scholarly journals Processive translocation of cohesive and non-cohesive cohesin in vivo

2021 ◽  
Author(s):  
Marc R Gartenberg ◽  
Melinda S Borrie
Keyword(s):  
Direct Evidence ◽  
Chromosome Structure ◽  
Maximum Size ◽  
Upstream Gene ◽  
Architectural Element ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
M Phase

Cohesin is a central architectural element of chromosome structure that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops. In vitro, cohesin translocates along DNA and extrudes loops in an ATP-dependent fashion. In vivo, cohesin redistributes in response to transcription as if pushed by RNA polymerase. Direct evidence of processive genomic translocation by the complex, however, is lacking. Here, obstacles of increasing size were tethered to DNA in yeast to detect translocation. The obstacles were built from a GFP-lacI core fused to one or more mCherries. Cohesin translocation was initiated from an upstream gene. A chimera with four mCherries blocked cohesin passage in late G1. During M phase, the threshold barrier to passage depended on the state of cohesion: non-cohesive complexes were also blocked by four mCherries whereas cohesive complexes were blocked by only three mCherries. That synthetic barriers alter cohesin redistribution demonstrates that the complex translocates processively on chromatin in vivo. The approach provides a relative measure of the maximum size of the protein chamber(s) that embraces DNA during cohesin translocation. The data indicate that the cohesive embrace is more restrictive than the embrace of non-cohesive complexes.

scholarly journals The Highly Conserved tRNAHis Guanylyltransferase Thg1p Interacts with the Origin Recognition Complex and Is Required for the G2/M Phase Transition in the Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 4 (4) ◽  
pp. 832-835 ◽  
Author(s):  
Terri S. Rice ◽  
Min Ding ◽  
David S. Pederson ◽  
Nicholas H. Heintz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Origin Recognition Complex ◽  
Nuclear Division ◽  
Permissive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
M Phase ◽  
And Migration ◽  
Origin Recognition

ABSTRACT Here we show that the Saccharomyces cerevisiae tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex in vivo and in vitro and that overexpression of hemagglutinin-Thg1p selectively impedes growth of orc2-1(Ts) cells at the permissive temperature. Studies with conditional mutants indicate that Thg1p couples nuclear division and migration to cell budding and cytokinesis in yeast.

scholarly journals Live View of Gonadotropin-Releasing Hormone Containing Neuron Migration

Endocrinology ◽  
2005 ◽  
Vol 146 (1) ◽  
pp. 463-468 ◽  
Author(s):  
Elizabeth P. Bless ◽  
Heather J. Walker ◽  
Kwok W. Yu ◽  
J. Gabriel Knoll ◽  
Suzanne M. Moenter ◽  
...  
Keyword(s):  
Real Time ◽  
Direct Evidence ◽  
Gonadotropin Releasing Hormone ◽  
Releasing Hormone ◽  
Reproductive Axis ◽  
Gnrh Neurons ◽  
Migratory Route ◽  
The Brain

Neurons that synthesize GnRH control the reproductive axis and migrate over long distances and through different environments during development. Prior studies provided strong clues for the types of molecules encountered and movements expected along the migratory route. However, our studies provide the first real-time views of the behavior of GnRH neurons in the context of an in vitro preparation that maintains conditions comparable to those in vivo. The live views provide direct evidence of the changing behavior of GnRH neurons in their different environments, showing that GnRH neurons move with greater frequency and with more changes in direction after they enter the brain. Perturbations of guiding fibers distal to moving GnRH neurons in the nasal compartment influenced movement without detectable changes in the fibers in the immediate vicinity of moving GnRH neurons. This suggests that the use of fibers by GnRH neurons for guidance may entail selective signaling in addition to mechanical guidance. These studies establish a model to evaluate the influences of specific molecules that are important for their migration.

scholarly journals Association of profilin with filament-free regions of human leukocyte and platelet membranes and reversible membrane binding during platelet activation.

