bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract mechanical connection and abelian reconstruction foralmost Kähler manifolds
Authors: Pekarsky Sergey , Marsden Jerrold E.
Published: September 2002

When the phase space P
of a Hamiltonian
G‐system (P, ω, G, J, H)
has an almost Kähler structure a preferred
connection, called abstract mechanical connection, can
be defined by declaring horizontal spaces at each point to be
metric orthogonal to the tangent to the group orbit. Explicit
formulas for the corresponding connection one‐form
𝒜 are derived in terms of the momentum map,
symplectic and complex structures. Such connection can play the
role of the reconstruction connection (due to the work of A.
Blaom), thus
significantly simplifying computations of the corresponding
dynamic and geometric phases for an Abelian group G. These
ideas are illustrated using the example of the resonant
three‐wave interaction. Explicit formulas for the connection
one‐form and the phases are given together with some new results
on the symmetry reduction of the Poisson structure.
Blowup of solutions of a nonlinear wave equation
Authors: Benaissa Abbes , Messaoudi Salim A.
Published: September 2002

We establish a blowup result to an initial boundary value problem for the nonlinear wave equation utt − M(‖B1/2u‖ 2) Bu + kut = |u| p−2, x ∈ Ω, t > 0.
Unsteady heat transfer of a monatomic gas between two coaxial circular cylinders
Authors: Abourabia A. M. , Mahmoud M. A. , Abdel Kareem W. S.
Published: September 2002

We consider a kinetic‐theory treatment of the cylindrical unsteady heat transfer. A model kinetic equation of the BGK (Bhatnager‐Gross‐Krook) type is solved using the method of moments with a two‐sided distribution function. We study the relations between the different macroscopic properties of the gas as the temperature, density, and heat flux with both the radial distance r and the time t. Also we study the problem from the viewpoint of irreversible thermodynamics and estimate the entropy, entropy production, entropy flux, thermodynamic forces, kinetic coefficients, the change in internal energy, and verify Onsager′s relation for nonequilibrium thermodynamic properties of the system.
Integral representations for Padé‐type operators
Authors: Daras Nicholas J.
Published: September 2002

The main purpose of this paper is to consider an explicit form of the Padé‐type operators. To do so, we consider the representation of Padé‐type approximants to the Fourier series of the harmonic functions in the open disk and of the L p‐functions on the circle by means of integral formulas, and, then we define the corresponding Padé‐type operators. We are also oncerned with the properties of these integral operators and, in this connection, we prove some convergence results.
Alpha-Tocopherol Supplementation in Healthy Individuals Reduces Low-Density Lipoprotein Oxidation but Not Atherosclerosis
Authors: Hodis Howard N. , Mack Wendy J. , LaBree Laurie , Mahrer Peter R. , Sevanian Alex , Liu Chao-ran , Liu Ci-hua , Hwang Juliana , Selzer Robert H. , Azen Stanley P.
Published: September 2002

Background—

Epidemiological studies have demonstrated an inverse relationship between vitamin E intake and cardiovascular disease (CVD) risk. In contrast, randomized controlled trials have reported conflicting results as to whether vitamin E supplementation reduces atherosclerosis progression and CVD events.

Methods and Results—

The study population consisted of men and women ≥40 years old with an LDL cholesterol level ≥3.37 mmol/L (130 mg/dL) and no clinical signs or symptoms of CVD. Eligible participants were randomized to DL-α-tocopherol 400 IU per day or placebo and followed every 3 months for an average of 3 years. The primary trial end point was the rate of change in the common carotid artery far-wall intima-media thickness (IMT) assessed by computer image-processed B-mode ultrasonograms. A mixed effects model using all determinations of IMT was used to test the hypothesis of treatment differences in IMT change rates. Compared with placebo, α-tocopherol supplementation significantly raised plasma vitamin E levels (
P
<0.0001), reduced circulating oxidized LDL (
P
=0.03), and reduced LDL oxidative susceptibility (
P
<0.01). However, vitamin E supplementation did not reduce the progression of IMT over a 3-year period compared with subjects randomized to placebo.

