cells (hESCs) is a short G1 period without reducing periods of time allocated to S phase, G2, and mitosis. The rules that govern proliferation in hESCs remain to be comprehensively established. However, several lines of evidence suggest a key role for the naïve transcriptome of hESCs, which is competent to stringently regulate the ESC cell cycle. This supports the requirements of pluripotent cells to self propagate while suppressing expression of genes that confer lineage commitment and/or tissue specificity. However, for the first time, we consider unique dimensions to the architectural organization and assembly of regulatory machinery for gene expression in nuclear microenviornments that define parameters of pluripotency. From both fundamental biological and clinical perspectives, understanding control of the abbreviated embryonic stem cell cycle can provide options to coordinate control of proliferation versus differentiation. Wound healing, tissue engineering, and cell-based therapy to mitigate developmental aberrations illustrate applications that benefit from knowledge of the biology of the pluripotent cell cycle. PMID:22552993
Proteomic analysis of the bacterial cell cycle

PubMed Central

Grünenfelder, Björn; Rummel, Gabriele; Vohradsky, Jiri; Röder, Daniel; Langen, Hanno; Jenal, Urs

2001-01-01

A global approach was used to analyze protein synthesis and stability during the cell cycle of the bacterium Caulobacter crescentus. Approximately one-fourth (979) of the estimated C. crescentus gene products were detected by two-dimensional gel electrophoresis, 144 of which showed differential cell cycle expression patterns. Eighty-one of these proteins were identified by mass spectrometry and were assigned to a wide variety of functional groups. Pattern analysis revealed that coexpression groups were functionally clustered. A total of 48 proteins were rapidly degraded in the course of one cell cycle. More than half of these unstable proteins were also found to be synthesized in a cell cycle-dependent manner, establishing a strong correlation between rapid protein turnover and the periodicity of the bacterial cell cycle. This is, to our knowledge, the first evidence for a global role of proteolysis in bacterial cell cycle control. PMID:11287652
Regulation of steroid hormone receptors and coregulators during the cell cycle highlights potential novel function in addition to roles as transcription factors

PubMed Central

Zheng, Yingfeng; Murphy, Leigh C.

2016-01-01

Cell cycle progression is tightly controlled by several kinase families including Cyclin-Dependent Kinases, Polo-Like Kinases, and Aurora Kinases. A large amount of data show that steroid hormone receptors and various components of the cell cycle, including cell cycle regulated kinases, interact, and this often results in altered transcriptional activity of the receptor. Furthermore, steroid hormones, through their receptors, can also regulate the transcriptional expression of genes that are required for cell cycle regulation. However, emerging data suggest that steroid hormone receptors may have roles in cell cycle progression independent of their transcriptional activity. The following is a review of how steroid receptors and their coregulators can regulate or be regulated by the cell cycle machinery, with a particular focus on roles independent of transcription in G2/M. PMID:26778927
Regulation of cell division cycle progression by bcl-2 expression: a potential mechanism for inhibition of programmed cell death

PubMed Central

1996-01-01

Expression of the bcl-2 gene has been shown to effectively confer resistance to programmed cell death under a variety of circumstances. However, despite a wealth of literature describing this phenomenon, very little is known about the mechanism of resistance. In the experiments described here, we show that bcl-2 gene expression can result in an inhibition of cell division cycle progression. These findings are based upon the analysis of cell cycle distribution, cell cycle kinetics, and relative phosphorylation of the retinoblastoma tumor suppressor protein, using primary tissues in vivo, ex vivo, and in vitro, as well as continuous cell lines. The effects of bcl-2 expression on cell cycle progression appear to be focused at the G1 to S phase transition, which is a critical control point in the decision between continued cell cycle progression or the induction programmed cell death. In all systems tested, bcl-2 expression resulted in a substantial 30-60% increase in the length of G1 phase; such an increase is very substantial in the context of other regulators of cell cycle progression. Based upon our findings, and the related findings of others, we propose a mechanism by which bcl-2 expression might exert its well known inhibition of programmed cell death by regulating the kinetics of cell cycle progression at a critical control point. PMID:8642331
Targeted Approaches to Overcoming Endocrine Resistance in Breast Cancer

DTIC Science & Technology

2011-08-01

NM_001012271 BUB1 BUB1 budding uninhibited by benzimidazoles 1 homolog AF053305 CDC20 Cell division cycle 20 homolog BG256659 CDC25B Cell division cycle...by benzimidazoles 1 homolog), BIRC5/ Survivin, CDCA8 (cell division cycle-associated protein 8), AURKB (aurora kinase B), CDC25B (cell division cycle
Circadian clock regulation of the cell cycle in the zebrafish intestine.

PubMed

Peyric, Elodie; Moore, Helen A; Whitmore, David

2013-01-01

The circadian clock controls cell proliferation in a number of healthy tissues where cell renewal and regeneration are critical for normal physiological function. The intestine is an organ that typically undergoes regular cycles of cell division, differentiation and apoptosis as part of its role in digestion and nutrient absorption. The aim of this study was to explore circadian clock regulation of cell proliferation and cell cycle gene expression in the zebrafish intestine. Here we show that the zebrafish gut contains a directly light-entrainable circadian pacemaker, which regulates the daily timing of mitosis. Furthermore, this intestinal clock controls the expression of key cell cycle regulators, such as cdc2, wee1, p21, PCNA and cdk2, but only weakly influences cyclin B1, cyclin B2 and cyclin E1 expression. Interestingly, food deprivation has little impact on circadian clock function in the gut, but dramatically reduces cell proliferation, as well as cell cycle gene expression in this tissue. Timed feeding under constant dark conditions is able to drive rhythmic expression not only of circadian clock genes, but also of several cell cycle genes, suggesting that food can entrain the clock, as well as the cell cycle in the intestine. Rather surprisingly, we found that timed feeding is critical for high amplitude rhythms in cell cycle gene expression, even when zebrafish are maintained on a light-dark cycle. Together these results suggest that the intestinal clock integrates multiple rhythmic cues, including light and food, to function optimally.
Circadian Clock Regulation of the Cell Cycle in the Zebrafish Intestine

PubMed Central

Peyric, Elodie; Moore, Helen A.; Whitmore, David

2013-01-01

The circadian clock controls cell proliferation in a number of healthy tissues where cell renewal and regeneration are critical for normal physiological function. The intestine is an organ that typically undergoes regular cycles of cell division, differentiation and apoptosis as part of its role in digestion and nutrient absorption. The aim of this study was to explore circadian clock regulation of cell proliferation and cell cycle gene expression in the zebrafish intestine. Here we show that the zebrafish gut contains a directly light-entrainable circadian pacemaker, which regulates the daily timing of mitosis. Furthermore, this intestinal clock controls the expression of key cell cycle regulators, such as cdc2, wee1, p21, PCNA and cdk2, but only weakly influences cyclin B1, cyclin B2 and cyclin E1 expression. Interestingly, food deprivation has little impact on circadian clock function in the gut, but dramatically reduces cell proliferation, as well as cell cycle gene expression in this tissue. Timed feeding under constant dark conditions is able to drive rhythmic expression not only of circadian clock genes, but also of several cell cycle genes, suggesting that food can entrain the clock, as well as the cell cycle in the intestine. Rather surprisingly, we found that timed feeding is critical for high amplitude rhythms in cell cycle gene expression, even when zebrafish are maintained on a light-dark cycle. Together these results suggest that the intestinal clock integrates multiple rhythmic cues, including light and food, to function optimally. PMID:24013905
ARTD1 regulates cyclin E expression and consequently cell-cycle re-entry and G1/S progression in T24 bladder carcinoma cells.

PubMed

Léger, Karolin; Hopp, Ann-Katrin; Fey, Monika; Hottiger, Michael O

2016-08-02

ADP-ribosylation is involved in a variety of biological processes, many of which are chromatin-dependent and linked to important functions during the cell cycle. However, any study on ADP-ribosylation and the cell cycle faces the problem that synchronization with chemical agents or by serum starvation and subsequent growth factor addition already activates ADP-ribosylation by itself. Here, we investigated the functional contribution of ARTD1 in cell cycle re-entry and G1/S cell cycle progression using T24 urinary bladder carcinoma cells, which synchronously re-enter the cell cycle after splitting without any additional stimuli. In synchronized cells, ARTD1 knockdown, but not inhibition of its enzymatic activity, caused specific down-regulation of cyclin E during cell cycle re-entry and G1/S progression through alterations of the chromatin composition and histone acetylation, but not of other E2F-1 target genes. Although Cdk2 formed a functional complex with the residual cyclin E, p27(Kip 1) protein levels increased in G1 upon ARTD1 knockdown most likely due to inappropriate cyclin E-Cdk2-induced phosphorylation-dependent degradation, leading to decelerated G1/S progression. These results provide evidence that ARTD1 regulates cell cycle re-entry and G1/S progression via cyclin E expression and p27(Kip 1) stability independently of its enzymatic activity, uncovering a novel cell cycle regulatory mechanism.

Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion

PubMed Central

JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K.; Keyomarsi, Khandan

2016-01-01

ABSTRACT Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen. PMID:27049344
Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion.

PubMed

JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan

2016-06-17

Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.
High-throughput synchronization of mammalian cell cultures by spiral microfluidics.

PubMed

Lee, Wong Cheng; Bhagat, Ali Asgar S; Lim, Chwee Teck

2014-01-01

The development of mammalian cell cycle synchronization techniques has greatly advanced our understanding of many cellular regulatory events and mechanisms specific to different phases of the cell cycle. In this chapter, we describe a high-throughput microfluidic-based approach for cell cycle synchronization. By exploiting the relationship between cell size and its phase in the cell cycle, large numbers of synchronized cells can be obtained by size fractionation in a spiral microfluidic channel. Protocols for the synchronization of primary cells such as mesenchymal stem cells, and immortal cell lines such as Chinese hamster ovarian cells (CHO-CD36) and HeLa cells are provided as examples.
Cell cycle progression is an essential regulatory component of phospholipid metabolism and membrane homeostasis

PubMed Central

Sanchez-Alvarez, Miguel; Zhang, Qifeng; Finger, Fabian; Wakelam, Michael J. O.; Bakal, Chris

2015-01-01

We show that phospholipid anabolism does not occur uniformly during the metazoan cell cycle. Transition to S-phase is required for optimal mobilization of lipid precursors, synthesis of specific phospholipid species and endoplasmic reticulum (ER) homeostasis. Average changes observed in whole-cell phospholipid composition, and total ER lipid content, upon stimulation of cell growth can be explained by the cell cycle distribution of the population. TORC1 promotes phospholipid anabolism by slowing S/G2 progression. The cell cycle stage-specific nature of lipid biogenesis is dependent on p53. We propose that coupling lipid metabolism to cell cycle progression is a means by which cells have evolved to coordinate proliferation with cell and organelle growth. PMID:26333836
Cell cycle progression is an essential regulatory component of phospholipid metabolism and membrane homeostasis.

PubMed

Sanchez-Alvarez, Miguel; Zhang, Qifeng; Finger, Fabian; Wakelam, Michael J O; Bakal, Chris

2015-09-01

We show that phospholipid anabolism does not occur uniformly during the metazoan cell cycle. Transition to S-phase is required for optimal mobilization of lipid precursors, synthesis of specific phospholipid species and endoplasmic reticulum (ER) homeostasis. Average changes observed in whole-cell phospholipid composition, and total ER lipid content, upon stimulation of cell growth can be explained by the cell cycle distribution of the population. TORC1 promotes phospholipid anabolism by slowing S/G2 progression. The cell cycle stage-specific nature of lipid biogenesis is dependent on p53. We propose that coupling lipid metabolism to cell cycle progression is a means by which cells have evolved to coordinate proliferation with cell and organelle growth. © 2015 The Authors.
Coordinating cell proliferation and differentiation: Antagonism between cell cycle regulators and cell type-specific gene expression

PubMed Central

Ruijtenberg, Suzan; van den Heuvel, Sander

2016-01-01

ABSTRACT Cell proliferation and differentiation show a remarkable inverse relationship. Precursor cells continue division before acquiring a fully differentiated state, while terminal differentiation usually coincides with proliferation arrest and permanent exit from the division cycle. Mechanistic insight in the temporal coordination between cell cycle exit and differentiation has come from studies of cells in culture and genetic animal models. As initially described for skeletal muscle differentiation, temporal coordination involves mutual antagonism between cyclin-dependent kinases that promote cell cycle entry and transcription factors that induce tissue-specific gene expression. Recent insights highlight the contribution of chromatin-regulating complexes that act in conjunction with the transcription factors and determine their activity. In particular SWI/SNF chromatin remodelers contribute to dual regulation of cell cycle and tissue-specific gene expression during terminal differentiation. We review the concerted regulation of the cell cycle and cell type-specific transcription, and discuss common mutations in human cancer that emphasize the clinical importance of proliferation versus differentiation control. PMID:26825227
Redox Changes During the Cell Cycle in the Embryonic Root Meristem of Arabidopsis thaliana.

PubMed

de Simone, Ambra; Hubbard, Rachel; de la Torre, Natanael Viñegra; Velappan, Yazhini; Wilson, Michael; Considine, Michael J; Soppe, Wim J J; Foyer, Christine H

2017-12-20

The aim of this study was to characterize redox changes in the nuclei and cytosol occurring during the mitotic cell cycle in the embryonic roots of germinating Arabidopsis seedlings, and to determine how redox cycling was modified in mutants with a decreased capacity for ascorbate synthesis. Using an in vivo reduction-oxidation (redox) reporter (roGFP2), we show that transient oxidation of the cytosol and the nuclei occurred at G1 in the synchronized dividing cells of the Arabidopsis root apical meristem, with reduction at G2 and mitosis. This redox cycle was absent from low ascorbate mutants in which nuclei were significantly more oxidized than controls. The cell cycle-dependent increase in nuclear size was impaired in the ascorbate-deficient mutants, which had fewer cells per unit area in the root proliferation zone. The transcript profile of the dry seeds and size of the imbibed seeds was strongly influenced by low ascorbate but germination, dormancy release and seed aging characteristics were unaffected. These data demonstrate the presence of a redox cycle within the plant cell cycle and that the redox state of the nuclei is an important factor in cell cycle progression. Controlled oxidation is a key feature of the early stages of the plant cell cycle. However, sustained mild oxidation restricts nuclear functions and impairs progression through the cell cycle leading to fewer cells in the root apical meristem. Antioxid. Redox Signal. 27, 1505-1519.
Dihydroartemisinin inhibits indoxyl sulfate (IS)-promoted cell cycle progression in mesangial cells by targeting COX-2/mPGES-1/PGE2 cascade.