1989 ◽  
Vol 109 (4) ◽  
pp. 1571-1579 ◽  
Author(s):  
J H Hartwig ◽  
K A Chambers ◽  
K L Hopcia ◽  
D J Kwiatkowski
Keyword(s):  
Platelet Activation ◽  
Direct Evidence ◽  
Human Leukocyte ◽  
Cell Types ◽  
Response To Treatment ◽  
Membrane Association ◽  
Actin Monomer ◽  
High Affinity

Profilin is a conserved, widely distributed actin monomer binding protein found in eukaryotic cells. Mammalian profilin reversibly sequesters actin monomers in a high affinity profilactin complex. In vitro, the complex is dissociated in response to treatment with the polyphosphoinositides, phosphatidylinositol monophosphate, and phosphatidylinositol 4,5-bisphosphate. Here, we demonstrate the ultrastructural immunolocalization of profilin in human leukocytes and platelets. In both cell types, a significant fraction of profilin is found associated with regions of cell membrane devoid of actin filaments and other discernible structures. After platelet activation, the membrane association of profilin reversibly increases. This study represents the first direct evidence for an interaction between profilin and phospholipids in vivo.

scholarly journals Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Marloes I. Hofstee ◽  
Martijn Riool ◽  
Igors Terjajevs ◽  
Keith Thompson ◽  
Martin J. Stoddart ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Cells ◽  
Early Stage ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Maximum Size ◽  
Human Neutrophils ◽  
Content Type

ABSTRACT Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

scholarly journals Identification of PLK1 as a New Therapeutic Target in Mucinous Ovarian Carcinoma

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 672 ◽  
Author(s):  
Roberta Affatato ◽  
Laura Carrassa ◽  
Rosaria Chilà ◽  
Monica Lupi ◽  
Valentina Restelli ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Epithelial Ovarian Cancer ◽  
High Throughput Screening ◽  
Therapeutic Option ◽  
Xenograft Model ◽  
Antimitotic Drugs ◽  
M Phase ◽  
Sirna Library

Mucinous epithelial ovarian cancer (mEOC) is a rare subset of epithelial ovarian cancer. When diagnosed at a late stage, its prognosis is very poor, as it is quite chemo-resistant. To find new therapeutic options for mEOC, we performed high-throughput screening using a siRNA library directed against human protein kinases in a mEOC cell line, and polo-like kinase1 (PLK1) was identified as the kinase whose downregulation interfered with cell proliferation. Both PLK1 siRNA and two specific PLK1 inhibitors (onvansertib and volasertib) were able to inhibit cell growth, induce apoptosis and block cells in the G2/M phase of the cell cycle. We evaluated, in vitro, the combinations of PLK1 inhibitors and different chemotherapeutic drugs currently used in the treatment of mEOC, and we observed a synergistic effect of PLK1 inhibitors and antimitotic drugs. When translated into an in vivo xenograft model, the combination of onvansertib and paclitaxel resulted in stronger tumor regressions and in a longer mice survival than the single treatments. These effects were associated with a higher induction of mitotic block and induction of apoptosis, similarly to what was observed in vitro. These data suggest that the combination onvansertib/paclitaxel could represent a new active therapeutic option in mEOC.

Casticin Induced Apoptosis in A375.S2 Human Melanoma Cells through the Inhibition of NF-κB and Mitochondria-Dependent Pathways In Vitro and Inhibited Human Melanoma Xenografts in a Mouse Model In Vivo

2016 ◽  
Vol 44 (03) ◽  
pp. 637-661 ◽  
Author(s):  
Yin-Wen Shiue ◽  
Chi-Cheng Lu ◽  
Yu-Ping Hsiao ◽  
Ching-Lung Liao ◽  
Jing-Pin Lin ◽  
...  
Keyword(s):  
Human Melanoma ◽  
Oxygen Species ◽  
Protein Levels ◽  
Phase Arrest ◽  
Viable Cells ◽  
M Phase ◽  
Induced Apoptosis ◽  
Western Blotting Assay