Conclusions—

The results are consistent with previous randomized controlled trials and extend the null results of vitamin E supplementation to the progression of IMT in healthy men and women at low risk for CVD.
Normal outcome of pregnancy in chronic myeloid leukemia treated with interferon‐α in 1sttrimester: Report of 3 cases and review of the literature
Authors: Mubarak A.A.S. , Kakil I.R. , Awidi A. , Al‐Homsi U. , Fawzi Z. , Kelta M. , Al‐Hassan A.
Published: September 2002

Three patients with Chronic Myeloid Leukemia (CML) in chronic phase received Interferon‐α during pregnancy, starting from the 1sttrimester. No maternal complications were reported. The 3 patients delivered normal looking babies apart from one baby who was found to have transient mild thrombocytopenia. Subsequently these children were followed for 30, 12, and 4 months and all had normal growth and development. Am. J. Hematol. 69:115–118, 2002. © 2002 Wiley‐Liss, Inc.
Using homework in therapy for depression
Authors: Garland Anne , Scott Jan
Published: September 2002

There is a growing body of research evidence that demonstrates that completion of homework assignments is significantly correlated with outcome in cognitive therapy. The cognitive model of depression sees homework as an intrinsic aspect of the therapy process. Homework serves a number of purposes, including generalizing learning from the session into everyday life and fostering the independent practice of skills acquired during treatment. We review a number of commonly occurring problems that arise when seeking to engage the client in homework. The negotiation and implementation of homework assignments is a core clinical skill, and we present a range of strategies the clinician can use to optimize its effectiveness. It is vital that practitioners have an awareness of their own role and expectations in developing homework assignments. © Wiley Periodicals, Inc. J Clin Psychol/In Session 58: 489–498, 2002.
Interannual variability of European winter weather: the potential vorticity insight
Authors: Massacand Alexia C. , Davies Huw C.
Published: September 2002

Consideration of well‐established teleconnection patterns account for only a moderate part of European winter variations. Here January interannual variability is examined from a potential vorticity perspective and this yields a reappraisal of the pattern structure and link to European winter weather. The influence of a novel hemispheric pattern in particular is detected. Copyright © 2001 Royal Meteorological Society.
Stimulation of human embryonic lung fibroblasts by TGF‐β and PDGF acting in synergism. The role of Cell Density.
Authors: Stathakos D. , Psarras S. , Kletsas D.
Published: September 2002

The concerted action of TGF‐β and PDGF on a diploid human embryonic lung fibroblast cell strain (Flow 2002) grown in an homologous environment is investigated here. In sparse cultures, TGF‐β stimulates DNA synthesis over a broad concentration range (0.1‐10 ng/ml). Furthermore, it acts in synergism with PDGF, a phenomenon which persists also during in vitro aging of the cells. Preincubation of TGF‐β with the fibroblasts up to 12 hours reduces the subsequent PDGF binding to the cells, while prolonged preincubation restores PDGF binding to control levels. Finally, in cultures of higher cell densities, TGF‐β ceases to stimulate DNA synthesis, whereas PDGF continues even at cell confluency, retains its stimulatory activity suggesting different roles for the two growth factors during the wound healing process.
Transfected Chinese hamster ovary cells as model system for cytokine immunocytochemistry and in situ hybridisation.
Authors: Mueller Regula , Junker Udo , Wagner Kathrin , Heusser Christoph , Bullock Gillian
Published: September 2002

The presence of cytokine producing cells is most easily revealed by techniques measuring the secreted cytokines in culture supernatants or body fluids. However, these techniques only measure the bulk cytokine release by a given, often mixed cell population. To demonstrate cytokine production at the single cell level, immunocytochemistry (ICC) and in situ hybridisation (ISH) are now widely used techniques. To establish these techniques, an easily accessible model system is needed which permits the evaluation of different ICC and ISH protocols. It can be used to demonstrate the specificity of the antibodies and may serve as a positive control for samples of unknown cytokine content. Here we propose the use of Chinese hamster ovary (CHO) cells transfected to express one specific cytokine as such a model system. Its usefulness is demonstrated by the characterisation of six monoclonal antibodies to human interleukin‐4 and the establishment of two in situ hybridisation protocols.
Retraction of cultured endothelial cell monolayer by human breast cancer cells, MCF‐7.
Authors: Yoshioka Kiyoko , Mukai Mutsuko , Shinkai Kiyoko , Akedo Hitoshi
Published: September 2002

