Integrative functional genomics analysis of sustained polyploidy phenotypes in breast cancer cells identifies an oncogenic profile for GINS2

Aneuploidy is among the most obvious differences between normal and cancer cells. However, mechanisms contributing to development and maintenance of aneuploid cell growth are diverse and incompletely understood. Functional genomics analyses have shown that aneuploidy in cancer cells is correlated with diffuse gene expression signatures and that aneuploidy can arise by a variety of mechanisms, including cytokinesis failures, DNA endoreplication and possibly through polyploid intermediate states. Here, we used a novel cell spot microarray technique to identify genes with a loss-of-function effect inducing polyploidy and/or allowing maintenance of polyploid cell growth of breast cancer cells. Integrative genomics profiling of candidate genes highlighted GINS2 as a potential oncogene frequently overexpressed in clinical breast cancers as well as in several other cancer types. Multivariate analysis indicated GINS2 to be an independent prognostic factor for breast cancer outcome (p = 0.001). Suppression of GINS2 expression effectively inhibited breast cancer cell growth and induced polyploidy. In addition, protein level detection of nuclear GINS2 accurately distinguished actively proliferating cancer cells suggesting potential use as an operational biomarker.

Some observations on the behaviour of colchicine-treated cells during recovery in Allium CEPA

The accidental discovery of a cell with haploid number of diplochromosomes in squashes of colchicine-treated root tips led to a search for haploid anaphases in treated roots allowed to recover in water. No haploid anaphases were seen. Apart from the divisional stages of diploid and tetraploid nuclei, cells with two pro-, meta- and ana-phases were observed. The formation of distinct cell boundaries by each nucleus of an originally multinucleate cell indicates their potentialities in this direction.

Expression of lactate transporters MCT1, MCT2, MCT4 and the ancillary protein CD147 in horse muscle and red blood cells

Monocarboxylate transporters (MCTs) transport lactate and protons across cell membranes. During intense exercise, lactate and protons accumulate in the exercising muscle and are transported to the plasma. In the horse, MCTs are responsible for the majority of lactate and proton removal from exercising muscle, and are therefore also the main mechanism to hinder the decline in pH in muscle cells. Two isoforms, MCT1 and MCT4, which need an ancillary protein CD147, are expressed in equine muscle. In the horse, as in other species, MCT1 is predominantly expressed in oxidative fibres, where its likely role is to transport lactate into the fibre to be used as a fuel at rest and during light work, and to remove lactate during intensive exercise when anaerobic energy production is needed. The expression of CD147 follows the fibre type distribution of MCT1. These proteins were detected in both the cytoplasm and sarcolemma of muscle cells in the horse breeds studied: Standardbred and Coldblood trotters. In humans, training increases the expression of both MCT1 and MCT4. In this study, the proportion of oxidative fibres in the muscle of Norwegian-Swedish Coldblood trotters increased with training. Simultaneously, the expression of MCT1 and CD147, measured immunohistochemically, seemed to increase more in the cytoplasm of oxidative fibres than in the fast fibre type IIB. Horse MCT4 antibody failed to work in immunohistochemistry. In the future, a quantitative method should be introduced to examine the effect of training on muscle MCT expression in the horse. Lactate can be taken up from plasma by red blood cells (RBCs). In horses, two isoforms, MCT1 and MCT2, and the ancillary protein CD147 are expressed in RBC membranes. The horse is the only species studied in which RBCs have been found to express MCT2, and the physiological role of this protein in RBCs is unknown. The majority of horses express all three proteins, but 10-20% of horses express little or no MCT1 or CD147. This leads to large interindividual variation in the capacity to transport lactate into RBCs. Here, the expression level of MCT1 and CD147 was bimodally distributed in three studied horse breeds: Finnhorse, Standardbred and Thoroughbred. The level of MCT2 expression was distributed unimodally. The expression level of lactate transporters could not be linked to performance markers in Thoroughbred racehorses. In the future, better performance indexes should be developed to better enable the assessment of whether the level of MCT expression affects athletic performance. In human subjects, several mutations in MCT1 have been shown to cause decreased lactate transport activity in muscle and signs of myopathy. In the horse, two amino acid sequence variations, one of which was novel, were detected in MCT1 (V432I and K457Q). The mutations found in horses were in different areas compared to mutations found in humans. One mutation (M125V) was detected in CD147. The mutations found could not be linked with exercise-induced myopathy. MCT4 cDNA was sequenced for the first time in the horse, but no mutations could be detected in this protein.