Casticin, a polymethoxyflavone occurring in natural plants, has been shown to have anticancer activities. In the present study, we aims to investigate the anti-skin cancer activity of casticin on melanoma cells in vitro and the antitumor effect of casticin on human melanoma xenografts in nu/nu mice in vivo. A flow cytometric assay was performed to detect expression of viable cells, cell cycles, reactive oxygen species production, levels of [Formula: see text] and caspase activity. A Western blotting assay and confocal laser microscope examination were performed to detect expression of protein levels. In the in vitro studies, we found that casticin induced morphological cell changes and DNA condensation and damage, decreased the total viable cells, and induced G2/M phase arrest. Casticin promoted reactive oxygen species (ROS) production, decreased the level of [Formula: see text], and promoted caspase-3 activities in A375.S2 cells. The induced G2/M phase arrest indicated by the Western blotting assay showed that casticin promoted the expression of p53, p21 and CHK-1 proteins and inhibited the protein levels of Cdc25c, CDK-1, Cyclin A and B. The casticin-induced apoptosis indicated that casticin promoted pro-apoptotic proteins but inhibited anti-apoptotic proteins. These findings also were confirmed by the fact that casticin promoted the release of AIF and Endo G from mitochondria to cytosol. An electrophoretic mobility shift assay (EMSA) assay showed that casticin inhibited the NF-[Formula: see text]B binding DNA and that these effects were time-dependent. In the in vivo studies, results from immuno-deficient nu/nu mice bearing the A375.S2 tumor xenograft indicated that casticin significantly suppressed tumor growth based on tumor size and weight decreases. Early G2/M arrest and mitochondria-dependent signaling contributed to the apoptotic A375.S2 cell demise induced by casticin. In in vivo experiments, A375.S2 also efficaciously suppressed tumor volume in a xenotransplantation model. Therefore, casticin might be a potential therapeutic agent for the treatment of skin cancer in the future.

scholarly journals O2 Dependence of in vivo Brain Cytochrome Redox Responses and Energy Metabolism in Bloodless Rats

1988 ◽  
Vol 8 (2) ◽  
pp. 163-172 ◽  
Author(s):  
Avis L. Sylvia ◽  
Claude A. Piantadosi
Keyword(s):  
Energy Metabolism ◽  
Direct Evidence ◽  
Redox Status ◽  
Energy Deficit ◽  
Brain Oxygenation ◽  
Control Value ◽  
Partial Pressures ◽  
Fluorocarbon Emulsion

Oxygen-dependent changes in brain cytochrome redox state and cerebrocortical energy metabolism were evaluated in fluorocarbon-circulated rats at hematocrits of <1%. Redox levels of three respiratory chain cytochrome complexes, b, c, and a,a3 (cytochrome c oxidase), were continuously measured directly through the intact skulls of animals using reflectance spectrophotometry. The in vivo redox status of cytochromes at different Fio2 was directly compared with in vitro measured changes in cortical metabolites known to reflect energy production, i.e., glucose, pyruvate, lactate, phosphocreatine (PCr), ADP, and ATP. Lowering the Fio2 to <1.0 caused the cytochromes to become increasingly more reduced. This was associated with increased tissue accumulation of pyruvate and lactate and a concomitant increase in the lactate/pyruvate (L/P) ratio. At Fio2 = 0.6, cytochromes b, c, and a,a3 were 57, 53, and 46% reduced, respectively. There was no apparent cerebral energy deficit since changes in cortical PCr, ADP, and ATP concentrations were not statistically significant. Bloodless animals did not survive below Fio2 = 0.5. At this Fio2, the inability of the animals to sustain arterial pressure correlated ( r = 0.87) with depletion of PCr and further increases in the L/P ratio ( r = 0.66). Yet, the cortical ATP content was reduced by only 9% of control value. These data provide direct evidence that fluorocarbon emulsion (FC-43) sustains brain oxygenation and energy metabolism at high partial pressures of molecular o2. At lower Fio2, however, mitochondrial o2 uptake becomes limited as a function of decreasing perfusion pressure.

scholarly journals The periplasmic chaperone Skp is required for successful Salmonella Typhimurium infection in a murine typhoid model

Microbiology ◽  
2011 ◽  
Vol 157 (3) ◽  
pp. 848-858 ◽  
Author(s):  
Gary Rowley ◽  
Henrieta Skovierova ◽  
Andrew Stevenson ◽  
Bronislava Rezuchova ◽  
Dagmar Homerova ◽  
...  
Keyword(s):  
Essential Gene ◽  
Outer Membrane Proteins ◽  
Polymyxin B ◽  
Effector Proteins ◽  
Upstream Gene ◽  
Increased Sensitivity ◽  
Periplasmic Chaperone ◽  
And Function