PubMed

Mungun, Harr-Keshauve; Li, Shuzhen; Zhang, Yue; Huang, Songming; Jia, Zhanjun; Ding, Guixia; Zhang, Aihua

2018-01-01

Dihydroartemisinin (DHA) is a semisynthetic derivative of artemisinin and has been used as an antimalarial drug. Recently, roles of artemisinin and its derivatives in treating diseases besides antimalarial effect were documented. Thus, this study was undertaken to investigate the role of DHA in indoxyl sulfate (IS)-promoted cell cycle progression in glomerular mesangial cells, as well as the potential mechanisms. Under the basal condition, DHA significantly retarded the cell cycle progression as shown by decreased cell percentage in S phase and increased cell percentage in G1/G0 phases in line with reduced cell cycle proteins cyclin A2 and cyclin D1. Interestingly, DHA also inactivated the COX-2/mPGES-1/PGE 2 cascade which has been shown to play a critical role in promoting the mesangial cell cycle progression by our previous studies. Next, we investigated the role of DHA in IS-triggered cell cycle progression in this mesangial cell line. As expected, DHA treatment significantly retarded IS-induced cell cycle progression and inhibited the activation of COX-2/mPGES-1/PGE 2 cascade induced by IS. In summary, these data indicated that DHA inhibited the cell cycle progression in glomerular mesangial cells under normal condition or IS challenge possibly through the inhibition of COX-2/mPGES-1/PGE 2 cascade, suggesting a potential of DHA in treating glomerular diseases with mesangial cell proliferation.
A 'new' Cromer-related high frequency antigen probably antithetical to WES.

PubMed

Daniels, G L; Green, C A; Darr, F W; Anderson, H; Sistonen, P

1987-01-01

An antibody to a high frequency antigen, made in a WES+ Black antenatal patient (Wash.), failed to react with the red cells of a presumed WES+ homozygote and is, therefore, probably antithetical to anti-WES. Like anti-WES, it reacted with papain, ficin, trypsin or neuraminidase treated cells but not with alpha-chymotrypsin or pronase treated cells and was specifically inhibited by concentrated serum. It also reacted more strongly in titration with WES- cells than with WES+ cells. The antibody is Cromer-related as it failed to react with Inab phenotype (IFC-) cells and reacted only weakly with Dr(a-) cells. Wash. cells and those of the other possible WES+ homozygote are Cr(a+) Tc(a+b-c-) Dr(a+) IFC+ but reacted only very weakly with anti-Esa.
Mitochondrial Bioenergetics and Dysfunction in Failing Heart.

PubMed

Sheeran, Freya L; Pepe, Salvatore

2017-01-01

Energy insufficiency has been recognized as a key feature of systolic heart failure. Although mitochondria have long been known to sustain myocardial work energy supply, the capacity to therapeutically target mitochondrial bioenergetics dysfunction is hampered by a complex interplay of multiple perturbations that progressively compound causing myocardial failure and collapse. Compared to non-failing human donor hearts, activity rates of complexes I and IV, nicotinamide nucleotide transhydrogenase (NADPH-transhydrogenase, Nnt) and the Krebs cycle enzymes isocitrate dehydrogenase, malate dehydrogenase and aconitase are markedly decreased in end-stage heart failure. Diminished REDOX capacity with lower total glutathione and coenzyme Q 10 levels are also a feature of chronic left ventricular failure. Decreased enzyme activities in part relate to abundant and highly specific oxidative, nitrosylative, and hyperacetylation modifications. In this brief review we highlight that energy deficiency in end-stage failing human left ventricle predominantly involves concomitantly impaired activities of key electron transport chain and Krebs cycle enzymes rather than altered expression of respective genes or proteins. Augmented oxidative modification of these enzyme subunit structures, and the formation of highly reactive secondary metabolites, implicates dysfunction due to diminished capacity for management of mitochondrial reactive oxygen species, which contribute further to progressive decreases in bioenergetic capacity and contractile function in human heart failure.
Durability investigation of a group of strain gage pressure transducers

NASA Technical Reports Server (NTRS)

Lederer, P. S.; Hilten, J. S.

1972-01-01

A durability investigation was conducted on a group of eighteen bonded-wire strain gage pressure transducers with ranges of 0 to 15 psig and 0 to 100 psig using an improved version of a previously developed technique. Some of the transducers were subjected to 40 million pressure cycles at a 5-Hz rate at laboratory ambient conditions, others were cycled at a temperature of 150 F (65.6 C). The largest change in sensitivity observed was 0.22% for a 100-psig transducer subjected to 40 million pressure cycles at 150 F. The largest change in zero pressure output observed was 0.91% FS for the same transducer. None of the transducers failed completely as a result of cycling at or below full scale pressure.
A genome-wide resource of cell cycle and cell shape genes of fission yeast

PubMed Central

Hayles, Jacqueline; Wood, Valerie; Jeffery, Linda; Hoe, Kwang-Lae; Kim, Dong-Uk; Park, Han-Oh; Salas-Pino, Silvia; Heichinger, Christian; Nurse, Paul

2013-01-01

To identify near complete sets of genes required for the cell cycle and cell shape, we have visually screened a genome-wide gene deletion library of 4843 fission yeast deletion mutants (95.7% of total protein encoding genes) for their effects on these processes. A total of 513 genes have been identified as being required for cell cycle progression, 276 of which have not been previously described as cell cycle genes. Deletions of a further 333 genes lead to specific alterations in cell shape and another 524 genes result in generally misshapen cells. Here, we provide the first eukaryotic resource of gene deletions, which describes a near genome-wide set of genes required for the cell cycle and cell shape. PMID:23697806
Systems-level feedback regulation of cell cycle transitions in Ostreococcus tauri.

PubMed

Kapuy, Orsolya; Vinod, P K; Bánhegyi, Gábor; Novák, Béla

2018-05-01

Ostreococcus tauri is the smallest free-living unicellular organism with one copy of each core cell cycle genes in its genome. There is a growing interest in this green algae due to its evolutionary origin. Since O. tauri is diverged early in the green lineage, relatively close to the ancestral eukaryotic cell, it might hold a key phylogenetic position in the eukaryotic tree of life. In this study, we focus on the regulatory network of its cell division cycle. We propose a mathematical modelling framework to integrate the existing knowledge of cell cycle network of O. tauri. We observe that feedback loop regulation of both G1/S and G2/M transitions in O. tauri is conserved, which can make the transition bistable. This is essential to make the transition irreversible as shown in other eukaryotic organisms. By performing sequence analysis, we also predict the presence of the Greatwall/PP2A pathway in the cell cycle of O. tauri. Since O. tauri cell cycle machinery is conserved, the exploration of the dynamical characteristic of the cell division cycle will help in further understanding the regulation of cell cycle in higher eukaryotes. Copyright © 2018 Elsevier Masson SAS. All rights reserved.
Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging

PubMed Central

Gao, Jing; Wang, Feng; Liu, Yanhou; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

2015-01-01

Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction. PMID:25762114
Serial Charging Test on High Capacity Li-Ion Cells for the Orbiter Advanced Hydraulic Power System

NASA Technical Reports Server (NTRS)

Jeevarajan, Judith A.; Irlbeck, Brad

2006-01-01

Although it looks like module level voltage drives the cutoff for charge, the actual cutoff is due to unbalanced cell voltages that drive the module voltage up. Individual cell voltage drives the cutoff for discharge Low resistance cells are the first to reach the low-voltage cutoff Cell-to-Cell voltage differences are generally small and show similar trends for each cycle Increase for a distinct window during charge and at the end of discharge Increase in max to min cell voltage difference with time/cycles Decrease in max to min cell voltage difference during high current pulses with time/cycles Individual cell voltage trends (with respect to other cells) are very repeatable from cycle to cycle, although voltage slowly degrades with time/cycles (resistance growth) Much more difference observed near end of discharge Little change in order of cell voltage (cell with highest voltage to cell with lowest voltage) Temp sensor on the side of cell (between 2 cells) shows much greater rise during discharge than for single cell tests (18 C vs 5 C) Conclusion: Serial Charging of this string of cells is feasible as it has only a minor impact on useful capacity
Comparison of Genome-Wide Binding of MyoD in Normal Human Myogenic Cells and Rhabdomyosarcomas Identifies Regional and Local Suppression of Promyogenic Transcription Factors

PubMed Central

MacQuarrie, Kyle L.; Yao, Zizhen; Fong, Abraham P.; Diede, Scott J.; Rudzinski, Erin R.; Hawkins, Douglas S.

2013-01-01

Rhabdomyosarcoma is a pediatric tumor of skeletal muscle that expresses the myogenic basic helix-loop-helix protein MyoD but fails to undergo terminal differentiation. Prior work has determined that DNA binding by MyoD occurs in the tumor cells, but myogenic targets fail to activate. Using MyoD chromatin immunoprecipitation coupled to high-throughput sequencing and gene expression analysis in both primary human muscle cells and RD rhabdomyosarcoma cells, we demonstrate that MyoD binds in a similar genome-wide pattern in both tumor and normal cells but binds poorly at a subset of myogenic genes that fail to activate in the tumor cells. Binding differences are found both across genomic regions and locally at specific sites that are associated with binding motifs for RUNX1, MEF2C, JDP2, and NFIC. These factors are expressed at lower levels in RD cells than muscle cells and rescue myogenesis when expressed in RD cells. MEF2C is located in a genomic region that exhibits poor MyoD binding in RD cells, whereas JDP2 exhibits local DNA hypermethylation in its promoter in both RD cells and primary tumor samples. These results demonstrate that regional and local silencing of differentiation factors contributes to the differentiation defect in rhabdomyosarcomas. PMID:23230269
Cell cycle gene expression networks discovered using systems biology: Significance in carcinogenesis

PubMed Central

Scott, RE; Ghule, PN; Stein, JL; Stein, GS

2015-01-01

The early stages of carcinogenesis are linked to defects in the cell cycle. A series of cell cycle checkpoints are involved in this process. The G1/S checkpoint that serves to integrate the control of cell proliferation and differentiation is linked to carcinogenesis and the mitotic spindle checkpoint with the development of chromosomal instability. This paper presents the outcome of systems biology studies designed to evaluate if networks of covariate cell cycle gene transcripts exist in proliferative mammalian tissues including mice, rats and humans. The GeneNetwork website that contains numerous gene expression datasets from different species, sexes and tissues represents the foundational resource for these studies (www.genenetwork.org). In addition, WebGestalt, a gene ontology tool, facilitated the identification of expression networks of genes that co-vary with key cell cycle targets, especially Cdc20 and Plk1 (www.bioinfo.vanderbilt.edu/webgestalt). Cell cycle expression networks of such covariate mRNAs exist in multiple proliferative tissues including liver, lung, pituitary, adipose and lymphoid tissues among others but not in brain or retina that have low proliferative potential. Sixty-three covariate cell cycle gene transcripts (mRNAs) compose the average cell cycle network with p = e−13 to e−36. Cell cycle expression networks show species, sex and tissue variability and they are enriched in mRNA transcripts associated with mitosis many of which are associated with chromosomal instability. PMID:25808367
Silencing cytokeratin 18 gene inhibits intracellular replication of Trypanosoma cruzi in HeLa cells but not binding and invasion of trypanosomes.

PubMed

Claser, Carla; Curcio, Marli; de Mello, Samanta M; Silveira, Eduardo V; Monteiro, Hugo P; Rodrigues, Mauricio M

2008-12-17

As an obligatory intracellular parasite, Trypanosoma cruzi, the etiological agent of Chagas' disease, must invade and multiply within mammalian cells. Cytokeratin 18 (CK18) is among the host molecules that have been suggested as a mediator of important events during T. cruzi-host cell interaction. Based on that possibility, we addressed whether RNA interference (RNAi)-mediated down regulation of the CK18 gene could interfere with the parasite life cycle in vitro. HeLa cells transiently transfected with CK18-RNAi had negligible levels of CK18 transcripts, and significantly reduced levels of CK18 protein expression as determined by immunoblotting or immunofluorescence. CK18 negative or positive HeLa cells were invaded equally as well by trypomastigotes of different T. cruzi strains. Also, in CK18 negative or positive cells, parasites recruited host cells lysosomes and escaped from the parasitophorous vacuole equally as well. After that, the growth of amastigotes of the Y or CL-Brener strains, was drastically arrested in CK18 RNAi-treated cells. After 48 hours, the number of amastigotes was several times lower in CK18 RNAi-treated cells when compared to control cells. Simultaneous staining of parasites and CK18 showed that in HeLa cells infected with the Y strain both co-localize. Although the amastigote surface protein-2 contains the domain VTVXNVFLYNR previously described to bind to CK18, in several attempts, we failed to detect binding of a recombinant protein to CK-18. The study demonstrates that silencing CK18 by transient RNAi, inhibits intracellular multiplication of the Y and CL strain of T. cruzi in HeLa cells, but not trypanosome binding and invasion.
Silencing cytokeratin 18 gene inhibits intracellular replication of Trypanosoma cruzi in HeLa cells but not binding and invasion of trypanosomes

PubMed Central

Claser, Carla; Curcio, Marli; de Mello, Samanta M; Silveira, Eduardo V; Monteiro, Hugo P; Rodrigues, Mauricio M

2008-01-01

Background As an obligatory intracellular parasite, Trypanosoma cruzi, the etiological agent of Chagas' disease, must invade and multiply within mammalian cells. Cytokeratin 18 (CK18) is among the host molecules that have been suggested as a mediator of important events during T. cruzi-host cell interaction. Based on that possibility, we addressed whether RNA interference (RNAi)-mediated down regulation of the CK18 gene could interfere with the parasite life cycle in vitro. HeLa cells transiently transfected with CK18-RNAi had negligible levels of CK18 transcripts, and significantly reduced levels of CK18 protein expression as determined by immunoblotting or immunofluorescence. Results CK18 negative or positive HeLa cells were invaded equally as well by trypomastigotes of different T. cruzi strains. Also, in CK18 negative or positive cells, parasites recruited host cells lysosomes and escaped from the parasitophorous vacuole equally as well. After that, the growth of amastigotes of the Y or CL-Brener strains, was drastically arrested in CK18 RNAi-treated cells. After 48 hours, the number of amastigotes was several times lower in CK18 RNAi-treated cells when compared to control cells. Simultaneous staining of parasites and CK18 showed that in HeLa cells infected with the Y strain both co-localize. Although the amastigote surface protein-2 contains the domain VTVXNVFLYNR previously described to bind to CK18, in several attempts, we failed to detect binding of a recombinant protein to CK-18. Conclusion The study demonstrates that silencing CK18 by transient RNAi, inhibits intracellular multiplication of the Y and CL strain of T. cruzi in HeLa cells, but not trypanosome binding and invasion. PMID:19087356
Porcine epidemic diarrhea virus through p53-dependent pathway causes cell cycle arrest in the G0/G1 phase.

