Regulation of Nuclear Envelope Assembly/Disassembly by MAP Kinase

AbstractMouse eggs arrested in metaphase II display high levels of cdc2/cyclin B1 and MAP protein kinase activities. Following fertilization there is a time-dependent decrease in the activity of each of these protein kinases. The decline in cdc2/cyclin B1 protein kinase correlates with the resumption of meiosis and the emission of the second polar body and precedes the decline in MAP kinase activity, which correlates temporally with the formation of the male and female pronuclear envelopes. These results suggest that high levels of MAP kinase activity are incompatible with the presence of a pronuclear envelope. To test this possibility, we expressed in mouse eggs a constitutively active form of MAP kinase kinase (MEK) whose only known target is p42/p44 MAP kinase. We show that following fertilization cdc2/cyclin B1 kinase activity declines and a second polar body is emitted. The endogenous MAP kinase remains active, however, and no pronuclear envelopes form. Thus, high levels of MAP kinase activity by itself in mouse eggs appear incompatible with the presence of a pronuclear envelope

The fundamental problem of command : plan and compliance in a partially centralised economy

When a principal gives an order to an agent and advances resources for its implementation, the temptations for the agent to shirk or steal from the principal rather than comply constitute the fundamental problem of command. Historically, partially centralised command economies enforced compliance in various ways, assisted by nesting the fundamental problem of exchange within that of command. The Soviet economy provides some relevant data. The Soviet command system combined several enforcement mechanisms in an equilibrium that shifted as agents learned and each mechanism's comparative costs and benefits changed. When the conditions for an equilibrium disappeared, the system collapsed.Comparative Economic Studies (2005) 47, 296–314. doi:10.1057/palgrave.ces.810011

Chromatin-mediated cortical granule redistribution is responsible for the formation of the cortical granule-free domain in mouse eggs

AbstractA cortical granule-free domain (CGFD) overlies the metaphase chromatin in fully mature mouse eggs. Although a chromatin-induced localized release of cortical granules (CG) during maturation is thought to be a major contributing factor to its formation, there are indications that CG redistribution may also be involved in generating the CGFD. We performed experiments to determine the relative contributions of CG exocytosis and redistribution in generating the CGFD. We found that the CGFD-inducing activity was not specific to female germ cell chromatin and was heat stable but sensitive to DNase and protease treatment. Surprisingly, chelation of egg intracellular Ca2+ levels did not prevent CGFD formation in response to microinjection of exogenous chromatin, suggesting that development of the CGFD was not a result of CG exocytosis. This finding was confirmed by the lack of CG exudate on the plasma membrane surface of the injected eggs and the absence of conversion of ZP2 to ZP2f during formation of the new CGFD. Moreover, clamping intracellular Ca2+ did not prevent the formation of the CGFD during oocyte maturation, but did inhibit the maturation-associated release of CGs between metaphase I and II. Results of these experiments suggest that CG redistribution is the dominant factor in formation of the CGFD

Biochemistry and Molecular Biology MicroRNA Signature in Wound Healing Following Excimer Laser Ablation: Role of miR-133b on TGFb1, CTGF, SMA, and COL1A1 Expression Levels in Rabbit Corneal Fibroblasts

PURPOSE. The role of microRNA (miRNA) regulation in corneal wound healing and scar formation has yet to be elucidated. This study analyzed the miRNA expression pattern involved in corneal wound healing and focused on the effect of miR-133b on expression of several profibrotic genes. METHODS. Laser-ablated mouse corneas were collected at 0 and 30 minutes and 2 days. Ribonucleic acid was collected from corneas and analyzed using cell differentiation and development miRNA PCR arrays. Luciferase assay was used to determine whether miR-133b targeted the 3 0 untranslated region (UTR) of transforming growth factor b1 (TGFb1) and connective tissue growth factor (CTGF) in rabbit corneal fibroblasts (RbCF). Quantitative realtime PCR (qRT-PCR) and Western blots were used to determine the effect of miR-133b on CTGF, smooth muscle actin (SMA), and collagen (COL1A1) in RbCF. Migration assay was used to determine the effect of miR-133b on RbCF migration. RESULTS. At day 2, 37 of 86 miRNAs had substantial expression fold changes. miR-133b had the greatest fold decrease at À14.33. Pre-miR-133b targeted the 3 0 UTR of CTGF and caused a significant decrease of 38% (P < 0.01). Transforming growth factor b1-treated RbCF had a significant decrease of miR-133b of 49% (P < 0.01), whereas CTGF, SMA, and COL1A1 had significant increases of 20%, 54%, and 37% (P < 0.01), respectively. The RbCF treated with TGFb1 and pre-miR133b showed significant decreases in expression of CTGF, SMA, and COL1A1 of 30%, 37%, and 28% (P < 0.01), respectively. Finally, there was significant decrease in migration of miR-133b-treated RbCF. CONCLUSIONS. Significant changes occur in key miRNAs during early corneal wound healing, suggesting novel miRNA targets to reduce scar formation. Keywords: CTGF, microRNA, corneal wound healing, gene expression A fter corneal trauma, stromal wound healing is the result of a complex cascade of multiple factors including growth factors, cytokines, chemokines, proteases, and, most recently discovered, microRNAs (miRNAs). Directly after epithelial damage, the process of healing is initiated by multiple cytokines and growth factors released by the epithelial cells, keratocytes/ corneal fibroblast, and/or the lacrimal gland