A human breast cancer cell line (MCF‐7), when sealed on confluent bovine pulmonary aortic endothelial cell (CPAE) monolayers, induced morphological changes (retraction) in CPAE cells. The area of retraction depended on the incubation time and the number of MCF‐7 cells, suggesting that MCF‐7 cells had the capacity to retract CPAE cells. This capacity was reduced by 60% by pretreatment of MCF‐7 cells with 17β‐estradiol (E) and progesterone (Pg). The extent of retraction was not affected by the addition of various protease inhibitors. CPAE retraction was induced also by adding conditioned medium (CM) from the culture of MCF‐7 cells. Considerably less activity was detected in the CM obtained from MCF‐7 cells cultured in the presence of E and Pg. The retraction was reversed in 24 h by culturing the monolayer in fresh medium without CM and was not induced by trypsin treatment of the CM.
Sensitivity of 3T3 cells to low serum concentration and the associated problems of serum withdrawal.
Authors: Yin Zhikang , Wheatley Denys N.
Published: September 2002

The sensitivity of cells to serum deprivation depends upon whether they are transformed. Most supplies of 3T3 cells are of this type and are considerably less sensitive than untransformed cells. In addition, the apparently simple manoeuvre of reducing serum levels has considerable effects on cell fragility, viability, growth rate and metabolism, which were found to be due to small changes in pH, substrate availability, cell density and other parameters, many of which cannot be attributed to the absence of growth factors from the medium. Supplementation of medium with bovine serum albumin (BSA) to compensate for low normal serum protein did not aid growth nor offset the disturbances caused by low serum levels themselves. Problems associated with the altered precursor availability for DNA, RNA and protein synthesis are also discussed.
Effects of hypothermia on the survival and cryopreservation of minipig ileal cells and Chinese hamster ovary cells.
Authors: Kaeffer B. , Uriel I. Garcia , Bottreau E.
Published: September 2002

Temperature of culture can be used to modulate cellular metabolism for improving small intestinal cell culture and cryopreservation. An hypothermia pretreatment (2 days at 25°C and 3 hours recovery at 37°C) improved hamster cell survival to freeze‐thaw damage (p < 0.01) but decreased the survival of 2 immortal pig ileal cell lines even though epithelioid IPI‐2I cells were more tolerant to hypothermia than IPI‐I fibroblasts. Epithelioid cells survived 3 days at 25°C with unaltered expression of cytokeratin‐18 whereas colonies of fibroblasts did not survive more than a day at 25°C (p < 0.001). These results suggest that hypothermia‐tolerance of pig ileal cell lines might differ according to cell lineage calling for further experiments on small intestinal primary cell culture.
Aphidicolin induces myogenic differentiation in the human rhabdomyosarcoma cell line KFR.
Authors: Cinatl Jindrich , Cinatl Jaroslav , Driever Pablo Hernáiz , Rabenau Holger , Novak Milos , Stefanik Milan , Kornhuber Bernhard , Doerr Hans‐Wilhelm
Published: September 2002

The effects of aphidicolin, a specific inhibitor of DNA polymerase α, on cell growth, DNA synthesis and myogenic differentiation in the human alveolar rhabdomyosarcoma cell line KFR were studied. The treatment with aphidicolin at 5 × 10−6 M concentration, which completely inhibited DNA synthesis and cell growth, induced morphological differentiation of small mononuclear cells to elongated, multinucleated (myotube‐like) structures. The morphological differentiation was accompanied by the expression of skeletal muscle myosin; about 30% myosin‐positive cells were observed after 14 days of treatment, compared to 2.3% in untreated cultures. The results showed that aphidicolin induces differentiation of human rhabdomyosarcoma cells and that multinucleated myotube‐like elements may develop simply by cell fusion without cell division and DNA synthesis.
Speed‐mapping of arsenic distribution in the tissues of earthworms inhabiting arsenious soil.
Authors: Morgan A. J. , Winters C. , Yarwood A.
Published: September 2002