PubMed

Sun, Pei; Wu, Haoyang; Huang, Jiali; Xu, Ying; Yang, Feng; Zhang, Qi; Xu, Xingang

2018-05-22

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic Alphacoronavirus, has caused enormous economic losses in the swine industry. p53 protein exists in a wide variety of animal cells, which is involved in cell cycle regulation, apoptosis, cell differentiation and other biological functions. In this study, we investigated the effects of PEDV infection on the cell cycle of Vero cells and p53 activation. The results demonstrated that PEDV infection induces cell cycle arrest at G0/G1 phase in Vero cells, while UV-inactivated PEDV does not cause cell cycle arrest. PEDV infection up-regulates the levels of p21, cdc2, cdk2, cdk4, Cyclin A protein and down-regulates Cyclin E protein. Further research results showed that inhibition of p53 signaling pathway can reverse the cell cycle arrest in G0/G1 phase induced by PEDV infection and cancel out the up-regulation of p21 and corresponding Cyclin/cdk mentioned above. In addition, PEDV infection of the cells synchronized in various stages of cell cycle showed that viral subgenomic RNA and virus titer were higher in the cells released from G0/G1 phase synchronized cells than that in the cells released from the G1/S phase and G2/M phase synchronized or asynchronous cells after 18 h p.i.. This is the first report to demonstrate that the p53-dependent pathway plays an important role in PEDV induced cell cycle arrest and beneficially contributes to viral infection. Copyright © 2018 Elsevier B.V. All rights reserved.

Pharmacodynamic Modeling of Cell Cycle Effects for Gemcitabine and Trabectedin Combinations in Pancreatic Cancer Cells

PubMed Central

Miao, Xin; Koch, Gilbert; Ait-Oudhia, Sihem; Straubinger, Robert M.; Jusko, William J.

2016-01-01

Combinations of gemcitabine and trabectedin exert modest synergistic cytotoxic effects on two pancreatic cancer cell lines. Here, systems pharmacodynamic (PD) models that integrate cellular response data and extend a prototype model framework were developed to characterize dynamic changes in cell cycle phases of cancer cell subpopulations in response to gemcitabine and trabectedin as single agents and in combination. Extensive experimental data were obtained for two pancreatic cancer cell lines (MiaPaCa-2 and BxPC-3), including cell proliferation rates over 0–120 h of drug exposure, and the fraction of cells in different cell cycle phases or apoptosis. Cell cycle analysis demonstrated that gemcitabine induced cell cycle arrest in S phase, and trabectedin induced transient cell cycle arrest in S phase that progressed to G2/M phase. Over time, cells in the control group accumulated in G0/G1 phase. Systems cell cycle models were developed based on observed mechanisms and were used to characterize both cell proliferation and cell numbers in the sub G1, G0/G1, S, and G2/M phases in the control and drug-treated groups. The proposed mathematical models captured well both single and joint effects of gemcitabine and trabectedin. Interaction parameters were applied to quantify unexplainable drug-drug interaction effects on cell cycle arrest in S phase and in inducing apoptosis. The developed models were able to identify and quantify the different underlying interactions between gemcitabine and trabectedin, and captured well our large datasets in the dimensions of time, drug concentrations, and cellular subpopulations. PMID:27895579
Cell cycle pathway dysregulation in human keratinocytes during chronic exposure to low arsenite.

PubMed

Al-Eryani, Laila; Waigel, Sabine; Jala, Venkatakrishna; Jenkins, Samantha F; States, J Christopher

2017-09-15

Arsenic is naturally prevalent in the earth's crust and widely distributed in air and water. Chronic low arsenic exposure is associated with several cancers in vivo, including skin cancer, and with transformation in vitro of cell lines including immortalized human keratinocytes (HaCaT). Arsenic also is associated with cell cycle dysregulation at different exposure levels in multiple cell lines. In this work, we analyzed gene expression in HaCaT cells to gain an understanding of gene expression changes contributing to transformation at an early time point. HaCaT cells were exposed to 0 or 100nM NaAsO 2 for 7weeks. Total RNA was purified and analyzed by microarray hybridization. Differential expression with fold change≥|1.5| and p-value≤0.05 was determined using Partek Genomic Suite™ and pathway and network analyses using MetaCore™ software (FDR≤0.05). Cell cycle analysis was performed using flow cytometry. 644 mRNAs were differentially expressed. Cell cycle/cell cycle regulation pathways predominated in the list of dysregulated pathways. Genes involved in replication origin licensing were enriched in the network. Cell cycle assay analysis showed an increase in G2/M compartment in arsenite-exposed cells. Arsenite exposure induced differential gene expression indicating dysregulation of cell cycle control, which was confirmed by cell cycle analysis. The results suggest that cell cycle dysregulation is an early event in transformation manifested in cells unable to transit G2/M efficiently. Further study at later time points will reveal additional changes in gene expression related to transformation processes. Copyright © 2017 Elsevier Inc. All rights reserved.
Cross-talk between branched-chain amino acids and hepatic mitochondria is compromised in nonalcoholic fatty liver disease.

PubMed

Sunny, Nishanth E; Kalavalapalli, Srilaxmi; Bril, Fernando; Garrett, Timothy J; Nautiyal, Manisha; Mathew, Justin T; Williams, Caroline M; Cusi, Kenneth

2015-08-15

Elevated plasma branched-chain amino acids (BCAA) in the setting of insulin resistance have been relevant in predicting type 2 diabetes mellitus (T2DM) onset, but their role in the etiology of hepatic insulin resistance remains uncertain. We determined the link between BCAA and dysfunctional hepatic tricarboxylic acid (TCA) cycle, which is a central feature of hepatic insulin resistance and nonalcoholic fatty liver disease (NAFLD). Plasma metabolites under basal fasting and euglycemic hyperinsulinemic clamps (insulin stimulation) were measured in 94 human subjects with varying degrees of insulin sensitivity to identify their relationships with insulin resistance. Furthermore, the impact of elevated BCAA on hepatic TCA cycle was determined in a diet-induced mouse model of NAFLD, utilizing targeted metabolomics and nuclear magnetic resonance (NMR)-based metabolic flux analysis. Insulin stimulation revealed robust relationships between human plasma BCAA and indices of insulin resistance, indicating chronic metabolic overload from BCAA. Human plasma BCAA and long-chain acylcarnitines also showed a positive correlation, suggesting modulation of mitochondrial metabolism by BCAA. Concurrently, mice with NAFLD failed to optimally induce hepatic mTORC1, plasma ketones, and hepatic long-chain acylcarnitines, following acute elevation of plasma BCAA. Furthermore, elevated BCAA failed to induce multiple fluxes through hepatic TCA cycle in mice with NAFLD. Our data suggest that BCAA are essential to mediate efficient channeling of carbon substrates for oxidation through mitochondrial TCA cycle. Impairment of BCAA-mediated upregulation of the TCA cycle could be a significant contributor to mitochondrial dysfunction in NAFLD.
Cross-talk between branched-chain amino acids and hepatic mitochondria is compromised in nonalcoholic fatty liver disease

PubMed Central

Kalavalapalli, Srilaxmi; Bril, Fernando; Garrett, Timothy J.; Nautiyal, Manisha; Mathew, Justin T.; Williams, Caroline M.; Cusi, Kenneth

2015-01-01

Elevated plasma branched-chain amino acids (BCAA) in the setting of insulin resistance have been relevant in predicting type 2 diabetes mellitus (T2DM) onset, but their role in the etiology of hepatic insulin resistance remains uncertain. We determined the link between BCAA and dysfunctional hepatic tricarboxylic acid (TCA) cycle, which is a central feature of hepatic insulin resistance and nonalcoholic fatty liver disease (NAFLD). Plasma metabolites under basal fasting and euglycemic hyperinsulinemic clamps (insulin stimulation) were measured in 94 human subjects with varying degrees of insulin sensitivity to identify their relationships with insulin resistance. Furthermore, the impact of elevated BCAA on hepatic TCA cycle was determined in a diet-induced mouse model of NAFLD, utilizing targeted metabolomics and nuclear magnetic resonance (NMR)-based metabolic flux analysis. Insulin stimulation revealed robust relationships between human plasma BCAA and indices of insulin resistance, indicating chronic metabolic overload from BCAA. Human plasma BCAA and long-chain acylcarnitines also showed a positive correlation, suggesting modulation of mitochondrial metabolism by BCAA. Concurrently, mice with NAFLD failed to optimally induce hepatic mTORC1, plasma ketones, and hepatic long-chain acylcarnitines, following acute elevation of plasma BCAA. Furthermore, elevated BCAA failed to induce multiple fluxes through hepatic TCA cycle in mice with NAFLD. Our data suggest that BCAA are essential to mediate efficient channeling of carbon substrates for oxidation through mitochondrial TCA cycle. Impairment of BCAA-mediated upregulation of the TCA cycle could be a significant contributor to mitochondrial dysfunction in NAFLD. PMID:26058864
Physical interaction of human T-cell leukemia virus type 1 Tax with cyclin-dependent kinase 4 stimulates the phosphorylation of retinoblastoma protein.

PubMed

Haller, Kerstin; Wu, Yalin; Derow, Elisabeth; Schmitt, Iris; Jeang, Kuan-Teh; Grassmann, Ralph

2002-05-01

The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) induces leukemia in transgenic mice and permanent T-cell growth in vitro. In transformed lymphocytes, it acts as an essential growth factor. Tax stimulates the cell cycle in the G(1) phase by activating the cyclin-dependent kinase (CDK) CDK4 and CDK6 holoenzyme complexes. Here we show that Tax directly interacts with CDK4. This binding to CDK4 was specific, since Tax did not bind to either CDK2 or CDK1. The interaction with CDK4/cyclin D complexes was observed in vitro, in transfected fibroblasts, in HTLV-1-infected T cells, and in adult T-cell leukemia-derived cultures. Binding studies with several point and deletion mutants indicated that the N terminus of Tax mediates the interaction with CDK4. The Tax/CDK complex represented an active holoenzyme which capably phosphorylates the Rb protein in vitro and is resistant to repression by the inhibitor p21(CIP). Binding-deficient Tax mutants failed to activate CDK4, indicating that direct association with Tax is required for enhanced kinase activity. Tax also increased the association of CDK4 with its positive cyclin regulatory subunit. Thus, protein-protein contact between Tax and the components of the cyclin D/CDK complexes provides a further mechanistic explanation for the mitogenic and immortalizing effects of this HTLV-1 oncoprotein.
Physical Interaction of Human T-Cell Leukemia Virus Type 1 Tax with Cyclin-Dependent Kinase 4 Stimulates the Phosphorylation of Retinoblastoma Protein

PubMed Central

Haller, Kerstin; Wu, Yalin; Derow, Elisabeth; Schmitt, Iris; Jeang, Kuan-Teh; Grassmann, Ralph

2002-01-01

The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) induces leukemia in transgenic mice and permanent T-cell growth in vitro. In transformed lymphocytes, it acts as an essential growth factor. Tax stimulates the cell cycle in the G1 phase by activating the cyclin-dependent kinase (CDK) CDK4 and CDK6 holoenzyme complexes. Here we show that Tax directly interacts with CDK4. This binding to CDK4 was specific, since Tax did not bind to either CDK2 or CDK1. The interaction with CDK4/cyclin D complexes was observed in vitro, in transfected fibroblasts, in HTLV-1-infected T cells, and in adult T-cell leukemia-derived cultures. Binding studies with several point and deletion mutants indicated that the N terminus of Tax mediates the interaction with CDK4. The Tax/CDK complex represented an active holoenzyme which capably phosphorylates the Rb protein in vitro and is resistant to repression by the inhibitor p21CIP. Binding-deficient Tax mutants failed to activate CDK4, indicating that direct association with Tax is required for enhanced kinase activity. Tax also increased the association of CDK4 with its positive cyclin regulatory subunit. Thus, protein-protein contact between Tax and the components of the cyclin D/CDK complexes provides a further mechanistic explanation for the mitogenic and immortalizing effects of this HTLV-1 oncoprotein. PMID:11971966
Architecture and inherent robustness of a bacterial cell-cycle control system.

PubMed

Shen, Xiling; Collier, Justine; Dill, David; Shapiro, Lucy; Horowitz, Mark; McAdams, Harley H

2008-08-12

A closed-loop control system drives progression of the coupled stalked and swarmer cell cycles of the bacterium Caulobacter crescentus in a near-mechanical step-like fashion. The cell-cycle control has a cyclical genetic circuit composed of four regulatory proteins with tight coupling to processive chromosome replication and cell division subsystems. We report a hybrid simulation of the coupled cell-cycle control system, including asymmetric cell division and responses to external starvation signals, that replicates mRNA and protein concentration patterns and is consistent with observed mutant phenotypes. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. Formal model-checking analysis of the digital circuit showed that the cell-cycle control is robust to intrinsic stochastic variations in reaction rates and nutrient supply, and that it reliably stops and restarts to accommodate nutrient starvation. Model checking also showed that mechanisms involving methylation-state changes in regulatory promoter regions during DNA replication increase the robustness of the cell-cycle control. The hybrid cell-cycle simulation implementation is inherently extensible and provides a promising approach for development of whole-cell behavioral models that can replicate the observed functionality of the cell and its responses to changing environmental conditions.
Arachidonic acid induces macrophage cell cycle arrest through the JNK signaling pathway.

PubMed

Shen, Ziying; Ma, Yunqing; Ji, Zhonghao; Hao, Yang; Yan, Xuan; Zhong, Yuan; Tang, Xiaochun; Ren, Wenzhi

2018-02-09

Arachidonic acid (AA) has potent pro-apoptotic effects on cancer cells at a low concentration and on macrophages at a very high concentration. However, the effects of AA on the macrophage cell cycle and related signaling pathways have not been fully investigated. Herein we aim to observe the effect of AA on macrophages cell cycle. AA exposure reduced the viability and number of macrophages in a dose- and time-dependent manner. The reduction in RAW264.7 cell viability was not caused by apoptosis, as indicated by caspase-3 and activated caspase-3 detection. Further research illustrated that AA exposure induced RAW264.7 cell cycle arrested at S phase, and some cell cycle-regulated proteins were altered accordingly. Moreover, JNK signaling was stimulated by AA, and the stimulation was partially reversed by a JNK signaling inhibitor in accordance with cell cycle-related factors. In addition, nuclear and total Foxo1/3a and phosphorylated Foxo1/3a were elevated by AA in a dose- and time-dependent manner, and this elevation was suppressed by the JNK signaling inhibitor. Our study demonstrated that AA inhibits macrophage viability by inducing S phase cell cycle arrest. The JNK signaling pathway and the downstream FoxO transcription factors are involved in AA-induced RAW264.7 cell cycle arrest.
Start-stop testing of two self-acting air-lubricated spiral groove thrust bearing coatings

NASA Technical Reports Server (NTRS)

Dunfee, J. D.; Shapiro, W.