Targeted Decorin Gene Therapy Delivered with Adeno-Associated Virus Effectively Retards Corneal Neovascularization \u3cem\u3ein vivo\u3c/em\u3e

Decorin, small leucine-rich proteoglycan, has been shown to modulate angiogenesis in nonocular tissues. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the rabbit stroma with adeno-associated virus serotype 5 (AAV5) impedes corneal neovascularization (CNV) in vivo without significant side effects. An established rabbit CNV model was used. Targeted decorin gene therapy in the rabbit stroma was delivered with a single topical AAV5 titer (100 μl; 5x10^12 vg/ml) application onto the stroma for two minutes after removing corneal epithelium. The levels of CNV were examined with stereomicroscopy, H&E staining, lectin, collagen type IV, CD31 immunocytochemistry and CD31 immunoblotting. Real-time PCR quantified mRNA expression of pro- and anti-angiogenic genes. Corneal health in live animals was monitored with clinical, slit-lamp and optical coherence tomography biomicroscopic examinations. Selective decorin delivery into stroma showed significant 52% (p\u3c0.05), 66% (p\u3c0.001), and 63% (p\u3c0.01) reduction at early (day 5), mid (day 10), and late (day 14) stages of CNV in decorin-delivered rabbit corneas compared to control (no decorin delivered) corneas in morphometric analysis. The H&E staining, lectin, collagen type IV, CD31 immunostaining (57–65, p\u3c0.5), and CD31 immunoblotting (62–67%, p\u3c0.05) supported morphometric findings. Quantitative PCR studies demonstrated decorin gene therapy down-regulated expression of VEGF, MCP1 and angiopoietin (pro-angiogenic) and up-regulated PEDF (anti-angiogenic) genes. The clinical, biomicroscopy and transmission electron microscopy studies revealed that AAV5– mediated decorin gene therapy is safe for the cornea. Tissue-targeted AAV5-mediated decorin gene therapy decreases CNV with no major side effects, and could potentially be used for treating patients

A case study of boundary layer ventilation by convection and coastal processes

It is often assumed that ventilation of the atmospheric boundary layer is weak in the absence of fronts, but is this always true? In this paper we investigate the processes responsible for ventilation of the atmospheric boundary layer during a nonfrontal day that occurred on 9 May 2005 using the UK Met Office Unified Model. Pollution sources are represented by the constant emission of a passive tracer everywhere over land. The ventilation processes observed include shallow convection, turbulent mixing followed by large-scale ascent, a sea breeze circulation and coastal outflow. Vertical distributions of tracer are validated qualitatively with AMPEP (Aircraft Measurement of chemical Processing Export fluxes of Pollutants over the UK) CO aircraft measurements and are shown to agree impressively well. Budget calculations of tracers are performed in order to determine the relative importance of these ventilation processes. Coastal outflow and the sea breeze circulation were found to ventilate 26% of the boundary layer tracer by sunset of which 2% was above 2 km. A combination of coastal outflow, the sea breeze circulation, turbulent mixing and large-scale ascent ventilated 46% of the boundary layer tracer, of which 10% was above 2 km. Finally, coastal outflow, the sea breeze circulation, turbulent mixing, large-scale ascent and shallow convection together ventilated 52% of the tracer into the free troposphere, of which 26% was above 2 km. Hence this study shows that significant ventilation of the boundary layer can occur in the absence of fronts (and thus during high-pressure events). Turbulent mixing and convection processes can double the amount of pollution ventilated from the boundary layer