We have examined the distribution of arsenic in the metal‐sequestering chloragogenous tissue of a population of the earthworm, Lumbricus rubellus, exposed to the metalloid in its natural environment. The localization technique was qualitative digital X‐ray speed mapping (2 minute total acquisition time) of fresh air dried tissue smears. Arsenic was located in association with sulphur in a discrete chloragocytic compartment; it was not detected in the phosphate‐rich chloragosome granules. In earthworm tissues arsenic was distributed according to its sulphydryl‐seeking trivalent form, not as the phosphate‐resembling pentavalent form.
Demonstration of a β‐type adaptin at the plant plasma membrane
Authors: Drucker Martin , Herkt Burghard , Robinson David G.
Published: September 2002

Highly purified plasma membrane was prepared from etiolated zucchini hypocotyls, developing pea cotyledons and suspension‐cultured soybean cells by phase partitioning and sucrose density gradient centrifugation. The brain β‐adaptin monoclonal antibody B1/M6, which recognizes a similar adaptin in zucchini clathrin coated vesicles, cross‐reacted with an electrophoretically identical polypeptide in the plant plasma membrane fractions. Immunogold labelling of thin sections of soybean cells confirmed the presence of the β‐adaptin at the plasma membrane. The presence of this adaptin at the Golgi apparatus could not be demonstrated unequivocally.
Factors influencing myofibroblast differentiation during wound healing and fibrosis.
Authors: Desmoulière A.
Published: September 2002

Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilaments bundles and the expression of α‐SM actin, the actin isoform typical of contractile vascular SM cells. Myofibroblasts have been suggested to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. When granulation tissue evolves into a scar, myofibroblasts containing α‐SM actin disappear, probably as a result of apoptosis. In contrast, myofibroblasts expressing α‐SM actin persist in excessive scarring and in fibrotic conditions. The mechanisms leading to the development of myofibroblastic features remain to be investigated. Studies on the factors regulating the phenotype of myofibroblasts will be necessary for understanding their behavior in vivo, and possibly modifying this behavior during the different clinical settings.
Photoreactive (caged) cyclic AMP analogs induce DNA synthesis in mammary epithelial cells
Authors: Yuh In‐Sub , Sheffield Lewis G.
Published: September 2002

Dimethoxy‐2‐nitrobenzyl adenosine‐3′,5′ cyclic monophosphate (DMNB) is a metabolically active, photolabile cyclic AMP analog that yields free cyclic AMP upon UV hydrolysis. The analog is useful in that it permits short term, transient elevations of intracellular cyclic AMP. Addition of DMNB (1‐10 μM) to mouse mammary epithelial cells, followed by UV irradiation of cells, caused a significant increase in DNA synthesis over that observed with controls, UV irradiation alone or DMNB alone. In subsequent studies, DMNB exhibited a modest, but statistically significant, interaction with epidermal growth factor in promoting DNA synthesis. Effects of DMNB were observed if DNA synthesis was measured as either 3H‐thymidine incorporation into DNA or as percent S‐phase cells. These results indicate that previously observed effects of agents such as cholera toxin and phosphodiesterase resistant cyclic AMP analogs on mammary epithelial proliferation can be mimicked, at least in part, by a short term pulse of cyclic AMP.
THE NON‐HUMAN PRIMATE ENDOCRINE PANCREAS: DEVELOPMENT, REGENERATION POTENTIAL AND METAPLASIA
Authors: WOLFE‐COOTE SONIA , LOUW JOHAN , WOODROOF COLIN , DU TOIT DON F.
Published: September 2002