1974-01-01

Start-stop tests were conducted on air-lubricated spiral-groove thrust bearings. Application of a matrix-bonded molybdenum disulfide (MoS2) coating over a porous chrome oxide coating resulted in significantly lower friction, compared to bearings coated with chrome oxide only. The MoS2 coated bearing sustained 15,000 start-stop cycles at a maximum of 3600 rpm. Each cycle was 15 seconds on, 30 seconds off. The chrome oxide coated bearing failed by local welding after 2030 cycles. Both types of coatings exhibited early failures under higher thrust loads when operating films were insufficient to sustain the load without overheating.
A stochastic spatiotemporal model of a response-regulator network in the Caulobacter crescentus cell cycle

NASA Astrophysics Data System (ADS)

Li, Fei; Subramanian, Kartik; Chen, Minghan; Tyson, John J.; Cao, Yang

2016-06-01

The asymmetric cell division cycle in Caulobacter crescentus is controlled by an elaborate molecular mechanism governing the production, activation and spatial localization of a host of interacting proteins. In previous work, we proposed a deterministic mathematical model for the spatiotemporal dynamics of six major regulatory proteins. In this paper, we study a stochastic version of the model, which takes into account molecular fluctuations of these regulatory proteins in space and time during early stages of the cell cycle of wild-type Caulobacter cells. We test the stochastic model with regard to experimental observations of increased variability of cycle time in cells depleted of the divJ gene product. The deterministic model predicts that overexpression of the divK gene blocks cell cycle progression in the stalked stage; however, stochastic simulations suggest that a small fraction of the mutants cells do complete the cell cycle normally.
Cell Cycle Deregulation in the Neurons of Alzheimer’s Disease

PubMed Central

Moh, Calvin; Kubiak, Jacek Z.; Bajic, Vladan P.; Zhu, Xiongwei; Smith, Mark A.

2018-01-01

The cell cycle consists of four main phases: G1, S, G2, and M. Most cells undergo these cycles up to 40–60 times in their life. However, neurons remain in a nondividing, nonreplicating phase, G0. Neurons initiate but do not complete cell division, eventually entering apoptosis. Research has suggested that like cancer, Alzheimer’s disease (AD) involves dysfunction in neuronal cell cycle reentry, leading to the development of the two-hit hypothesis of AD. The first hit is abnormal cell cycle reentry, which typically results in neuronal apoptosis and prevention of AD. However, with the second hit of chronic oxidative damage preventing apoptosis, neurons gain “immortality” analogous to tumor cells. Once both of these hits are activated, AD can develop and produce senile plaques and neurofibrillary tangles throughout brain tissue. In this review, we propose a mechanism for neuronal cell cycle reentry and the development of AD. PMID:21630160
Comprehensive Mass Cytometry Analysis of Cell Cycle, Activation, and Coinhibitory Receptors Expression in CD4 T Cells from Healthy and HIV-Infected Individuals.

PubMed

Corneau, Aurélien; Cosma, Antonio; Even, Sophie; Katlama, Christine; Le Grand, Roger; Frachet, Véronique; Blanc, Catherine; Autran, Brigitte

2017-01-01

Mass cytometry allows large multiplex analysis of cell cycle stages together with differentiation, activation, and exhaustion markers, allowing further assessment of the quiescence status of resting CD4 T cells. Peripheral blood CD4 T lymphocytes from 8 individuals, 4 healthy donors, and 4 HIV-infected on antiretroviral treatment (T) were stained with the same 26 monoclonal antibodies and dyes targeting surface and intracellular markers of differentiation, activation, exhaustion, and cell cycle stages. Samples were run on a CYTOF-2. Patterns of naïve [TN] CD4 T cells strongly differed from all other memory subsets central-memory (CM), transitional-memory (TM), effector-memory (EM), and terminally differentiated RA-expressing (TEMRA) subsets, while stem-cell memory (SCM) and T follicular-helper cells (TfH) were close to CM and TM cells with the highest percentages in cell cycle. EM and TEMRA were the most altered by HIV infection, with an increased frequency of activated and cycling cells. Activation markers and coinhibitory receptor expression differed among cell cycle stages, with HLA-DR fitting better than CD25 or CD38 with cycle, and opposite PD-1 gradients along differentiation and cell cycle. "Resting" DR-CD25- CD4+ T cells contained similar amounts of cells in G1 than the activated DR ± CD25± ones but three fold lower cells in S-G2-M. This broad multiplex mass cytometry analysis demonstrates some subsets of the so-called "resting" CD25-DR- CD4+ T cells contain noticeable amounts of cells into cycle or expressing coinhibitory receptors, opening new avenues for a redefinition of resting peripheral blood CD4 T cells harboring the HIV reservoirs. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.
Redistribution of cell cycle by arsenic trioxide is associated with demethylation and expression changes of cell cycle related genes in acute promyelocytic leukemia cell line (NB4).

PubMed

Hassani, Saeed; Khaleghian, Ali; Ahmadian, Shahin; Alizadeh, Shaban; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir; Ghaffari, Seyed H

2018-01-01

PML-RARα perturbs the normal epigenetic setting, which is essential to oncogenic transformation in acute promyelocytic leukemia (APL). Transcription induction and recruitment of DNA methyltransferases (DNMTs) by PML-RARα and subsequent hypermethylation are components of this perturbation. Arsenic trioxide (ATO), an important drug in APL therapy, concurrent with degradation of PML-RARα induces cell cycle change and apoptosis. How ATO causes cell cycle alteration has remained largely unexplained. Here, we investigated DNA methylation patterns of cell cycle regulatory genes promoters, the effects of ATO on the methylated genes and cell cycle distribution in an APL cell line, NB4. Analysis of promoter methylation status of 22 cell cycle related genes in NB4 revealed that CCND1, CCNE1, CCNF, CDKN1A, GADD45α, and RBL1 genes were methylated 60.7, 84.6, 58.6, 8.7, 33.4, and 73.7%, respectively, that after treatment with 2 μM ATO for 48 h, turn into 0.6, 13.8, 0.1, 6.6, 10.7, and 54.5% methylated. ATO significantly reduced the expression of DNMT1, 3A, and 3B. ATO induced the expression of CCND1, CCNE1, and GADD45α genes, suppressed the expression of CCNF and CDKN1A genes, which were consistent with decreased number of cells in G1 and S phases and increased number of cells in G2/M phase. In conclusion, demethylation and alteration in the expression level of the cell cycle related genes may be possible mechanisms in ATO-induced cell cycle arrest in APL cells. It may suggest that ATO by demethylation of CCND1 and CCNE1 and their transcriptional activation accelerates G1 and S transition into the G2/M cell cycle arrest.
Cell cycle-dependent protein fingerprint from a single cancer cell: image cytometry coupled with single-cell capillary sieving electrophoresis.

PubMed

Hu, Shen; Le, Zhang; Krylov, Sergey; Dovichi, Norman J

2003-07-15

Study of cell cycle-dependent protein expression is important in oncology, stem cell research, and developmental biology. In this paper, we report the first protein fingerprint from a single cell with known phase in the cell cycle. To determine that phase, we treated HT-29 colon cancer cells with Hoescht 33342, a vital nuclear stain. A microscope was used to measure the fluorescence intensity from one treated cell; in this form of image cytometry, the fluorescence intensity is proportional to the cell's DNA content, which varies in a predictable fashion during the cell cycle. To generate the protein fingerprint, the cell was aspirated into the separation capillary and lysed. Proteins were fluorescently labeled with 3-(2-furoylquinoline-2-carboxaldehyde, separated by capillary sieving electrophoresis, and detected by laser-induced fluorescence. This form of electrophoresis is the capillary version of SDS-PAGE. The single-cell electropherogram partially resolved approximately 25 components in a 30-min separation, and the dynamic range of the detector exceeded 5000. There was a large cell-to-cell variation in protein expression, averaging 40% relative standard deviation across the electropherogram. The dominant source of variation was the phase of the cell in the cell cycle; on average, approximately 60% of the cell-to-cell variance in protein expression was associated with the cell cycle. Cells in the G1 and G2/M phases of the cell cycle had 27 and 21% relative standard deviations in protein expression, respectively. Cells in the G2/M phase generated signals that were twice the amplitude of the signals generated by G1 phase cells, as expected for cells that are soon to divide into two daughter cells. When electropherograms were normalized to total protein content, the expression of only one component was dependent on cell cycle at the 99% confidence limit. That protein is tentatively identified as cytokeratin 18 in a companion paper.
Cell cycle-dependent induction of autophagy, mitophagy and reticulophagy.

PubMed

Tasdemir, Ezgi; Maiuri, M Chiara; Tajeddine, Nicolas; Vitale, Ilio; Criollo, Alfredo; Vicencio, José Miguel; Hickman, John A; Geneste, Olivier; Kroemer, Guido

2007-09-15

When added to cells, a variety of autophagy inducers that operate through distinct mechanisms and target different organelles for autophagic destruction (mitochondria in mitophagy, endoplasmic reticulum in reticulophagy) rarely induce autophagic vacuolization in more than 50% or the cells. Here we show that this heterogeneity may be explained by cell cycle-specific effects. The BH3 mimetic ABT737, lithium, rapamycin, tunicamycin or nutrient depletion stereotypically induce autophagy preferentially in the G(1) and S phases of the cell cycle, as determined by simultaneous monitoring of cell cycle markers and the cytoplasmic aggregation of GFP-LC3 in autophagic vacuoles. These results point to a hitherto neglected crosstalk between autophagic vacuolization and cell cycle regulation.
Forecasting of the electrical actuators condition using stator’s current signals

NASA Astrophysics Data System (ADS)

Kruglova, T. N.; Yaroshenko, I. V.; Rabotalov, N. N.; Melnikov, M. A.

2017-02-01

This article describes a forecasting method for electrical actuators realized through the combination of Fourier transformation and neural network techniques. The method allows finding the value of diagnostic functions in the iterating operating cycle and the number of operational cycles in time before the BLDC actuator fails. For forecasting of the condition of the actuator, we propose a hierarchical structure of the neural network aiming to reduce the training time of the neural network and improve estimation accuracy.
Exquisite sensitivity of TP53 mutant and basal breast cancers to a dose-dense epirubicin-cyclophosphamide regimen.

PubMed

Bertheau, Philippe; Turpin, Elisabeth; Rickman, David S; Espié, Marc; de Reyniès, Aurélien; Feugeas, Jean-Paul; Plassa, Louis-François; Soliman, Hany; Varna, Mariana; de Roquancourt, Anne; Lehmann-Che, Jacqueline; Beuzard, Yves; Marty, Michel; Misset, Jean-Louis; Janin, Anne; de Thé, Hugues

2007-03-01

In breast cancers, only a minority of patients fully benefit from the different chemotherapy regimens currently in use. Identification of markers that could predict the response to a particular regimen would thus be critically important for patient care. In cell lines or animal models, tumor protein p53 (TP53) plays a critical role in modulating the response to genotoxic drugs. TP53 is activated in response to DNA damage and triggers either apoptosis or cell-cycle arrest, which have opposite effects on cell fate. Yet, studies linking TP53 status and chemotherapy response have so far failed to unambiguously establish this paradigm in patients. Breast cancers with a TP53 mutation were repeatedly shown to have a poor outcome, but whether this reflects poor response to treatment or greater intrinsic aggressiveness of the tumor is unknown. In this study we analyzed 80 noninflammatory breast cancers treated by frontline (neoadjuvant) chemotherapy. Tumor diagnoses were performed on pretreatment biopsies, and the patients then received six cycles of a dose-dense regimen of 75 mg/m(2) epirubicin and 1,200 mg/m(2) cyclophosphamide, given every 14 days. After completion of chemotherapy, all patients underwent mastectomies, thus allowing for a reliable assessment of chemotherapy response. The pretreatment biopsy samples were used to determine the TP53 status through a highly efficient yeast functional assay and to perform RNA profiling. All 15 complete responses occurred among the 28 TP53-mutant tumors. Furthermore, among the TP53-mutant tumors, nine out of ten of the highly aggressive basal subtypes (defined by basal cytokeratin [KRT] immunohistochemical staining) experienced complete pathological responses, and only TP53 status and basal subtype were independent predictors of a complete response. Expression analysis identified many mutant TP53-associated genes, including CDC20, TTK, CDKN2A, and the stem cell gene PROM1, but failed to identify a transcriptional profile associated with complete responses among TP53 mutant tumors. In patients with unresponsive tumors, mutant TP53 status predicted significantly shorter overall survival. The 15 patients with responsive TP53-mutant tumors, however, had a favorable outcome, suggesting that this chemotherapy regimen can overcome the poor prognosis generally associated with mutant TP53 status. This study demonstrates that, in noninflammatory breast cancers, TP53 status is a key predictive factor for response to this dose-dense epirubicin-cyclophosphamide regimen and further suggests that the basal subtype is exquisitely sensitive to this association. Given the well-established predictive value of complete responses for long-term survival and the poor prognosis of basal and TP53-mutant tumors treated with other regimens, this chemotherapy could be particularly suited for breast cancer patients with a mutant TP53, particularly those with basal features.
Repressive histone methylation regulates cardiac myocyte cell cycle exit.

PubMed

El-Nachef, Danny; Oyama, Kyohei; Wu, Yun-Yu; Freeman, Miles; Zhang, Yiqiang; Robb MacLellan, W

2018-05-22

Mammalian cardiac myocytes (CMs) stop proliferating soon after birth and subsequent heart growth comes from hypertrophy, limiting the adult heart's regenerative potential after injury. The molecular events that mediate CM cell cycle exit are poorly understood. To determine the epigenetic mechanisms limiting CM cycling in adult CMs (ACMs) and whether trimethylation of lysine 9 of histone H3 (H3K9me3), a histone modification associated with repressed chromatin, is required for the silencing of cell cycle genes, we developed a transgenic mouse model where H3K9me3 is specifically removed in CMs by overexpression of histone demethylase, KDM4D. Although H3K9me3 is found across the genome, its loss in CMs preferentially disrupts cell cycle gene silencing. KDM4D binds directly to cell cycle genes and reduces H3K9me3 levels at these promotors. Loss of H3K9me3 preferentially leads to increased cell cycle gene expression resulting in enhanced CM cycling. Heart mass was increased in KDM4D overexpressing mice by postnatal day 14 (P14) and continued to increase until 9-weeks of age. ACM number, but not size, was significantly increased in KDM4D expressing hearts, suggesting CM hyperplasia accounts for the increased heart mass. Inducing KDM4D after normal development specifically in ACMs resulted in increased cell cycle gene expression and cycling. We demonstrated that H3K9me3 is required for CM cell cycle exit and terminal differentiation in ACMs. Depletion of H3K9me3 in adult hearts prevents and reverses permanent cell cycle exit and allows hyperplastic growth in adult hearts in vivo. Copyright © 2017. Published by Elsevier Ltd.
Novel piplartine-containing ruthenium complexes: synthesis, cell growth inhibition, apoptosis induction and ROS production on HCT116 cells.