Microarray Analysis of Gene Expression Patterns during Healing of Rat Corneas after Excimer Laser Photorefractive Keratectomy

PURPOSE. To characterize changes over time in the genomic expression profile of rat corneas after excimer laser photorefractive keratectomy (PRK), in an effort to better understand the cellular response to injury and the dynamic changes that occur in gene expression patterns as a wound heals. METHODS. The corneal gene expression profile of 1176 genes at 3 and 7 days after PRK was determined and compared with untreated corneal gene expression patterns by interrogating commercially available cDNA arrays with labeled target cDNA prepared from pooled total RNA harvested from the respective treatment group of adult male rats. The gene expression patterns were inferred based on the hybridization intensities of the probes on the cDNA arrays. The hybridization signals were globally normalized and filtered. The data were analyzed by using hierarchical and k-means clustering algorithms before and after normalization of variances. RESULTS. Of the 1176 cDNA elements on the array, 588 consistently produced similar results in replicate experiments and comprised the data set analyzed in this work. In total, 73 genes were identified, with expression levels that differed by at least threefold at either 3 or 7 days after PRK. At 3 days after PRK, 70 genes were identified with expression levels that differed by more than threefold, compared with the expression level in untreated animals. The expression of 42 genes increased by threefold or more, whereas expression of 28 genes decreased by threefold or more. By day 7 after PRK, the number of genes displaying more than a threefold difference in expression pattern was reduced to 27 genes, 20 of which showed elevated levels, whereas 7 exhibited decreased levels. Hierarchical clustering of the 588 studied genes produced 10 clusters with correlation coefficients of 0.9 or greater. To determine whether any of the clusters were overrepresented by genes with related functions, the cumulative hypergeometric probability was calculated by obtaining the observed number of functionally related genes within each of the 10 clusters. Seven of the clusters were statistically overrepresented by one or more categories of functionally related genes, such as cell cycle regulators, transcription factors, and metabolic pathway genes. Clustering analysis of 56 genes generally considered to influence corneal wound healing produced 10 gene clusters with correlation coefficients of at least 0.9. Expression of 23 of these 56 genes increased at day 3, then decreased at day 7 to levels similar to those on day 0. These included several growth factors (VEGF, FGF, IGF-I), proteases (PAI-1, PAI-2A) and protease inhibitors (TIMP-2 and TIMP-3). Expression of nine genes increased on both days 3 and 7 compared with expression on day 0 (e.g., TGFB1, TGFBIIR, M6P/IGFR-2), and no genes decreased on both days 3 and 7, compared with day 0. CONCLUSIONS. Microarray analysis of 1176 identified 588 genes with reproducible patterns of expression in rat corneas on days 3 and 7 after PRK and 73 genes with a threefold change in expression compared with untreated corneas. Hierarchical clustering of these 588 genes identified 10 clusters of genes with very similar patterns of expression. Clustering of genes with similar patterns of expression implies a common regulatory pathway for the genes within a cluster, and identifies potential new targets for regulating corneal wound healing. (Invest Ophthalmol Vis Sci. 2002;43:1772-1782 C orneal wound healing is a complex biological process that involves the integrated actions of many genes that code for proteins with diverse functions, such as cytokines, growth factors, receptors, extracellular matrix proteins, integrins, proteases, inhibitors and cell cycle genes. 1,2 When expression of these genes is regulated correctly, corneal wounds heal appropriately. However, when intrinsic or extrinsic factors disrupt the expression of these genes, corneal wounds may either fail to heal adequately or may heal with excessive scar formation. Thus, there is a need to understand, as broadly as possible, how the expression of key genes changes during corneal wound healing so that therapies can be logically designed to prevent complications from occurring. Previous studies investigating gene expression during corneal wound healing have analyzed mRNA levels of a small number of selected growth factors, receptors, and extracellular matrix genes that are thought to regulate or participate in corneal wound healing. For example, quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) of rat corneas after photorefractive keratectomy (PRK) showed prolonged elevation of mRNAs for the three isoforms of transforming growth factor-␤ (TGF␤1, TGF␤2, TGF␤3), the TGF␤ type II receptor (TGF␤R-II), and extracellular matrix proteins (types I, III, and VI collagen and fibronectin). 