An investigation into the development of the Vervet monkey endocrine pancreas revealed a sequence of occurrence of pancreatic peptides that differed from previous reports in mice, dog and human with PP and somatostatin occurring before glucagon and insulin. All four pancreatic peptides were identified, immunohistochemically, in only one of the pancreatic primordial buds, before fusion of the two buds to form the pancreas. This questions the hypothesis that the heterogeneous endocrine cell distribution seen in the adult pancreas is due to the contribution of only PP cells by the ventral bud and non‐PP cells by the dorsal bud. Co‐localization of glucagon and PP was observed extensively in the developing pancreas and the predominant expression of one over the other in an apparently organized non‐random manner accounted for the glucagon‐ and PP‐rich areas seen in the developing pancreas. A small number of cells immunoreactive to glucagon and PP were also observed in the adult. Reports of plasticity of differentiation of other pancreatic cells led us to investigate regeneration potential of the adult monkey pancreas. Partial obstruction of the Vervet monkey main pancreatic duct, by cellophane wrapping, resulted in duct cell proliferation and differentiation to form new endocrine tissue in a way that mimics normal organogenesis. Focal areas of hepatocytes were found in the regenerated pancreas of one monkey, illustrating further the latent developmental capabilities of adult pancreas cells. These findings could lead to interesting new therapies for pancreas and liver disease.
DISRUPTION OF CELL—CELL ADHESION IN THE PRESENCE OF SODIUM BUTYRATE ACTIVATES EXPRESSION OF THE 92kDa TYPE IV COLLAGENASE IN MDCK CELLS
Authors: FIORINO ANTHONY S. , ZVIBEL ISABEL
Published: September 2002

We have investigated the effects of altered cell shape on the regulation of the 92kDa type IV collagenase. In MDCK cells, anti‐E‐cadherin antibodies alter cell shape by disrupting normal cell—cell contacts, while sodium butyrate causes a marked flattening and spreading of cells. The disruption of cell—cell contacts led to a faint expression of the 92kDa collagenase. This effect was enhanced by sodium butyrate, which by itself did not induce collagenase expression. In contrast, stromelysin expression was not regulated in these conditions. Although mRNA expression was enhanced, the secreted collagenase activity was not altered in these conditions in either cell line. Examination of cytoskeletal and extracellular matrix proteins and cell—cell and cell—matrix adhesion proteins by immunofluorescence and Western blot revealed a disruption of the actin network, tight junctions, and fibronectin deposition by anti E‐cadherin antibodies, and alterations in actin, cytokeratin 8, cytokeratin 14, laminin and β1 integrin induced by sodium butyrate. Thus, the induction of collagenase expression in epithelial cells by disrupted cell—cell adhesion and sodium butyrate is associated with changes in cell shape and structure.
ASSOCIATION OF MURINE SPLENOCYTE CD3 COMPLEX TO THE CYTOSKELETON: ABSENCE OF MODULATION BY EXOGENOUS FATTY ACIDS
Authors: PECK MICHAEL D. , LI ZHIMING , JY WENCHE , CHU ARTHUR J. , BOURGUIGNON LILLY Y. W.
Published: September 2002

The cytoplasmic regions of the CD3 complex are presumably involved in signal transduction following ligand—receptor binding. We investigated the effects of incubating either stearic or oleic acid on the association of murine lymphocyte CD3 complex with the cytoskeleton. Both cytochalasin D, an inhibitor of microfilament formation, and W7, an inhibitor of calmodulin, inhibited capping of CD3. The association of CD3 with the cytoskeleton was confirmed by confocal laser scanning microscopy studies, which showed co‐localization of the cross‐linked CD3 receptors and the membrane attachment proteins ankyrin and fodrin. Although exogenous oleic acid increased plasma membrane fluidity, neither expression nor capping of CD3 receptors was increased. Nonetheless, oleic acid did increase uptake of tritiated thymidine after binding of anti‐CD3 antibodies. Lymphoproliferation was progressively inhibited by both cytochalasin D and W7, confirming the importance of intact cytoskeleton for cellular activation.
HYPERTHERMIC STRESS AFFECTS GLUCOCORTICOID RECEPTOR‐MEDIATED TRANSCRIPTION IN RAT LIVER
Authors: VIDOVIĆ STOJKO , ČVORO ALEKSANDRA , DUNDJERSKI JADRANKA , TRAJKOVIĆ DIVNA , MATIĆ GORDANA
Published: September 2002