PubMed

D'Sousa Costa, Cinara O; Araujo Neto, João H; Baliza, Ingrid R S; Dias, Rosane B; Valverde, Ludmila de F; Vidal, Manuela T A; Sales, Caroline B S; Rocha, Clarissa A G; Moreira, Diogo R M; Soares, Milena B P; Batista, Alzir A; Bezerra, Daniel P

2017-11-28

Piplartine (piperlongumine) is a plant-derived molecule that has been receiving intense interest due to its anticancer characteristics that target the oxidative stress. In the present paper, two novel piplartine-containing ruthenium complexes [Ru(piplartine)(dppf)(bipy)](PF 6 ) 2 (1) and [Ru(piplartine)(dppb)(bipy)](PF 6 ) 2 (2) were synthesized and investigated for their cellular and molecular responses on cancer cell lines. We found that both complexes are more potent than metal-free piplartine in a panel of cancer cell lines on monolayer cultures, as well in 3D model of cancer multicellular spheroids formed from human colon carcinoma HCT116 cells. Mechanistic studies uncovered that the complexes reduced the cell growth and caused phosphatidylserine externalization, internucleosomal DNA fragmentation, caspase-3 activation and loss of the mitochondrial transmembrane potential on HCT116 cells. Moreover, the pre-treatment with Z-VAD(OMe)-FMK, a pan-caspase inhibitor, reduced the complexes-induced apoptosis, indicating cell death by apoptosis through caspase-dependent and mitochondrial intrinsic pathways. Treatment with the complexes also caused a marked increase in the production of reactive oxygen species (ROS), including hydrogen peroxide, superoxide anion and nitric oxide, and decreased reduced glutathione levels. Application of N-acetyl-cysteine, an antioxidant, reduced the ROS levels and apoptosis induced by the complexes, indicating activation of ROS-mediated apoptosis pathway. RNA transcripts of several genes, including gene related to the cell cycle, apoptosis and oxidative stress, were regulated under treatment. However, the complexes failed to induce DNA intercalation. In conclusion, the complexes are more potent than piplartine against different cancer cell lines and are able to induce caspase-dependent and mitochondrial intrinsic apoptosis on HCT116 cells by ROS-mediated pathway.
Mitochondrial protection impairs BET bromodomain inhibitor-mediated cell death and provides rationale for combination therapeutic strategies.

PubMed

Lasorsa, E; Smonksey, M; Kirk, J S; Rosario, S; Hernandez-Ilizaliturri, F J; Ellis, L

2015-12-10

Inhibitors of the bromodomain and extraterminal domain family (BETI) have recently entered phase I clinical trials. In patients with advanced leukemia's, potent antileukemia activity was displayed with minimum dose-limiting toxicity. In preclinical models of hematological malignancies, including aggressive B-cell lymphomas, BETI induced cell-cycle arrest and apoptosis. However, the underlying cell death mechanisms are still not well understood. Dissecting the mechanisms required by BETI to mediate cell death would provide strong direction on how to best utilize BETI to treat patients with aggressive hematological malignancies. Herein, we provide understanding of the molecular mechanisms underlying BETI-mediated cell death using I-BET762. Induction of cell death occurred in primary murine and human B-cell lymphomas through apoptosis. Genetic dissection using Eμ-myc B-cell lymphoma compound mutants demonstrated that I-BET762-induced apoptosis does not require the p53 pathway. Furthermore, deletion of Apaf1, and thus the absence of a functional apoptosome, is associated with a delayed drug response but do not provide long-term resistance. Prolonged treatment of this model in fact fails to suppress the therapeutic efficacy of the drug and is associated with biochemical features of autophagy. However, lack of mitochondrial permeability completely inhibited I-BET762-mediated tumor cell death, indicating mitochondrial damage as key events for its activity. Combination of I-BET762 with BH3-only mimetics ABT-263 or obatoclax, restored sensitivity to I-BET762 lymphoma killing; however, success was determined by expression of Bcl-2 family antiapoptotic proteins. Our study provides critical insight for clinical decisions regarding the appropriate strategy for using BETI as a single agent or in combination to treat patients with aggressive B-cell lymphomas.

Morus alba Leaf Lectin (MLL) Sensitizes MCF-7 Cells to Anoikis by Inhibiting Fibronectin Mediated Integrin-FAK Signaling through Ras and Activation of P38 MAPK

PubMed Central

Saranya, Jayaram; Shilpa, Ganesan; Raghu, Kozhiparambil G.; Priya, Sulochana

2017-01-01

Lectins are a unique class of carbohydrate binding proteins/glycoproteins, and many of them possess anticancer properties. They can induce cell cycle arrest and apoptosis, inhibit protein synthesis, telomerase activity and angiogenesis in cancer cells. In the present study, we have demonstrated the effect of Morus alba leaf lectin (MLL) on anoikis induction in MCF-7 cells. Anoikis induction in cancer cells has a significant role in preventing early stage metastasis. MLL treatment in monolayers of MCF-7 cells caused significant detachment of cells in a time and concentration dependent manner. The detached cells failed to re-adhere and grew even to culture plates coated with different matrix proteins. DNA fragmentation, membrane integrity studies, annexin V staining, caspase 9 activation and upregulation of Bax/Bad confirmed that the detached cells underwent apoptosis. Upregulation of matrix metalloproteinase 9 (MMP-9) caused a decrease in fibronectin (FN) production which facilitated the cells to detach by blocking the FN mediated downstream signaling. On treatment with MLL, we have observed downregulation of integrin expression, decreased phosphorylation of focal adhesion kinase (FAK), loss in FAK-integrin interaction and active Ras. MLL treatment downregulated the levels of phosphorylated Akt and PI3K. Also, we have studied the effect of MLL on two stress activated protein kinases p38 MAPK and JNK. p38 MAPK activation was found to be elevated, but there was no change in the level of JNK. Thus our study substantiated the possible antimetastatic effect of MLL by inducing anoikis in MCF-7 cells by activation of caspase 9 and proapoptotic Bax/Bad by blockage of FN mediated integrin/FAK signaling and partly by activation of p38 MAPK. PMID:28223935
The prolyl isomerase Pin1 increases β-cell proliferation and enhances insulin secretion.

PubMed

Nakatsu, Yusuke; Mori, Keiichi; Matsunaga, Yasuka; Yamamotoya, Takeshi; Ueda, Koji; Inoue, Yuki; Mitsuzaki-Miyoshi, Keiko; Sakoda, Hideyuki; Fujishiro, Midori; Yamaguchi, Suguru; Kushiyama, Akifumi; Ono, Hiraku; Ishihara, Hisamitsu; Asano, Tomoichiro

2017-07-14

The prolyl isomerase Pin1 binds to the phosphorylated Ser/Thr-Pro motif of target proteins and enhances their cis-trans conversion. This report is the first to show that Pin1 expression in pancreatic β cells is markedly elevated by high-fat diet feeding and in ob/ob mice. To elucidate the role of Pin1 in pancreatic β cells, we generated β-cell-specific Pin1 KO (βPin1 KO) mice. These mutant mice showed exacerbation of glucose intolerance but had normal insulin sensitivity. We identified two independent factors underlying impaired insulin secretion in the βPin1 KO mice. Pin1 enhanced pancreatic β-cell proliferation, as indicated by a reduced β-cell mass in βPin1 KO mice compared with control mice. Moreover, a diet high in fat and sucrose failed to increase pancreatic β-cell growth in the βPin1 KO mice, an observation to which up-regulation of the cell cycle protein cyclin D appeared to contribute. The other role of Pin1 was to activate the insulin-secretory step: Pin1 KO β cells showed impairments in glucose- and KCl-induced elevation of the intracellular Ca 2+ concentration and insulin secretion. We also identified salt-inducible kinase 2 (SIK2) as a Pin1-binding protein that affected the regulation of Ca 2+ influx and found Pin1 to enhance SIK2 kinase activity, resulting in a decrease in p35 protein, a negative regulator of Ca 2+ influx. Taken together, our observations demonstrate critical roles of Pin1 in pancreatic β cells and that Pin1 both promotes β-cell proliferation and activates insulin secretion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Laser scanning cytometry (LCS) allows detailed analysis of the cell cycle in PI stained human fibroblasts (TIG-7).

PubMed

Kawasaki, M; Sasaki, K; Satoh, T; Kurose, A; Kamada, T; Furuya, T; Murakami, T; Todoroki, T

1997-01-01

We have demonstrated a method for the in situ determination of the cell cycle phases of TIG-7 fibroblasts using a laser scanning cytometer (LSC) which has not only a function equivalent to flow cytometry (FCM) but also has a capability unique in itself. LSC allows a more detailed analysis of the cell cycle in cells stained with propidium iodide (PI) than FCM. With LSC it is possible to discriminate between mitotic cells and G2 cells, between post-mitotic cells and G1 cells, and between quiescent cells and cycling cells in a PI fluorescence peak (chromatin condensation) vs. fluorescence value (DNA content) cytogram for cells stained with PI. These were amply confirmed by experiments using colcemid and adriamycin. We were able to identify at least six cell subpopulations for PI stained cells using LSC; namely G1, S, G2, M, postmitotic and quiescent cell populations. LSC analysis facilitates the monitoring of effects of drugs on the cell cycle.
Checkpoints couple transcription network oscillator dynamics to cell-cycle progression.

PubMed

Bristow, Sara L; Leman, Adam R; Simmons Kovacs, Laura A; Deckard, Anastasia; Harer, John; Haase, Steven B

2014-09-05

The coupling of cyclin dependent kinases (CDKs) to an intrinsically oscillating network of transcription factors has been proposed to control progression through the cell cycle in budding yeast, Saccharomyces cerevisiae. The transcription network regulates the temporal expression of many genes, including cyclins, and drives cell-cycle progression, in part, by generating successive waves of distinct CDK activities that trigger the ordered program of cell-cycle events. Network oscillations continue autonomously in mutant cells arrested by depletion of CDK activities, suggesting the oscillator can be uncoupled from cell-cycle progression. It is not clear what mechanisms, if any, ensure that the network oscillator is restrained when progression in normal cells is delayed or arrested. A recent proposal suggests CDK acts as a master regulator of cell-cycle processes that have the potential for autonomous oscillatory behavior. Here we find that mitotic CDK is not sufficient for fully inhibiting transcript oscillations in arrested cells. We do find that activation of the DNA replication and spindle assembly checkpoints can fully arrest the network oscillator via overlapping but distinct mechanisms. Further, we demonstrate that the DNA replication checkpoint effector protein, Rad53, acts to arrest a portion of transcript oscillations in addition to its role in halting cell-cycle progression. Our findings indicate that checkpoint mechanisms, likely via phosphorylation of network transcription factors, maintain coupling of the network oscillator to progression during cell-cycle arrest.
Regulation of the Embryonic Cell Cycle During Mammalian Preimplantation Development.

PubMed

Palmer, N; Kaldis, P

2016-01-01

The preimplantation development stage of mammalian embryogenesis consists of a series of highly conserved, regulated, and predictable cell divisions. This process is essential to allow the rapid expansion and differentiation of a single-cell zygote into a multicellular blastocyst containing cells of multiple developmental lineages. This period of development, also known as the germinal stage, encompasses several important developmental transitions, which are accompanied by dramatic changes in cell cycle profiles and dynamics. These changes are driven primarily by differences in the establishment and enforcement of cell cycle checkpoints, which must be bypassed to facilitate the completion of essential cell cycle events. Much of the current knowledge in this area has been amassed through the study of knockout models in mice. These mouse models are powerful experimental tools, which have allowed us to dissect the relative dependence of the early embryonic cell cycles on various aspects of the cell cycle machinery and highlight the extent of functional redundancy between members of the same gene family. This chapter will explore the ways in which the cell cycle machinery, their accessory proteins, and their stimuli operate during mammalian preimplantation using mouse models as a reference and how this allows for the usually well-defined stages of the cell cycle to be shaped and transformed during this unique and critical stage of development. © 2016 Elsevier Inc. All rights reserved.
Targeting of cytosolic phospholipase A2α impedes cell cycle re-entry of quiescent prostate cancer cells.

PubMed

Yao, Mu; Xie, Chanlu; Kiang, Mei-Yee; Teng, Ying; Harman, David; Tiffen, Jessamy; Wang, Qian; Sved, Paul; Bao, Shisan; Witting, Paul; Holst, Jeff; Dong, Qihan

2015-10-27

Cell cycle re-entry of quiescent cancer cells has been proposed to be involved in cancer progression and recurrence. Cytosolic phospholipase A2α (cPLA2α) is an enzyme that hydrolyzes membrane glycerophospholipids to release arachidonic acid and lysophospholipids that are implicated in cancer cell proliferation. The aim of this study was to determine the role of cPLA2α in cell cycle re-entry of quiescent prostate cancer cells. When PC-3 and LNCaP cells were rendered to a quiescent state, the active form of cPLA2α with a phosphorylation at Ser505 was lower compared to their proliferating state. Conversely, the phospho-cPLA2α levels were resurgent during the induction of cell cycle re-entry. Pharmacological inhibition of cPLA2α with Efipladib upon induction of cell cycle re-entry inhibited the re-entry process, as manifested by refrained DNA synthesis, persistent high proportion of cells in G0/G1 and low percentage of cells in S and G2/M phases, together with a stagnant recovery of Ki-67 expression. Simultaneously, Efipladib prohibited the emergence of Skp2 while maintained p27 at a high level in the nuclear compartment during cell cycle re-entry. Inhibition of cPLA2α also prevented an accumulation of cyclin D1/CDK4, cyclin E/CDK2, phospho-pRb, pre-replicative complex proteins CDC6, MCM7, ORC6 and DNA synthesis-related protein PCNA during induction of cell cycle re-entry. Moreover, a pre-treatment of the prostate cancer cells with Efipladib during induction of cell cycle re-entry subsequently compromised their tumorigenic capacity in vivo. Hence, cPLA2α plays an important role in cell cycle re-entry by quiescent prostate cancer cells.
Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lo, Yuan-Hung; Ho, Po-Chun; Chen, Min-Shan

Highlights: Black-Right-Pointing-Pointer Proliferating Cell Nuclear Antigen (PCNA) is phosphorylated at Y114. Black-Right-Pointing-Pointer Phospho-Y114 of PCNA is not required for cell proliferation for normal growth. Black-Right-Pointing-Pointer MCE during adipogenesis is abolished in the lack of the phosphorylation. Black-Right-Pointing-Pointer Homozygous Y114F mice are resistant to high fat diet induced obesity. Black-Right-Pointing-Pointer Our results shed light on the interface between proliferation and differentiation. -- Abstract: Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesismore » remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNA{sup F/F}) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNA{sup F/F} MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT mice contain significantly more adipocytes than those isolated from PCNA{sup F/F} mice. This study identifies a critical role for PCNA in adipose tissue development, and for the first time identifies a role of the core DNA replication machinery at the interface between proliferation and differentiation.« less
LPS-induced inflammatory response triggers cell cycle reactivation in murine neuronal cells through retinoblastoma proteins induction.