USING FLUORESCENT AND NON-FLUORESCENT STEROLS TO STUDY RAFTS AND INTRACELLULAR CHOLESTEROL TRANSPORT IN MAMMALIAN CELLS

Cholesterol is an essential component in the membranes of most eukaryotic cells, in which it mediates many functions including membrane fluidity, permeability and the formation of ordered membrane domains. In this work a fluorescent and a non-fluorescent cholesterol analog were characterized as tools to study cholesterol. Next, these analogs were used to study two specific cell biological processes that involve cholesterol, i.e. the structure and function of ordered membrane domains/rafts and intracellular cholesterol transport. The most common method for studying ordered membrane domains is by disrupting them by cholesterol depletion. Because cholesterol depletion affects many cellular functions besides those mediated by membrane domains, this procedure is highly unspecific. The cellular exchange of cholesterol by desmosterol as a tool to study ordered membrane domains was characterized. It turned out that the ability of desmosterol to form and stabilize membrane domains in vitro was weaker compared to cholesterol. This result was reinforced by atomistic scale simulations that indicated that desmosterol has a lower ordering effect on phospholipid acyl chains. Three procedures were established for exchanging cellular cholesterol by desmosterol. In cells in which desmosterol was the main sterol, insulin signaling was attenuated. The results suggest that this was caused by desmosterol destabilizing membrane rafts. Contrary to its effect on ordered membrane domains it was found that replacing cholesterol by desmosterol does not change cell growth/viability, subcellular sterol distribution, Golgi integrity, secretory pathway, phospholipid composition and membrane fluidity. Together these results suggest that exchanging cellular cholesterol by desmosterol provides a selective tool for perturbing rafts. Next, the importance of cholesterol for the structure and function of caveolae was analyzed by exchanging the cellular cholesterol by desmosterol. The sterol exchange reduced the stability of caveolae as determined by detergent resistance of caveolin-1 and heat resistance of caveolin-1 oligomers. Also the sterol exchange led to aberrations in the caveolar structure; the morphology of caveolae was altered and there was a larger variation in the amount of caveolin-1 molecules per caveola. These results demonstrate that cholesterol is important for caveolar stability and structural homogeneity. In the second part of this work a fluorescent cholesterol analog was characterized as a tool to study cholesterol transport. Tight control of the intracellular cholesterol distribution is essential for many cellular processes. An important mechanism by which cells regulate their membrane cholesterol content is by cholesterol traffic, mostly from the plasma membrane to lipid droplets. The fluorescent sterol probe BODIPY-cholesterol was characterized as a tool to analyze cholesterol transport between the plasma membrane, the endoplasmic reticulum (ER) and lipid droplets. The behavior of BODIPY-cholesterol was compared to that of natural sterols, using both biochemical and live-cell microcopy assays. The results show that the transport kinetics of BODIPY-cholesterol between the plasma membrane, the ER and lipid droplets is similar to that of unesterified cholesterol. Next, BODIPY-cholesterol was utilized to analyze the importance of oxysterol binding protein related proteins (ORPs) for cholesterol transport between the plasma membrane, the ER, and lipid droplets in mammalian cells. By overexpressing all human ORPs it turned out that especially ORP1S and ORP2 enhanced sterol transport from the plasma membrane to lipid droplets. Our results suggest that the increased sterol transport takes place between the plasma membrane and ER and not between the ER and lipid droplets. Simultaneous knockdown of ORP1S and ORP2 resulted in a moderate but significant inhibition of sterol traffic from the plasma membrane to ER and lipid droplets, suggesting a physiological role for these ORPs in this process. The two phenylalanines in an acidic tract (FFAT) motif in ORPs, which mediates interaction with vesicle associated membrane protein associated proteins (VAPs) in the ER, was not necessary for mediating sterol transport. However, VAP silencing slowed down sterol transport, most likely by destabilizing ORPs containing a FFAT motif.

The reaction of the nuclei of the vegetative cells and zygotes of Saccharomyces carlsbergensis to some staining techniques

Vegetative cells and zygotes of Saccharomyces carlsbergensis fixed in iodine formaldehyde acetic acid solution and stained after acid hydrolysis in hæmatoxylin, Feulgen and Giemsa show a remarkable similarity in the size and orientation of the structures in the nuclear matrix with reference to the nuclear membrane. The nucleolus described by Guilliermond may either be the chromocenter or the nucleolar equivalent.

Role and function of nonmuscle alpha-actinin-1 and -4 in regulating distinct subcategories of actin stress fibers in mammalian cells