Binding capacity of the cytoplasmic and nuclear glucocorticoid receptor (GR) and the activity of tyrosine aminotransferase (TAT) were examined in the liver of intact and adrenalectomized rats exposed to 41°C whole body hyperthermic stress. In glucocorticoid‐deprived animals, stress‐induced decrease in the cytoplasmic steroid binding was followed by parallel increases in its nuclear binding and TAT activity, suggesting a stimulation of TAT gene transcription by the GR in the absence of the ligand. In intact animals, however, a diminution of the steroid binding in the cytosol, its unchanged nuclear binding and an impairment of TAT activity were observed upon the stress. The results imply that stress could elicit different structural or functional alterations of unligandedvsliganded GR.
SENSITIVITY OF DAUDI CELLS TO TUMOR NECROSIS FACTOR ALPHA: EARLY CHANGES IN POLYPHOSPHOINOSITIDE METABOLISM
Authors: SANTAVENERE E. , DI PIETRO R. , DI DOMENICANTONIO L. , RANA R.
Published: September 2002

The effects of recombinant Tumor Necrosis Factor α (r‐TNF α) on polyphosphoinositide metabolism were examined in a Burkitt Lymphoma cell line (Daudi cells). After 1h of in vitro treatment with r‐TNF α, the incorporation of32Pi into phosphatidylinositol 4,5‐phosphate (PtdInsP2), phosphatidylinositol 4‐phosphate (PtdInsP) and phosphatidylinositol (PtdIns) was reduced compared with controls, confirming previous findings observed in other cell lines of a specific PtdIns breakdown following r‐TNF α treatment. The novelty of this study is therefore the demonstration of early changes in polyphosphoinositide metabolism during the antiproliferative response elicited by this cytokine in Daudi cells.
CHOLINE BASE EXCHANGE ACTIVITY IN RAT HEPATOCYTE NUCLEI AND NUCLEAR MEMBRANES
Authors: ALBI E. , VIOLA‐MAGNI M.P
Published: September 2002

Previous investigations have demonstrated the presence of phospholipids as a component of chromatin; however the mechanism of their synthesis, namely if they are synthesized in the nuclei or in the cytoplasm (microsomal fraction), from where they may eventually be transported to the nucleus, has not yet been clarified. The phosphatidylcholine, for example, can be formed, albeit in a limited amount, by an interconversion reaction between bases. The aim of the present research was to ascertain the presence of the enzyme complex responsible for this reaction in hepatocyte nuclei and in isolated nuclear membrane. The incorporation of [14C]‐choline in phosphatidylcholine was assayed in microsomes, hepatocyte nuclei, liver nuclei and nuclear membranes of rat liver. The reaction was Ca2+‐dependent and the specific activity was higher in microsomes but was present, albeit at a low level, also in nuclei and in nuclear membranes. Possible contaminations were excluded by specific microsomal markers and by the reaction time course. In fact, the nuclear reaction reached the maximum level slowly with respect to microsomes. Since the phosphatidylcholine extracted from the nuclei show an enrichment in unsaturated fatty acids of monoenoic fraction, such as oleic acid, the difference in reaction kinetics has been tentatively explained as due to the phosphatidylcholine fatty acid content. The presence of this base exchange enzyme complex may allow a fast change in chromatin phospholipid composition.
EXPRESSION OF A CYTOPLASMICALLY EPITOPE‐TAGGED, HUMAN GOLGI GLYCOSYLTRANSFERASE IN HOMOLOGOUS CELLS RESULTS IN MISLOCALIZATION OF MULTIPLE GOLGI PROTEINS
Authors: YANG WEI , STORRIE BRIAN
Published: September 2002