PubMed

D'Angelo, Barbara; Astarita, Carlo; Boffo, Silvia; Massaro-Giordano, Mina; Antonella Ianuzzi, Carmelina; Caporaso, Antonella; Macaluso, Marcella; Giordano, Antonio

2017-01-01

Cell cycle reactivation in adult neurons is an early hallmark of neurodegeneration. The lipopolysaccharide (LPS) is a well-known pro-inflammatory factor that provokes neuronal cell death via glial cells activation. The retinoblastoma (RB) family includes RB1/p105, retinoblastoma-like 1 (RBL1/p107), and retinoblastoma-like 2 (Rb2/p130). Several studies have indicated that RB proteins exhibit tumor suppressor activities, and play a central role in cell cycle regulation. In this study, we assessed LPS-mediated inflammatory effect on cell cycle reactivation and apoptosis of neuronally differentiated cells. Also, we investigated whether the LPS-mediated inflammatory response can influence the function and expression of RB proteins. Our results showed that LPS challenges triggered cell cycle reactivation of differentiated neuronal cells, indicated by an accumulation of cells in S and G2/M phase. Furthermore, we found that LPS treatment also induced apoptotic death of neurons. Interestingly, we observed that LPS-mediated inflammatory effect on cell cycle re-entry and apoptosis was concomitant with the aberrant expression of RBL1/p107 and RB1/p105. To the best of our knowledge, our study is the first to indicate a role of LPS in inducing cell cycle re-entry and/or apoptosis of differentiated neuronal cells, perhaps through mechanisms altering the expression of specific members of RB family proteins. This study provides novel information on the biology of post-mitotic neurons and could help in identifying novel therapeutic targets to prevent de novo cell cycle reactivation and/or apoptosis of neurons undergoing neurodegenerative processes.
Antigen-Specific CD8+ T Cells Fail To Respond to Shigella flexneri ▿

PubMed Central

Jehl, Stephanie P.; Doling, Amy M.; Giddings, Kara S.; Phalipon, Armelle; Sansonetti, Philippe J.; Goldberg, Marcia B.; Starnbach, Michael N.

2011-01-01

CD8+ T lymphocytes often play a primary role in adaptive immunity to cytosolic microbial pathogens. Surprisingly, CD8+ T cells are not required for protective immunity to the enteric pathogen Shigella flexneri, despite the ability of Shigella to actively secrete proteins into the host cytoplasm, a location from which antigenic peptides are processed for presentation to CD8+ T cells. To determine why CD8+ T cells fail to play a role in adaptive immunity to S. flexneri, we investigated whether antigen-specific CD8+ T cells are primed during infection but are unable to confer protection or, alternatively, whether T cells fail to be primed. To test whether Shigella is capable of stimulating an antigen-specific CD8+ T-cell response, we created an S. flexneri strain that constitutively secretes a viral CD8+ T-cell epitope via the Shigella type III secretion system and characterized the CD8+ T-cell response to this strain both in mice and in cultured cells. Surprisingly, no T cells specific for the viral epitope were stimulated in mice infected with this strain, and cells infected with the recombinant strain were not targeted by epitope-specific T cells. Additionally, we found that the usually robust T-cell response to antigens artificially introduced into the cytoplasm of cultured cells was significantly reduced when the antigen-presenting cell was infected with Shigella. Collectively, these results suggest that antigen-specific CD8+ T cells are not primed during S. flexneri infection and, as a result, afford little protection to the host during primary or subsequent infection. PMID:21357720
Saccharomyces cerevisiae Mob1p Is Required for Cytokinesis and Mitotic Exit

PubMed Central

Luca, Francis C.; Mody, Manali; Kurischko, Cornelia; Roof, David M.; Giddings, Thomas H.; Winey, Mark

2001-01-01

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G1 by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G1 transition to control cyclin-CDK inactivation and cytokinesis. PMID:11564880
Detection of Changes in the Medicago sativa Retinoblastoma-Related Protein (MsRBR1) Phosphorylation During Cell Cycle Progression in Synchronized Cell Suspension Culture.

PubMed

Ayaydin, Ferhan; Kotogány, Edit; Ábrahám, Edit; Horváth, Gábor V

2017-01-01

Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques that allow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell division can be achieved using different plant tissues; however, well-established cell suspension cultures provide large amount of biological sample for further analyses. Here, we describe the methodology of the establishment, propagation, and analysis of a Medicago sativa suspension culture that can be used for efficient synchronization of the cell division. A novel 5-ethynyl-2'-deoxyuridine (EdU)-based method is used for the estimation of cell fraction that enters DNA synthesis phase of the cell cycle and we also demonstrate the changes in the phosphorylation level of Medicago sativa retinoblastoma-related protein (MsRBR1) during cell cycle progression.
Nucleosome architecture throughout the cell cycle

PubMed Central

Deniz, Özgen; Flores, Oscar; Aldea, Martí; Soler-López, Montserrat; Orozco, Modesto

2016-01-01

Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity. PMID:26818620
Investigating steam penetration using thermometric methods in dental handpieces with narrow internal lumens during sterilizing processes with non-vacuum or vacuum processes.

PubMed

Winter, S; Smith, A; Lappin, D; McDonagh, G; Kirk, B

2017-12-01

Dental handpieces are required to be sterilized between patient use. Vacuum steam sterilization processes with fractionated pre/post-vacuum phases or unique cycles for specified medical devices are required for hollow instruments with internal lumens to assure successful air removal. Entrapped air will compromise achievement of required sterilization conditions. Many countries and professional organizations still advocate non-vacuum sterilization processes for these devices. To investigate non-vacuum downward/gravity displacement, type-N steam sterilization of dental handpieces, using thermometric methods to measure time to achieve sterilization temperature at different handpiece locations. Measurements at different positions within air turbines were undertaken with thermocouples and data loggers. Two examples of widely used UK benchtop steam sterilizers were tested: a non-vacuum benchtop sterilizer (Little Sister 3; Eschmann, Lancing, UK) and a vacuum benchtop sterilizer (Lisa; W&H, Bürmoos, Austria). Each sterilizer cycle was completed with three handpieces and each cycle in triplicate. A total of 140 measurements inside dental handpiece lumens were recorded. The non-vacuum process failed (time range: 0-150 s) to reliably achieve sterilization temperatures within the time limit specified by the international standard (15 s equilibration time). The measurement point at the base of the handpiece failed in all test runs (N = 9) to meet the standard. No failures were detected with the vacuum steam sterilization type B process with fractionated pre-vacuum and post-vacuum phases. Non-vacuum downward/gravity displacement, type-N steam sterilization processes are unreliable in achieving sterilization conditions inside dental handpieces, and the base of the handpiece is the site most likely to fail. Copyright © 2017 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
Host Range Restriction of Insect-Specific Flaviviruses Occurs at Several Levels of the Viral Life Cycle.

PubMed

Junglen, Sandra; Korries, Marvin; Grasse, Wolfgang; Wieseler, Janett; Kopp, Anne; Hermanns, Kyra; León-Juárez, Moises; Drosten, Christian; Kümmerer, Beate Mareike

2017-01-01

The genus Flavivirus contains emerging arthropod-borne viruses (arboviruses) infecting vertebrates, as well as insect-specific viruses (ISVs) (i.e., viruses whose host range is restricted to insects). ISVs are evolutionary precursors to arboviruses. Knowledge of the nature of the ISV infection block in vertebrates could identify functions necessary for the expansion of the host range toward vertebrates. Mapping of host restrictions by complementation of ISV and arbovirus genome functions could generate knowledge critical to predicting arbovirus emergence. Here we isolated a novel flavivirus, termed Niénokoué virus (NIEV), from mosquitoes sampled in Côte d'Ivoire. NIEV groups with insect-specific flaviviruses (ISFs) in phylogeny and grows in insect cells but not in vertebrate cells. We generated an infectious NIEV cDNA clone and a NIEV reporter replicon to study growth restrictions of NIEV in comparison to yellow fever virus (YFV), for which the same tools are available. Efficient RNA replication of the NIEV reporter replicon was observed in insect cells but not in vertebrate cells. Initial translation of the input replicon RNA in vertebrate cells was functional, but RNA replication did not occur. Chimeric YFV carrying the envelope proteins of NIEV was recovered via electroporation in C6/36 insect cells but did not infect vertebrate cells, indicating a block at the level of entry. Since the YF/NIEV chimera readily produced infectious particles in insect cells but not in vertebrate cells despite efficient RNA replication, restriction is also determined at the level of assembly/release. Taking the results together, the ability of ISF to infect vertebrates is blocked at several levels, including attachment/entry and RNA replication as well as assembly/release. IMPORTANCE Most viruses of the genus Flavivirus , e.g., YFV and dengue virus, are mosquito borne and transmitted to vertebrates during blood feeding of mosquitoes. Within the last decade, an increasing number of viruses with a host range exclusively restricted to insects in close relationship to the vertebrate-pathogenic flaviviruses were discovered in mosquitoes. To identify barriers that could block the arboviral vertebrate tropism, we set out to identify the steps at which the ISF replication cycle fails in vertebrates. Our studies revealed blocks at several levels, suggesting that flavivirus host range expansion from insects to vertebrates was a complex process that involved overcoming multiple barriers.
Host Range Restriction of Insect-Specific Flaviviruses Occurs at Several Levels of the Viral Life Cycle

PubMed Central

Junglen, Sandra; Korries, Marvin; Grasse, Wolfgang; Wieseler, Janett; Kopp, Anne; Hermanns, Kyra; León-Juárez, Moises; Drosten, Christian

2017-01-01

ABSTRACT The genus Flavivirus contains emerging arthropod-borne viruses (arboviruses) infecting vertebrates, as well as insect-specific viruses (ISVs) (i.e., viruses whose host range is restricted to insects). ISVs are evolutionary precursors to arboviruses. Knowledge of the nature of the ISV infection block in vertebrates could identify functions necessary for the expansion of the host range toward vertebrates. Mapping of host restrictions by complementation of ISV and arbovirus genome functions could generate knowledge critical to predicting arbovirus emergence. Here we isolated a novel flavivirus, termed Niénokoué virus (NIEV), from mosquitoes sampled in Côte d’Ivoire. NIEV groups with insect-specific flaviviruses (ISFs) in phylogeny and grows in insect cells but not in vertebrate cells. We generated an infectious NIEV cDNA clone and a NIEV reporter replicon to study growth restrictions of NIEV in comparison to yellow fever virus (YFV), for which the same tools are available. Efficient RNA replication of the NIEV reporter replicon was observed in insect cells but not in vertebrate cells. Initial translation of the input replicon RNA in vertebrate cells was functional, but RNA replication did not occur. Chimeric YFV carrying the envelope proteins of NIEV was recovered via electroporation in C6/36 insect cells but did not infect vertebrate cells, indicating a block at the level of entry. Since the YF/NIEV chimera readily produced infectious particles in insect cells but not in vertebrate cells despite efficient RNA replication, restriction is also determined at the level of assembly/release. Taking the results together, the ability of ISF to infect vertebrates is blocked at several levels, including attachment/entry and RNA replication as well as assembly/release. IMPORTANCE Most viruses of the genus Flavivirus, e.g., YFV and dengue virus, are mosquito borne and transmitted to vertebrates during blood feeding of mosquitoes. Within the last decade, an increasing number of viruses with a host range exclusively restricted to insects in close relationship to the vertebrate-pathogenic flaviviruses were discovered in mosquitoes. To identify barriers that could block the arboviral vertebrate tropism, we set out to identify the steps at which the ISF replication cycle fails in vertebrates. Our studies revealed blocks at several levels, suggesting that flavivirus host range expansion from insects to vertebrates was a complex process that involved overcoming multiple barriers. PMID:28101536
Cell reprogramming modelled as transitions in a hierarchy of cell cycles

NASA Astrophysics Data System (ADS)

Hannam, Ryan; Annibale, Alessia; Kühn, Reimer

2017-10-01

We construct a model of cell reprogramming (the conversion of fully differentiated cells to a state of pluripotency, known as induced pluripotent stem cells, or iPSCs) which builds on key elements of cell biology viz. cell cycles and cell lineages. Although reprogramming has been demonstrated experimentally, much of the underlying processes governing cell fate decisions remain unknown. This work aims to bridge this gap by modelling cell types as a set of hierarchically related dynamical attractors representing cell cycles. Stages of the cell cycle are characterised by the configuration of gene expression levels, and reprogramming corresponds to triggering transitions between such configurations. Two mechanisms were found for reprogramming in a two level hierarchy: cycle specific perturbations and a noise induced switching. The former corresponds to a directed perturbation that induces a transition into a cycle-state of a different cell type in the potency hierarchy (mainly a stem cell) whilst the latter is a priori undirected and could be induced, e.g. by a (stochastic) change in the cellular environment. These reprogramming protocols were found to be effective in large regimes of the parameter space and make specific predictions concerning reprogramming dynamics which are broadly in line with experimental findings.
Early induction of c-Myc is associated with neuronal cell death.

PubMed

Lee, Hyun-Pil; Kudo, Wataru; Zhu, Xiongwei; Smith, Mark A; Lee, Hyoung-gon

2011-11-14

Neuronal cell cycle activation has been implicated in neurodegenerative diseases such as Alzheimer's disease, while the initiating mechanism of cell cycle activation remains to be determined. Interestingly, our previous studies have shown that cell cycle activation by c-Myc (Myc) leads to neuronal cell death which suggests Myc might be a key regulator of cell cycle re-entry mediated neuronal cell death. However, the pattern of Myc expression in the process of neuronal cell death has not been addressed. To this end, we examined Myc induction by the neurotoxic agents camptothecin and amyloid-β peptide in a differentiated SH-SY5Y neuronal cell culture model. Myc expression was found to be significantly increased following either treatment and importantly, the induction of Myc preceded neuronal cell death suggesting it is an early event of neuronal cell death. Since ectopic expression of Myc in neurons causes the cell cycle activation and neurodegeneration in vivo, the current data suggest that induction of Myc by neurotoxic agents or other disease factors might be a key mediator in cell cycle activation and consequent cell death that is a feature of neurodegenerative diseases. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
A Toxoplasma MORN1 Null Mutant Undergoes Repeated Divisions but Is Defective in Basal Assembly, Apicoplast Division and Cytokinesis

PubMed Central

Lorestani, Alexander; Sheiner, Lilach; Yang, Kevin; Robertson, Seth D.; Sahoo, Nivedita; Brooks, Carrie F.; Ferguson, David J. P.; Striepen, Boris; Gubbels, Marc-Jan

2010-01-01

The membrane occupation and recognition nexus protein 1 (MORN1) is highly conserved among apicomplexan parasites and is associated with several structures that have a role in cell division. Here we dissected the role of MORN1 using the relatively simple budding process of Toxoplasma gondii as a model. Ablation of MORN1 in a conditional null mutant resulted in pronounced defects suggesting a central role for MORN1 in apicoplast segregation and in daughter cell budding. Lack of MORN1 resulted in double-headed parasites. These Janus-headed parasites form two complete apical complexes but fail to assemble a basal complex. Moreover, these parasites were capable of undergoing several more budding rounds resulting in the formation of up to 16-headed parasites conjoined at the basal end. Despite this segregation defect, the mother's cytoskeleton was completely disassembled in every budding round. Overall this argues that successful completion of the budding is not required for cell cycle progression. None of the known basal complex components, including a set of recently identified inner membrane complex (IMC) proteins, localized correctly in these multi-headed parasites. These data suggest that MORN1 is essential for assembly of the basal complex, and that lack of the basal complex abolishes the contractile capacity assigned to the basal complex late in daughter formation. Consistent with this hypothesis we observe that MORN1 mutants fail to efficiently constrict and divide the apicoplast. We used the null background provided by the mutant to dissect the function of subdomains of the MORN1 protein. This demonstrated that deletion of a single MORN domain already prevented the function of MORN1 whereas a critical role for the short linker between MORN domains 6 and 7 was identified. In conclusion, MORN1 is required for basal complex assembly and loss of MORN1 results in defects in apicoplast division and daughter segregation. PMID:20808817
Cancer cells mimic in vivo spatial-temporal cell-cycle phase distribution and chemosensitivity in 3-dimensional Gelfoam® histoculture but not 2-dimensional culture as visualized with real-time FUCCI imaging.