3 Similar results were reported for mRNA levels for collagens I, III, IV, and V using semiquantitative RT-PCR in rat corneas after PRK. 5 Other groups have investigated changes in protein levels in corneas during wound healing. Immunohistochemical analysis of cat corneas after PRK indicated there were increased levels of TGF␤1, TGF␤2, and TGF␤3 and the TGF␤R-I and TGF␤R-II receptor proteins in the subepithelial tissue of laser-ablated regions at 4 weeks after injury. 7 Analyses of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) in rat and rabbit corneas after PRK found increased levels of TIMP-2 and MMP-9, -7, and -13 proteins and mRNAs in migrating basal epithelial cells. 8 -12 Immunofluorescence analysis of radial keratotomy (RK) wounds in human corneas detected several extracellular matrix proteins that were not present in the stroma of normal corneas, including collagen types III, VIII, and XIV; the ␣1-␣2 chains of type IV collagen; tenascin-C; and fibrillin-1. MATERIALS AND METHODS Animal Models Animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the animal protocol was approved by the University of Florida Animal Care and Use Committee. Rat corneas underwent excimer laser ablation as described previously. RNA Isolation from Rat Corneas Total RNA was extracted from each group of 10 pooled corneas using guanidine isothiocyanate and phenol-chloroform according to standard methods. Briefly, pooled corneal tissue was homogenized in 0.75 mL RNA extraction solution (TRIzol; Gibco, Grand Island, NY) using a 5-mL frosted glass-on-glass tissue grinder (Duall; Konte Scientific Glassware, Vineland, NJ), RNA was extracted with chloroform, precipitated with isopropanol, washed with ethanol, and dissolved in RNase-free water treated with 0.1% diethylpyrocarbonate. The material was then treated with 1 U/L DNase I at 37°C for 30 minutes to remove any contaminating genomic DNA, and then extracted once with phenolchloroform-isoamyl alcohol (25:24:1 pH 4.5), followed by extraction with chloroform, precipitated with 2 M sodium acetate and 95% ethanol, washed with 80% ethanol, and resuspended in RNase-free water. The concentration of RNA were measured spectrophotometrically at 260 nm (GeneQuant; Amersham Pharmacia Biotech, Uppsala, Sweden). Absorbance ratios of 280 to 260 nm were consistently more than 1.8 for all samples. DNA Arrays and Interrogations Rat cDNA expression arrays (Atlas 1.2; Clontech Laboratories, Palo Alto, CA) were used in this work to study gene expression patterns in corneas after PRK. Each DNA array was manufactured by spotting a nylon membrane with cDNA fragments representing 1176 known genes in addition to three plasmid and bacteriophage DNA sequences, which were included as a negative control, and 9 housekeeping gene sequences, which were included as a positive control. To minimize cross-hybridization and nonspecific binding, cDNA fragments bound on the array were selected, so that they ranged in size from 200 to 600 bp and they did not contain repetitive elements. All clones used to generate the immobilized gene probes were sequenced by the manufacturer to verify identity, and the size of PCR products generated from all clones by gene-specific primers was confirmed by gel electrophoresis. For DNA array interrogation, the arrays were interrogated with ␣ 32 P-labeled cDNA prepared by reverse transcribing pools of total corneal RNA isolated. Using the manufacturer's protocol (Clontech Laboratories), 5 g total RNA was reverse transcribed with genespecific primers and reagents provided with the cDNA expression array. The radiolabeled target cDNAs were purified by column chromatography, denatured under basic conditions, neutralized, and added to 10-mL aliquots of hybridization solution (ExpressHyb; Clontech Laboratories) at 68°C, containing 100 g/mL heat-denatured and sheared salmon testes DNA (Sigma, St. Louis, MO), to obtain a final probe concentration of 5 ϫ 10 6 cpm/mL. The radiolabeled cDNA hybridization solutions were applied to prehybridize the array membranes (30 minutes in ExpressHyb with salmon testes DNA at 68°C without labeled probe) and were hybridized overnight at 68°C in glass bottles with constant rotational mixing in a hybridization incubator. After hybridization, the membranes were washed four times with 140 mL 2ϫ SSC (300 mM sodium chloride, 330 mM sodium citrate, pH 7.0), and 1% SDS solution at 68°C for 30 minutes each, followed by one wash for 30 minutes in 140 mL 0.1% SSC and 0.5% SDS, at 68°C. Finally, the membranes were rinsed for 5 minutes in a solution of 2ϫ SSC at room temperature, and exposed to phosphor screens. Each sample was applied to a new membrane--that is, the membranes were not stripped and rehybridized