Actin stress fibers are dynamic structures in the cytoskeleton, which respond to mechanical stimuli and affect cell motility, adhesion and invasion of cancer cells. In nonmuscle cells, stress fibers have been subcategorized to three distinct stress fiber types: dorsal and ventral stress fibers and transverse arcs. These stress fibers are dissimilar in their subcellular localization, connection to substratum as well as in their dynamics and assembly mechanisms. Still uncharacterized is how they differ in their function and molecular composition. Here, I have studied involvement of nonmuscle alpha-actinin-1 and -4 in regulating distinct stress fibers as well as their localization and function in human U2OS osteosarcoma cells. Except for the correlation of upregulation of alpha-actinin-4 in invasive cancer types very little is known about whether these two actinins are redundant or have specific roles. The availability of highly specific alpha-actinin-1 antibody generated in the lab, revealed localization of alpha-actinin-1 along all three categories of stress fibers while alphaactinin-4 was detected at cell edge, distal ends of stress fibers as well as perinuclear regions. Strikingly, by utilizing RNAi-mediated gene silencing of alpha-actinin-1 resulted in specific loss of dorsal stress fibers and relocalization of alpha-actinin-4 to remaining transverse arcs and ventral stress fibers. Unexpectedly, aberrant migration was not detected in cells lacking alpha-actinin-1 even though focal adhesions were significantly smaller and fewer. Whereas, silencing of alpha-actinin-4 noticeably affected overall cell migration. In summary, as part of my master thesis study I have been able to demonstrate distinct localization and functional patterns for both alpha-actinin-1 and -4. I have identified alpha-actinin-1 to be a selective dorsal stress fiber crosslinking protein as well as to be required for focal adhesion maturation, while alpha-actinin-4 was demonstrated to be fundamental for cell migration.

Diketopyrrolopyrrole-Based Donor-Acceptor Copolymers as Organic Sensitizers for Dye Sensitized Solar Cells

In this work diketopyrrolopyrrole based copolymers (PDPP-BBT and TDPP-BBT) containing a donor-acceptor structural unit have been explored as organic Sensitizers for quasi-solid state dye Sensitized solar cells. Polymer-sensitized solar cells (PSSC) fabricated utilizing PDPP-BBT and TDPP-BBT as the active layer resulted in a typical power conversion efficiency of 1.43% and 2.41%, respectively. The power conversion efficiency of PSSCs based on TDPP-BBT With use of TiCl4-modified TiO2 photoanode was about 3.06%, attributed to the reduced back recombination reaction and more charge carriers in the external Circuit.

Electro-Oxidation of Borohydride on Rhodium, Iridium, and Rhodium-Iridium Bimetallic Nanoparticles with Implications to Direct Borohydride Fuel Cells

Electrochemical oxidation of borohydride is studied on nanosized rhodium, iridium, and bimetallic rhodium-iridium catalysts supported onto Vulcan XC72R carbon. The catalysts are characterized by X-ray diffraction, transmission electron microscopy, and X-ray photoelectron spectroscopy in conjunction with cyclic voltammetry and polarization studies. The studies reveal that a 20 wt % bimetallic Rh-Ir catalyst supported onto carbon (Rh-Ir/C) is quite effective for the oxidation of borohydride. Direct borohydride fuel cell with Rh-Ir/C as the anode catalyst and Pt/C as the cathode catalyst exhibits a peak power density of 270 mW/cm(2) at a load current density of 290 mA/cm(2) as against 200 mW/cm(2) at 225 mA/cm(2) for Rh/C and 140 mW/cm(2) at 165 mA/cm(2) for Ir/C while operating at 80 degrees C. The synergistic catalytic activity for the bimetallic Rh-Ir nanoparticles toward borohydride oxidation is corroborated by density-functional theory calculations using electron-localization function. (C) 2010 The Electrochemical Society. [DOI:10.1149/1.3442372] All rights reserved.

Expression of cyclin E in endomitotic silk-gland cells from mulberry silkworm

The silk glands of mulberry silkworm Bombyx mori are endoreplicating tissues in which the genomic DNA undergoes multiple rounds of replication without mitosis and nuclear division. In the absence of normal mitotic division, the cell cycle essentially alternates between the G1 and S phases. Cyclin E is crucial for the G1/S transition in both mitotic and endoreplicating cycles. We have cloned and characterized cyclin E (cyclin box) from B. mori, which is nearly identical to the Drosophila cyclin E box except for an insertion of 21 amino acids. Two distinct cyclin E transcripts (1.7 and 2.1 kb) were detected in the silk-gland cells of B. mori and in the B. mori-derived embryonic cell line, BmN. Using anti Cyclin E antibodies two protein bands of 52 and 44 kDa were detected in silk glands and BmN cells at Comparable levels. Both BmN- and the silk-gland cells showed the presence of the interacting kinase Cdk2. Transcripts of the mitotic cyclin, cyclin B, were barely detectable in the endoreplicating silk-gland cells and amounted to only 4-7% of that seen in the mitotically dividing BmN cells. The near absence of cyclin B transcripts and the abundant expression of cyclin E in the silk glands correlate well with the alternation of only G1 and S phases without the intervening mitosis in these cells. (C) 2000 Elsevier Science B.V. All rights reserved.

Voltage delay during constant-current or constant-resistance discharge of Mg-MnO2 dry cells: A comparative study

A theoretical approach has been developed to relate the voltage delay transients of the Mg-MnO2 dry cell observed during discharge by two commonly employed modes, viz., (1) at constant current, and (2) across a constant resistance. The approach has been verified by comparison of experimentally obtained transients with those generated from theory. The method may be used to predict the delay parameters of the Mg-MnO2 dry cell under the two modes of discharge and can, in principle, be extended to lithium batteries.