PubMed

Yano, Shuya; Miwa, Shinji; Mii, Sumiyuki; Hiroshima, Yukihiko; Uehara, Fuminaru; Kishimoto, Hiroyuki; Tazawa, Hiroshi; Zhao, Ming; Bouvet, Michael; Fujiwara, Toshiyoshi; Hoffman, Robert M

2015-01-01

The phase of the cell cycle can determine whether a cancer cell can respond to a given drug. We previously reported monitoring of real-time cell cycle dynamics of cancer cells throughout a live tumor, intravitally in live mice, using a fluorescence ubiquitination-based cell-cycle indicator (FUCCI). Approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G0/G1 phase. Longitudinal real-time imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, had little effect on quiescent cancer cells, which are the vast majority of an established tumor. Moreover, resistant quiescent cancer cells restarted cycling after cessation of chemotherapy. These results suggested why most drugs currently in clinical use, which target cancer cells in S/G2/M, are mostly ineffective on solid tumors. In the present report, we used FUCCI imaging and Gelfoam® collagen-sponge-gel histoculture, to demonstrate in real time, that the cell-cycle phase distribution of cancer cells in Gelfoam® and in vivo tumors is highly similar, whereby only the surface cells proliferate and interior cells are quiescent in G0/G1. This is in contrast to 2D culture where most cancer cells cycle. Similarly, the cancer cells responded similarly to toxic chemotherapy in Gelfoam® culture as in vivo, and very differently than cancer cells in 2D culture which were much more chemosensitive. Gelfoam® culture of FUCCI-expressing cancer cells offers the opportunity to image the cell cycle of cancer cells continuously and to screen for novel effective therapies to target quiescent cells, which are the majority in a tumor and which would have a strong probability to be effective in vivo.
The origin of neap-spring tidal cycles

USGS Publications Warehouse

Kvale, E.P.

2006-01-01

The origin of oceanic tides is a basic concept taught in most introductory college-level sedimentology/geology, oceanography, and astronomy courses. Tides are typically explained in the context of the equilibrium tidal theory model. Yet this model does not take into account real tides in many parts of the world. Not only does the equilibrium tidal model fail to explicate amphidromic circulation, it also does not explain diurnal tides in low latitude positions. It likewise fails to explain the existence of tide-dominated areas where neap-spring cycles are synchronized with the 27.32-day orbital cycle of the Moon (tropical month), rather than with the more familiar 29.52-day cycle of lunar phases (synodic month). Both types of neap-spring cycles can be recognized in the rock record. A complete explanation of the origin of tides should include a discussion of dynamic tidal theory. In the dynamic tidal model, tides resulting from the motions of the Moon in its orbit around the Earth and the Earth in its orbit around the Sun are modeled as products of the combined effects of a series of phantom satellites. The movement of each of these satellites, relative to the Earth's equator, creates its own tidal wave that moves around an amphidromic point. Each of these waves is referred to as a tidal constituent. The geometries of the ocean basins determine which of these constituents are amplified. Thus, the tide-raising potential for any locality on Earth can be conceptualized as the result of a series of tidal constituents specific to that region. A better understanding of tidal cycles opens up remarkable opportunities for research on tidal deposits with implications for, among other things, a more complete understanding of the tidal dynamics responsible for sediment transport and deposition, changes in Earth-Moon distance through time, and the possible influences tidal cycles may exert on organisms. ?? 2006 Elsevier B.V. All rights reserved.

Serum Proteases Potentiate BMP-Induced Cell Cycle Re-entry of Dedifferentiating Muscle Cells during Newt Limb Regeneration.

PubMed

Wagner, Ines; Wang, Heng; Weissert, Philipp M; Straube, Werner L; Shevchenko, Anna; Gentzel, Marc; Brito, Goncalo; Tazaki, Akira; Oliveira, Catarina; Sugiura, Takuji; Shevchenko, Andrej; Simon, András; Drechsel, David N; Tanaka, Elly M

2017-03-27

Limb amputation in the newt induces myofibers to dedifferentiate and re-enter the cell cycle to generate proliferative myogenic precursors in the regeneration blastema. Here we show that bone morphogenetic proteins (BMPs) and mature BMPs that have been further cleaved by serum proteases induce cell cycle entry by dedifferentiating newt muscle cells. Protease-activated BMP4/7 heterodimers that are present in serum strongly induced myotube cell cycle re-entry with protease cleavage yielding a 30-fold potency increase of BMP4/7 compared with canonical BMP4/7. Inhibition of BMP signaling via muscle-specific dominant-negative receptor expression reduced cell cycle entry in vitro and in vivo. In vivo inhibition of serine protease activity depressed cell cycle re-entry, which in turn was rescued by cleaved-mimic BMP. This work identifies a mechanism of BMP activation that generates blastema cells from differentiated muscle. Copyright © 2017 Elsevier Inc. All rights reserved.
Duplication of the genome in normal and cancer cell cycles.

PubMed

Bandura, Jennifer L; Calvi, Brian R

2002-01-01

It is critical to discover the mechanisms of normal cell cycle regulation if we are to fully understand what goes awry in cancer cells. The normal eukaryotic cell tightly regulates the activity of origins of DNA replication so that the genome is duplicated exactly once per cell cycle. Over the last ten years much has been learned concerning the cell cycle regulation of origin activity. It is now clear that the proteins and cell cycle mechanisms that control origin activity are largely conserved from yeast to humans. Despite this conservation, the composition of origins of DNA replication in higher eukaryotes remains ill defined. A DNA consensus for predicting origins has yet to emerge, and it is of some debate whether primary DNA sequence determines where replication initiates. In this review we outline what is known about origin structure and the mechanism of once per cell cycle DNA replication with an emphasis on recent advances in mammalian cells. We discuss the possible relevance of these regulatory pathways for cancer biology and therapy.
Identification and characterization of the Myxococcus xanthus bsgA gene product.

PubMed Central

Gill, R E; Bornemann, M C

1988-01-01

The bsgA mutants of Myxococcus xanthus are blocked at a very early stage of the developmental program. They fail to produce fruiting bodies or to sporulate under normal conditions but can be rescued by extracellular complementation in mixtures with wild-type cells. A bsgA-lacZ gene fusion was constructed and expressed in Escherichia coli. The resulting fusion protein, which has beta-galactosidase enzyme activity, was partially purified by affinity chromatography and preparative polyacrylamide gel electrophoresis. The protein was used to immunize mice, which produced a hybridoma secreting monoclonal antibody that was specific for the bsgA gene product. The monoclonal antibody was used in Western blot (immunoblot) experiments to determine the apparent cellular location of the bsgA protein in M. xanthus and to compare the level of this protein at various times in the Myxococcus life cycle. Images PMID:2846515
The Formation of Tight Tumor Clusters Affects the Efficacy of Cell Cycle Inhibitors: A Hybrid Model Study

PubMed Central

Kim, MunJu; Reed, Damon; Rejniak, Katarzyna A.

2014-01-01

Cyclin-dependent kinases (CDKs) are vital in regulating cell cycle progression, and, thus, in highly proliferating tumor cells CDK inhibitors are gaining interest as potential anticancer agents. Clonogenic assay experiments are frequently used to determine drug efficacy against the survival and proliferation of cancer cells. While the anticancer mechanisms of drugs are usually described at the intracellular single-cell level, the experimental measurements are sampled from the entire cancer cell population. This approach may lead to discrepancies between the experimental observations and theoretical explanations of anticipated drug mechanisms. To determine how individual cell responses to drugs that inhibit CDKs affect the growth of cancer cell populations, we developed a spatially explicit hybrid agent-based model. In this model, each cell is equipped with internal cell cycle regulation mechanisms, but it is also able to interact physically with its neighbors. We model cell cycle progression, focusing on the G1 and G2/M cell cycle checkpoints, as well as on related essential components, such as CDK1, CDK2, cell size, and DNA damage. We present detailed studies of how the emergent properties (e.g., cluster formation) of an entire cell population depend on altered physical and physiological parameters. We analyze the effects of CDK1 and CKD2 inhibitors on population growth, time-dependent changes in cell cycle distributions, and the dynamic evolution of spatial cell patterns. We show that cell cycle inhibitors that cause cell arrest at different cell cycle phases are not necessarily synergistically super-additive. Finally, we demonstrate that the physical aspects of cell population growth, such as the formation of tight cell clusters versus dispersed colonies, alter the efficacy of cell cycle inhibitors, both in 2D and 3D simulations. This finding may have implications for interpreting the treatment efficacy results of in vitro experiments, in which treatment is applied before the cells can grow to produce clusters, especially because in vivo tumors, in contrast, form large masses before they are detected and treated. PMID:24607745
Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells.

PubMed

Guo, Jian-Ru; Chen, Qian-Qian; Lam, Christopher Wai-Kei; Zhang, Wei

2015-07-26

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells). MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells. Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.
The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle.

PubMed

Pramila, Tata; Wu, Wei; Miles, Shawna; Noble, William Stafford; Breeden, Linda L

2006-08-15

Transcription patterns shift dramatically as cells transit from one phase of the cell cycle to another. To better define this transcriptional circuitry, we collected new microarray data across the cell cycle of budding yeast. The combined analysis of these data with three other cell cycle data sets identifies hundreds of new highly periodic transcripts and provides a weighted average peak time for each transcript. Using these data and phylogenetic comparisons of promoter sequences, we have identified a late S-phase-specific promoter element. This element is the binding site for the forkhead protein Hcm1, which is required for its cell cycle-specific activity. Among the cell cycle-regulated genes that contain conserved Hcm1-binding sites, there is a significant enrichment of genes involved in chromosome segregation, spindle dynamics, and budding. This may explain why Hcm1 mutants show 10-fold elevated rates of chromosome loss and require the spindle checkpoint for viability. Hcm1 also induces the M-phase-specific transcription factors FKH1, FKH2, and NDD1, and two cell cycle-specific transcriptional repressors, WHI5 and YHP1. As such, Hcm1 fills a significant gap in our understanding of the transcriptional circuitry that underlies the cell cycle.
Analyzing the dynamics of cell cycle processes from fixed samples through ergodic principles.

PubMed

Wheeler, Richard John

2015-11-05

Tools to analyze cyclical cellular processes, particularly the cell cycle, are of broad value for cell biology. Cell cycle synchronization and live-cell time-lapse observation are widely used to analyze these processes but are not available for many systems. Simple mathematical methods built on the ergodic principle are a well-established, widely applicable, and powerful alternative analysis approach, although they are less widely used. These methods extract data about the dynamics of a cyclical process from a single time-point "snapshot" of a population of cells progressing through the cycle asynchronously. Here, I demonstrate application of these simple mathematical methods to analysis of basic cyclical processes--cycles including a division event, cell populations undergoing unicellular aging, and cell cycles with multiple fission (schizogony)--as well as recent advances that allow detailed mapping of the cell cycle from continuously changing properties of the cell such as size and DNA content. This includes examples using existing data from mammalian, yeast, and unicellular eukaryotic parasite cell biology. Through the ongoing advances in high-throughput cell analysis by light microscopy, electron microscopy, and flow cytometry, these mathematical methods are becoming ever more important and are a powerful complementary method to traditional synchronization and time-lapse cell cycle analysis methods. © 2015 Wheeler. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Environmental tests of metallization systems for terrestrial photovoltaic cells

NASA Technical Reports Server (NTRS)

Alexander, P., Jr.

1985-01-01

Seven different solar cell metallization systems were subjected to temperature cycling tests and humidity tests. Temperature cycling excursions were -50 deg C to 150 deg C per cycle. Humidity conditions were 70 deg C at 98% relative humidity. The seven metallization systems were: Ti/Ag, Ti/Pd/Ag, Ti/Pd/Cu, Ni/Cu, Pd/Ni/Solder, Cr/Pd/Ag, and thick film Ag. All metallization systems showed a slight to moderate decrease in cell efficiencies after subjection to 1000 temperature cycles. Six of the seven metallization systems also evidenced slight increases in cell efficiencies after moderate numbers of cycles, generally less than 100 cycles. The copper based systems showed the largest decrease in cell efficiencies after temperature cycling. All metallization systems showed moderate to large decreases in cell efficiencies after 123 days of humidity exposure. The copper based systems again showed the largest decrease in cell efficiencies after humidity exposure. Graphs of the environmental exposures versus cell efficiencies are presented for each metallization system, as well as environmental exposures versus fill factors or series resistance.
Cell cycle-tailored targeting of metastatic melanoma: Challenges and opportunities.

PubMed

Haass, Nikolas K; Gabrielli, Brian

2017-07-01

The advent of targeted therapies of metastatic melanoma, such as MAPK pathway inhibitors and immune checkpoint antagonists, has turned dermato-oncology from the "bad guy" to the "poster child" in oncology. Current targeted therapies are effective, although here is a clear need to develop combination therapies to delay the onset of resistance. Many antimelanoma drugs impact on the cell cycle but are also dependent on certain cell cycle phases resulting in cell cycle phase-specific drug insensitivity. Here, we raise the question: Have combination trials been abandoned prematurely as ineffective possibly only because drug scheduling was not optimized? Firstly, if both drugs of a combination hit targets in the same melanoma cell, cell cycle-mediated drug insensitivity should be taken into account when planning combination therapies, timing of dosing schedules and choice of drug therapies in solid tumors. Secondly, if the combination is designed to target different tumor cell subpopulations of a heterogeneous tumor, one drug effective in a particular subpopulation should not negatively impact on the other drug targeting another subpopulation. In addition to the role of cell cycle stage and progression on standard chemotherapeutics and targeted drugs, we discuss the utilization of cell cycle checkpoint control defects to enhance chemotherapeutic responses or as targets themselves. We propose that cell cycle-tailored targeting of metastatic melanoma could further improve therapy outcomes and that our real-time cell cycle imaging 3D melanoma spheroid model could be utilized as a tool to measure and design drug scheduling approaches. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Transcriptome changes and cAMP oscillations in an archaeal cell cycle.