The alternative sigma factor σ E (rpoE) is essential for survival in vivo of Salmonella Typhimurium but is dispensable during growth in the laboratory. We have been identifying σ E-regulated genes and studying their regulation and function to elucidate their potential role in the severe attenuation of S. Typhimurium rpoE mutants. In this study we identify five promoters that control the rseP, yaeT (bamA), skp region. A confirmed σ E-dependent promoter, yaeTp1, and a second downstream promoter, yaeTp2, are located within the upstream gene rseP and direct expression of the downstream genes. The only known function of RseP is σ E activation, and it is therefore not expected to be essential for S. Typhimurium in vitro. However, it proved impossible to delete the entire rseP gene due to the presence of internal promoters that regulate the essential gene yaeT. We could inactivate rseP by deleting the first third of the gene, leaving the yaeT promoters intact. Like the rpoE mutant, the rseP mutant exhibited severe attenuation in vivo. We were able to delete the entire coding sequence of skp, which encodes a periplasmic chaperone involved in targeting misfolded outer-membrane proteins to the β-barrel assembly machinery. The skp mutant was attenuated in mice after oral and parenteral infection. Virulence could be complemented by providing skp in trans but only by linking it to a heterologous σ E-regulated promoter. The reason the skp mutant is attenuated is currently enigmatic, but we know it is not through increased sensitivity to a variety of RpoE-activating host stresses, such as H2O2, polymyxin B and high temperature, or through altered secretion of effector proteins by either the Salmonella pathogenicity island (SPI)-1 or the SPI-2 type III secretion system.

scholarly journals Minireview: Recent Progress in Gonadotropin-Releasing Hormone Neuronal Migration

Endocrinology ◽  
2006 ◽  
Vol 147 (3) ◽  
pp. 1159-1165 ◽  
Author(s):  
Stuart A. Tobet ◽  
Gerald A. Schwarting
Keyword(s):  
Basal Forebrain ◽  
Direct Evidence ◽  
Neuronal Population ◽  
External Factors ◽  
Reproductive Axis ◽  
Gnrh Neurons ◽  
Molecular Phenotypes ◽  
The Brain

Neurons that synthesize GnRH are critical brain regulators of the reproductive axis, yet they originate outside the brain and must migrate over long distances and varied environments to get to their appropriate positions during development. Many studies, past and present, are providing clues for the types of molecules encountered and movements expected along the migratory route. Recent studies provide real-time views of the behavior of GnRH neurons in the context of in vitro preparations that model those in vivo. Live images provide direct evidence of the changing behavior of GnRH neurons in their different environments, showing that GnRH neurons move with greater frequency and with more alterations in direction after they enter the brain. The heterogeneity of molecular phenotypes for GnRH neurons likely ensures that multiple external factors will be found that regulate the migration of different portions of the GnRH neuronal population at different steps along the route. Molecules distributed in gradients both in the peripheral olfactory system and basal forebrain may be particularly influential in directing the appropriate movement of GnRH neurons along their arduous migration. Molecules that mediate the adhesion of GnRH neurons to changing surfaces may also play critical roles. It is likely that the multiple external factors converge on selective signal transduction pathways to engage the mechanical mechanisms needed to modulate GnRH neuronal movement and ultimately migration.

scholarly journals Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1.

1995 ◽  
Vol 15 (12) ◽  
pp. 7143-7151 ◽  
Author(s):  
K S Lee ◽  
Y L Yuan ◽  
R Kuriyama ◽  
R L Erikson
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Maximum Level ◽  
Specific Protein ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
M Phase ◽  
Nih 3T3

PLK (STPK13) encodes a murine protein kinase closely related to those encoded by the Drosophila melanogaster polo gene and the Saccharomyces cerevisiae CDC5 gene, which are required for normal mitotic and meiotic divisions. Affinity-purified antibody generated against the C-terminal 13 amino acids of Plk specifically recognizes a single polypeptide of 66 kDa in MELC, NIH 3T3, and HeLa cellular extracts. The expression levels of both poly(A)+ PLK mRNA and its encoded protein are most abundant about 17 h after serum stimulation of NIH 3T3 cells. Plk protein begins to accumulate at the S/G2 boundary and reaches the maximum level at the G2/M boundary in continuously cycling cells. Concurrent with cyclin B-associated cdc2 kinase activity, Plk kinase activity sharply peaks at the onset of mitosis. Plk enzymatic activity gradually decreases as M phase proceeds but persists longer than cyclin B-associated cdc2 kinase activity. Plk is localized to the area surrounding the chromosomes in prometaphase, appears condensed as several discrete bands along the spindle axis at the interzone in anaphase, and finally concentrates at the midbody during telophase and cytokinesis. Plk and CHO1/mitotic kinesin-like protein 1 (MKLP-1), which induces microtubule bundling and antiparallel movement in vitro, are colocalized during late M phase. In addition, CHO1/MKLP-1 appears to interact with Plk in vivo and to be phosphorylated by Plk-associated kinase activity in vitro.

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