We have compared the effect of mislocalization of a Golgi glycosyltransferase in heterologous and homologous cell systems on the distribution of other Golgi‐associated proteins. Myc‐spacer‐human N‐acetylglucosaminyltransferase I (NAGT‐I), an N‐terminally epitope‐tagged NAGT‐I, in which the first added negatively charged amino acid is in position 13, localizes to the endoplasmic reticulum (ER) by immunofluorescence when expressed in monkey (Vero) or human (HeLa) cells. When myc‐spacer‐human NAGT‐I was expressed in Vero cells, the distribution of the Golgi‐associated coat protein, β‐COP, was concentrated juxtanuclearly and undisturbed relative to control. When myc‐spacer‐human NAGT‐I was expressed in HeLa cells, however, both endogenous β‐COP and GalT were no longer concentrated in a juxtanuclear manner but were rather cytoplasmically distributed as was the myc‐tagged human NAGT‐I. Based on these observations, we suggest that extensive interactions between proteins that normally show overlapping distributions between the medial Golgi stack and trans Golgi/TGN are possible. Moreover, we suggest that small differences in sequence may play a large role in potentiating interactions of Golgi complex proteins.
EVIDENCE FOR A LOCALIZATION SIGNAL IN THE 3′UNTRANSLATED REGION OF MYOSIN HEAVY CHAIN MESSENGER RNA
Authors: WISEMAN JOHN W. , GLOVER L. ANNE , HESKETH JOHN E.
Published: September 2002

Localization signals in the 3′untranslated region (3′UTR) of myosin heavy chain mRNA were investigated using hybrid gene constructs. In myoblasts transfected with constructs containing either both coding sequences and 3′UTR of the rabbit β‐globin gene or the β‐globin coding sequences alonein situhybridization showed that globin transcripts were distributed throughout the cytoplasm with no localization. In contrast, in myoblasts transfected with β‐globin coding sequences linked to the myosin heavy chain 3′UTR there was strong perinuclear localization of the hybrid mRNA; this was maintained in myotubes. We conclude that myosin heavy chain 3′UTR contains a localization signal.
TIME‐ AND CONCENTRATION‐DEPENDENCE OF THE GROWTH‐PROMOTING ACTIVITY OF INSULIN AND HISTAMINE IN TETRAHYMENA. APPLICATION OF THE MTT‐METHOD FOR THE DETERMINATION OF CELL PROLIFERATION IN A PROTOZOAN MODEL
Authors: HEGYESI HARGITA , CSABA GYÖRGY
Published: September 2002

The unicellular ciliateTetrahymena pyriformiswas treated with different concentrations of insulin or histamine and at different time points the cell density was measured, using a tetrazolium‐based semiautomated colorimetric assay (MTT). The assay was suitable to determine the rate of cell proliferation of Tetrahymena. Insulin in each concentration significantly elevated the cell count up to 3h. After that, it was neutral or its effect was insignificant. Histamine at 10−5m concentration diminished cell count at 3, 5, 7 and 24h. At 10−6m concentration there was no difference and at 10−7m concentration it enhanced cell division up to 5h, after that there being no difference. The two hormones have cell division promoting activity for cells of higher animals and the experiments demonstrate this effect already at a unicellular level.
SOMATOSTATIN‐LIKE IMMUNOREACTIVE CELLS IN THE GROUND SQUIRREL RETINA
Authors: LUGO NIDZA , BLANCO ROSA E.
Published: September 2002

Immunocytochemical techniques were employed to locate somatostatin (SS)‐containing cells in the retina of the 13‐lined ground squirrel (Spermophilus tridecemlineatus). In normal retinas immunostain was limited to neuronal processes, yet distinctly labeled somata were detected in retinas of animals pretreated with colchicine. Labeled cell bodies were located in the outermost and innermost portions of the inner nuclear layer (INL) and in the ganglion cell layer (GCL). The largest population of SS‐like immunoreactive neurons was found in the innermost INL. These cells were identified as small and medium sized amacrine cells whose soma diameters ranged from 4 to 14μm. A smaller population of immunoreactive cells was observed in the outermost region of the INL. These cells, presumptive horizontal cells, were found mainly in peripheral regions of the retina. Immunoreactive cells in the GCL were of two types: displaced amacrines, and retinal ganglion cells. SS‐positive axons in the optic fiber layer suggest that some of the immunoreactive GCL neurons were ganglion cells, and it is our opinion that these cells belong to a class of associational ganglion cells previously identified in other species.
CONFOCAL ANALYSIS OF PRIMARY CILIA STRUCTURE AND COLOCALIZATION WITH THE GOLGI APPARATUS IN CHONDROCYTES AND AORTIC SMOOTH MUSCLE CELLS
Authors: POOLE C. ANTHONY , JENSEN CYNTHIA G. , SNYDER JUDITH A. , GRAY C. GEORGE , HERMANUTZ VALENTIN L. , WHEATLEY DENYS N.
Published: September 2002