PubMed

Baumann, Anke; Lange, Christian; Soppa, Jörg

2007-06-11

The cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote. A synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum. Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 microM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression. The analysis of cell cycle-specific transcriptome changes of H. salinarum allowed to identify a strategy of transcript level regulation that is different from all previously characterized species. The transcript levels of only 3% of all genes are regulated, a fraction that is considerably lower than has been reported for four eukaryotic species (6%-28%) and for the bacterium C. crescentus (19%). It was shown that cAMP is present in significant concentrations in an archaeon, and the phylogenetic profile of the adenylate cyclase indicates that this signaling molecule is widely distributed in archaea. The occurrence of cell cycle-dependent oscillations of the cAMP concentration in an archaeon and in several eukaryotic species indicates that cAMP level changes might be a phylogenetically old signal for cell cycle progression.
Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells

PubMed Central

Guo, Yongfeng; Flegel, Kerry; Kumar, Jayashree; McKay, Daniel J.

2016-01-01

ABSTRACT During development, cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. Here we examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. We find that a cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa wing. We show that in the wing, ecdysone signaling at the larva-to-puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva-to-puparium pulse during early metamorphosis, Broad expression plummets, allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing. PMID:27737823
The effect of valinomycin in fibroblasts from patients with fatty acid oxidation disorders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ndukwe Erlingsson, Uzochi Chimdinma; Iacobazzi, Francesco; Department of Basic Medical Sciences, University of Bari, Piazza Giulio Cesare 11, Policlinico, I-70124 Bari

Highlights: •Valinomycin can cause mitochondrial stress and stimulate fatty acid oxidation. •Cells with VLCAD deficiency fail to increase fatty acid oxidation in response to valinomycin. •Response to valinomycin can help in the diagnosis of VLCAD deficiency. -- Abstract: Disorders of the carnitine cycle and of the beta oxidation spiral impair the ability to obtain energy from fats at time of fasting and stress. This can result in hypoketotic hypoglycemia, cardiomyopathy, cardiac arrhythmia and other chronic medical problems. The in vitro study of fibroblasts from patients with these conditions is impaired by their limited oxidative capacity. Here we evaluate the capacitymore » of valinomycin, a potassium ionophore that increases mitochondrial respiration, to increase the oxidation of fatty acids in cells from patients with inherited fatty acid oxidation defects. The addition of valinomycin to fibroblasts decreased the accumulation of the lipophilic cation tetraphenylphosphonium (TPP{sup +}) at low concentrations due to the dissipation of the mitochondrial membrane potential. At higher doses, valinomycin increased TPP{sup +} accumulation due to the increased potassium permeability of the plasma membrane and subsequent cellular hyperpolarization. The incubation of normal fibroblasts with valinomycin increased [{sup 14}C]-palmitate oxidation (measured as [{sup 14}C]O{sub 2} release) in a dose-dependent manner. By contrast, valinomycin failed to increase palmitate oxidation in fibroblasts from patients with very long chain acyl CoA dehydrogenase (VLCAD) deficiency. This was not observed in fibroblasts from patients heterozygous for this condition. These results indicate that valinomycin can increase fatty acid oxidation in normal fibroblasts and could be useful to differentiate heterozygotes from patients affected with VLCAD deficiency.« less
Decision analysis defining optimal management of clinical stage 1 high-risk nonseminomatous germ cell testicular cancer with lymphovascular invasion.

PubMed

Avulova, Svetlana; Allen, Clayton; Morgans, Alicia; Moses, Kelvin A

2018-05-10

Risk of recurrent disease for men with clinical stage 1 high-risk nonseminomatous germ cell testicular cancer (CS1 NSGCT) with lymphovascular invasion (LVI) after orchiectomy is 50% and current treatment options (surveillance [S], retroperitoneal lymph node dissection [RPLND], or 1 cycle of BEP [BEP ×1]) are associated with a 99% disease specific survival, therefore practice patterns vary. We performed a decision analysis using updated data of long-term complications for men with CS1 NSGCT with LVI to quantify and assess relative treatment values. Decision analysis included previously defined utilities (via standard gamble) for posttreatment states of living from 0 (death from disease) to 1 (alive in perfect health) and updated morbidity probabilities. We quantified the values of S, RPLND, and BEP ×1 via the rollback method. Sensitivity analyses including a range of orchiectomy cure rates and utility values were performed. Estimated probabilities favoring treatment with RPLND (0.97) or BEP ×1 (0.97) were equivalent and superior to surveillance (0.88). Sensitivity analysis of orchiectomy cure rates (50%-100%) failed to find a cure rate that favored S over BEP ×1 or RPLND. Varying utility values for cure after S from 0.92 (previously defined utility) to 1 (perfect health), failed to find a viable utility state favoring S over BEP ×1 or RPLND. An orchiectomy cure rate of ≥82% would be required for S to equal treatment of either type. We demonstrate that for surveillance to be superior to treatment with BEP ×1 or RPLND, the orchiectomy cure rate must be at least 82%, which is not expected in a patient population with high-risk CS1 NSGCT. Copyright © 2018 Elsevier Inc. All rights reserved.
DNA replication checkpoint promotes G1-S transcription by inactivating the MBF repressor Nrm1

PubMed Central

de Bruin, R. A. M.; Kalashnikova, T. I.; Aslanian, A.; Wohlschlegel, J.; Chahwan, C.; Yates, J. R.; Russell, P.; Wittenberg, C.

2008-01-01

The cell cycle transcriptional program imposes order on events of the cell-cycle and is a target for signals that regulate cell-cycle progression, including checkpoints required to maintain genome integrity. Neither the mechanism nor functional significance of checkpoint regulation of the cell-cycle transcription program are established. We show that Nrm1, an MBF-specific transcriptional repressor acting at the transition from G1 to S phase of the cell cycle, is at the nexus between the cell cycle transcriptional program and the DNA replication checkpoint in fission yeast. Phosphorylation of Nrm1 by the Cds1 (Chk2) checkpoint protein kinase, which is activated in response to DNA replication stress, promotes its dissociation from the MBF transcription factor. This leads to the expression of genes encoding components that function in DNA replication and repair pathways important for cell survival in response to arrested DNA replication. PMID:18682565
The B-MYB Transcriptional Network Guides Cell Cycle Progression and Fate Decisions to Sustain Self-Renewal and the Identity of Pluripotent Stem Cells

PubMed Central

Zhan, Ming; Riordon, Daniel R.; Yan, Bin; Tarasova, Yelena S.; Bruweleit, Sarah; Tarasov, Kirill V.; Li, Ronald A.; Wersto, Robert P.; Boheler, Kenneth R.

2012-01-01

Embryonic stem cells (ESCs) are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B-MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity. PMID:22936984
The B-MYB transcriptional network guides cell cycle progression and fate decisions to sustain self-renewal and the identity of pluripotent stem cells.

PubMed

Zhan, Ming; Riordon, Daniel R; Yan, Bin; Tarasova, Yelena S; Bruweleit, Sarah; Tarasov, Kirill V; Li, Ronald A; Wersto, Robert P; Boheler, Kenneth R

2012-01-01

Embryonic stem cells (ESCs) are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B-MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity.
HCdc14A is involved in cell cycle regulation of human brain vascular endothelial cells following injury induced by high glucose, free fatty acids and hypoxia.

PubMed

Su, Jingjing; Zhou, Houguang; Tao, Yinghong; Guo, Zhuangli; Zhang, Shuo; Zhang, Yu; Huang, Yanyan; Tang, Yuping; Hu, Renming; Dong, Qiang

2015-01-01

Cell cycle processes play a vital role in vascular endothelial proliferation and dysfunction. Cell division cycle protein 14 (Cdc14) is an important cell cycle regulatory phosphatase. Previous studies in budding yeast demonstrated that Cdc14 could trigger the inactivation of mitotic cyclin-dependent kinases (Cdks), which are required for mitotic exit and cytokinesis. However, the exact function of human Cdc14 (hCdc14) in cell cycle regulation during vascular diseases is yet to be elucidated. There are two HCdc14 homologs: hCdc14A and hCdc14B. In the current study, we investigated the potential role of hCdc14A in high glucose-, free fatty acids (FFAs)-, and hypoxia-induced injury in cultured human brain vascular endothelial cells (HBVECs). Data revealed that high glucose, FFA, and hypoxia down-regulated hCdc14A expression remarkably, and also affected the expression of other cell cycle-related proteins such as cyclin B, cyclin D, cyclin E, and p53. Furthermore, the combined addition of the three stimuli largely blocked cell cycle progression, decreased cell proliferation, and increased apoptosis. We also determined that hCdc14A was localized mainly to centrosomes during interphase and spindles during mitosis using confocal microscopy, and that it could affect the expression of other cycle-related proteins. More importantly, the overexpression of hCdc14A accelerated cell cycle progression, enhanced cell proliferation, and promoted neoplastic transformation, whereas the knockdown of hCdc14A using small interfering RNA produced the opposite effects. Therefore, these findings provide novel evidence that hCdc14A might be involved in cell cycle regulation in cultured HBVECs during high glucose-, FFA-, and hypoxia-induced injury. Copyright © 2014 Elsevier Inc. All rights reserved.
Ruta graveolens L. induces death of glioblastoma cells and neural progenitors, but not of neurons, via ERK 1/2 and AKT activation.

PubMed

Gentile, Maria Teresa; Ciniglia, Claudia; Reccia, Mafalda G; Volpicelli, Floriana; Gatti, Monica; Thellung, Stefano; Florio, Tullio; Melone, Mariarosa A B; Colucci-D'Amato, Luca

2015-01-01

Glioblastoma multiforme is a highly aggressive brain tumor whose prognosis is very poor. Due to early invasion of brain parenchyma, its complete surgical removal is nearly impossible, and even after aggressive combined treatment (association of surgery and chemo- and radio-therapy) five-year survival is only about 10%. Natural products are sources of novel compounds endowed with therapeutic properties in many human diseases, including cancer. Here, we report that the water extract of Ruta graveolens L., commonly known as rue, induces death in different glioblastoma cell lines (U87MG, C6 and U138) widely used to test novel drugs in preclinical studies. Ruta graveolens' effect was mediated by ERK1/2 and AKT activation, and the inhibition of these pathways, via PD98058 and wortmannin, reverted its antiproliferative activity. Rue extract also affects survival of neural precursor cells (A1) obtained from embryonic mouse CNS. As in the case of glioma cells, rue stimulates the activation of ERK1/2 and AKT in A1 cells, whereas their blockade by pharmacological inhibitors prevents cell death. Interestingly, upon induction of differentiation and cell cycle exit, A1 cells become resistant to rue's noxious effects but not to those of temozolomide and cisplatin, two alkylating agents widely used in glioblastoma therapy. Finally, rutin, a major component of the Ruta graveolens water extract, failed to cause cell death, suggesting that rutin by itself is not responsible for the observed effects. In conclusion, we report that rue extracts induce glioma cell death, discriminating between proliferating/undifferentiated and non-proliferating/differentiated neurons. Thus, it can be a promising tool to isolate novel drugs and also to discover targets for therapeutic intervention.
Downregulation of the c-Fes protein-tyrosine kinase inhibits the proliferation of human renal carcinoma cells

PubMed Central

Kanda, Shigeru; Miyata, Yasuyoshi; Kanetake, Hiroshi; Smithgall, Thomas E.

2009-01-01

The c-Fes protein-tyrosine kinase is associated with growth and differentiation of hematopoietic, neuronal, vascular endothelial and epithelial cell types. In this study, we investigated whether small interfering RNA (siRNA)-mediated knockdown of c-Fes expression affected proliferation of the human renal carcinoma cell lines, ACHN and VMRC-RCW. Immunofluorescence microscopy showed that c-Fes was expressed in both the cytosol and nuclei of these cells, and siRNA treatment preferentially downregulated c-Fes expression in the cytosol. Knock-down of c-Fes inhibited cellular proliferation in a dose-dependent manner with minimal increase in cell death. c-Fes siRNA treatment also downregulated the phosphorylation of Akt1 on S473 and IKKα on T23, and cyclin D1 expression, enhanced the expression of IκBα, and prevented the nuclear localization of NFκB. Treatment with an NFκB inhibitory peptide (SN50) also blocked the proliferation and nuclear localization of NFκB in these cells. The effect of SN50 treatment was not enhanced by c-Fes siRNA, suggesting that downregulation of c-Fes expression inhibited cell cycle progression through the Akt1/NFκB pathway. In contrast to siRNA-mediated knockdown, ectopic expression of either wild-type or kinase-inactive c-Fes in renal carcinoma cells failed to alter their proliferation in vitro and in vivo. Thus, suppression of proliferation resulting from siRNA-mediated knockdown may depend upon an expression of c-Fes protein rather than its kinase activity. Taken together, our results indicate that downregulation of c-Fes expression may be a potential therapeutic strategy for advanced human renal cell carcinoma and inhibition of its kinase activity as an antiangiogenic therapy does not seem to induce the growth of human renal carcinoma cells. PMID:19082481
Peptidyl-prolyl cis/trans isomerase Pin1 regulates withaferin A-mediated cell cycle arrest in human breast cancer cells.

PubMed

Samanta, Suman K; Lee, Joomin; Hahm, Eun-Ryeong; Singh, Shivendra V

2018-07-01

We have reported previously that withaferin A (WA) prevents breast cancer development in mouse mammary tumor virus-neu (MMTV-neu) transgenic mice, but the mechanism is not fully understood. Unbiased proteomics of the mammary tumors from control- and WA-treated MMTV-neu mice revealed downregulation of peptidyl-prolyl cis/trans isomerase (Pin1) protein by WA administration. The present study extends these findings to elucidate the role of Pin1 in cancer chemopreventive mechanisms of WA. The mammary tumor level of Pin1 protein was lower by about 55% in WA-treated rats exposed to N-methyl-N-nitrosourea, compared to control. Exposure of MCF-7 and SK-BR-3 human breast cancer cells to WA resulted in downregulation of Pin1 protein. Ectopic expression of Pin1 attenuated G 2 and/or mitotic arrest resulting from WA treatment in both MCF-7 and SK-BR-3 cells. WA-induced apoptosis was increased by Pin1 overexpression in MCF-7 cells but not in the SK-BR-3 cell line. In addition, molecular docking followed by mass spectrometry indicated covalent interaction of WA with cysteine 113 of Pin1. Overexpression of Pin1 C113A mutant failed to attenuate WA-induced mitotic arrest or apoptosis in the MCF-7 cells. Furthermore, antibody array revealed upregulation of proapoptotic insulin-like growth factor binding proteins (IGFBPs), including IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6, in Pin1 overexpressing MCF-7 cells following WA treatment when compared to empty vector transfected control cells. These data support a crucial role of the Pin1 for mitotic arrest and apoptosis signaling by WA at least in the MCF-7 cells. © 2018 Wiley Periodicals, Inc.

Some links on this page may take you to non-federal websites. Their policies may differ from this site.