Chemical characteristics of air from differing source regions during the Pacific Exploratory Mission‐Tropics A (PEM‐Tropics A)

Ten‐day backward trajectories are used to determine the origins of air parcels arriving at airborne DC‐8 chemical measurement sites during NASA\u27s Pacific Exploratory Mission‐Tropics A (PEM‐T) that was conducted during August‐October 1996. Those sites at which the air had a common geographical origin and transport history are grouped together, and statistical measures of chemical characteristics are computed. Temporal changes in potential temperature are used to determine whether trajectories experience a significant convective influence during the 10‐day period. Those trajectories that do not experience a significant convective influence are divided into four geographical categories depending on their origins and paths. Air parcels originating over Africa and South America are characterized by enhanced mixing ratios of O3, CO, HNO3, and PAN. The backward trajectories travel at high altitudes (∼10–11 km), covering long distances due to strong upper‐tropospheric westerly winds. The observed enhancement of combustion‐related species is attributed to biomass burning from distant sources to the west, extending even to South America. The relatively large value of Be‐7 probably is due either to less efficient removal of aerosols from upper tropospheric air or to small stratospheric contributions. Aged marine parcels are found to have relatively small concentrations of burning‐related species. Although these trajectories arrive at a wide range of aircraft altitudes, they do not pass over a land mass during the preceding 10‐day period. Air passing over Australia but no other land mass exhibits a combustion signature; however, photochemical product species such as O3 and PAN are less enhanced than in the long‐range transport category. These trajectories travel shorter distances and are at lower altitudes (∼5–8 km) than those reaching Africa and/or South America. The combustion influence on these parcels is attributed to biomass burning emissions injected over Australia. That burning is less widespread than in Africa and South America. Finally, trajectories originating over Southeast Asia appear to receive a weak combustion influence. However, compared to Africa and South America, Southeast Asia has a relatively small incidence of biomass burning. There is little combustion input from Australia due to the high transport altitudes compared to the lower heights of the convection. The Southeast Asian parcels exhibit the greatest NOx to ∑NOi ratio of any category, perhaps due to lightning. Parcels experiencing a significant convective influence also are examined. Most of these parcels pass through widespread, persistent convection along either the South Pacific Convergence Zone or Intertropical Convergence Zone approximately 5 days prior to arriving at the aircraft locations. Thus the category mostly represents marine convection. Mixing ratios of peroxides and acids in the convective category are found to be smaller than in parcels not experiencing convection. Small mixing ratios of Be‐7 and Pb‐210 suggest particle removal by precipitation

Vorinostat: A Potent Agent to Prevent and Treat Laser-Induced Corneal Haze

PURPOSE—This study investigated the efficacy and safety of vorinostat, a deacetylase (HDAC) inhibitor, in the treatment of laser-induced corneal haze following photorefractive keratectomy (PRK) in rabbits in vivo and transforming growth factor beta 1 (TGFβ1) -induced corneal fibrosis in vitro. METHODS—Corneal haze in rabbits was produced with −9.00 diopters (D) PRK. Fibrosis in cultured human and rabbit corneal fibroblasts was activated with TGFβ1. Vorinostat (25 μm) was topically applied once for 5 minutes on rabbit cornea immediately after PRK for in vivo studies. Vorinostat (0 to 25 μm) was given to human/rabbit corneal fibroblasts for 5 minutes or 48 hours for in vitro studies. Slit-lamp microscopy, TUNEL assay, and trypan blue were used to determined vorinostat toxicity, whereas real-time polymerase chain reaction, immunocytochemistry, and immunoblotting were used to measure its efficacy. RESULTS—Single 5-minute vorinostat (25 μm) topical application on the cornea following PRK significantly reduced corneal haze (Pin vivoscreened 4 weeks after PRK. Vorinostat reduced TGFβ1-induced fibrosis in human and rabbit corneas in vitro in a dose-dependent manner without altering cellular viability, phenotype, or proliferation. CONCLUSIONS—Vorinostat is non-cytotoxic and safe for the eye and has potential to prevent laser-induced corneal haze in patients undergoing PRK for high myopia