Detyrosinated and acetylated α‐tubulins represent a stable pool of tubulin typically associated with microtubules of the centrosome and primary cilium of eukaryotic cells. Although primary cilium—centrosome and centrosome—Golgi relationships have been identified independently, the precise structural relationship between the primary cilium and Golgi has yet to be specifically defined. Confocal immunohistochemistry was used to localize detyrosinated (ID5) and acetylated (6‐11B‐1) tubulin antibodies in primary cilia of chondrocytes and smooth muscle cells, and to demonstrate their relationship to the Golgi complex identified by complementary lectin staining with wheat germ agglutinin. The results demonstrate the distribution and inherent structural variation of primary cilia tubulins, and the anatomical interrelationship between the primary cilium, the Golgi apparatus and the nucleus. We suggest that these interrelationships may form part of a functional feedback mechanism which could facilitate the directed secretion of newly synthesized connective tissue macromolecules.
ANALYSIS OF SPATIAL DISTRIBUTIONS OF CELLULAR MOLECULES DURING MECHANICAL STRESSING OF CELL SURFACE RECEPTORS USING CONFOCAL MICROSCOPY
Authors: RYCHLY JOACHIM , POMMERENKE HAGEN , DÜRR FRIEDA , SCHREIBER ERIK , NEBE BARBARA
Published: September 2002

Confocal laser scanning microscopy represents a suitable technique to study the localization of cellular components in three dimension. The authors used this technique to analyse cellular events related to mechanical stimulation of integrin receptors on the cell surface. By performing optical sections the distribution of integrin receptors on the apical surface of an osteoblastic cell was determined. Concerning intracellular compartimentalization of signal transduction events, it was demonstrated that mechanical stimulation of integrins induced their linkage to the cytoskeleton. Cytoskeletally associated proteins like vinculin and talin accumulated in the vicinity of the site where the mechanical stress was applied to integrins on the cell surface. Optical sections revealed that clustering of these proteins proceeded to the base of the cell with gradually decreasing extent. In summary, it was demonstrated that the local distribution of cellular components is an important factor in mechanically induced signal transduction.
EXPRESSION OF CATION EXCHANGER NHE AND ANION EXCHANGER AE ISOFORMS IN PRIMARY HUMAN BONE‐DERIVED OSTEOBLASTS
Authors: MOBASHERI A. , GOLDING S. , PAGAKIS S. N. , CORKEY K. , POCOCK A. E. , FERMOR B. , O’BRIEN M. J. , WILKINS R. J. , ELLORY J. C. , FRANCIS M. J. O.
Published: September 2002

The authors used isoform‐specific antibodies against cation (NHE) and anion (AE) exchange isoforms in order to establish their specific expression and localization in dispersed human bone‐derived cells. Immunocytochemical preparations of permeabilized osteoblasts probed with polyclonal antibodies were optically analysed by conventional immunofluorescence and con‐focal laser scanning microscopy. These techniques demonstrated the abundant presence of epitopes of the cation exchangers NHE1 and NHE3 and the anion exchanger AE2 in these cells. The NHE1 and NHE3 isoform proteins were predominantly located in subplasmalemmal and nucleoplasmic vesicles. The AE2 isoform was densely localized to a subcellular location characteristic of the Golgi complex. The molecular identity of the AE and NHE isoforms was investigated by RT‐PCR that confirmed the presence of NHE1 and NHE3 transcripts in addition to NHE4. RT‐PCR and diagnostic restriction analysis of amplified AE cDNA established preferential AE2 expression. Since AE2 has been shown to act as a sulfate transporter at low pH, it is possible that it performs this function in the osteoblast Golgi complex where sulfation reactions occur post‐translationally on numerous extracellular matrix macromolecules prior to secretion and mineralization. The Na+/H+exchanger proteins are regulated by mitogenic and non‐mitogenic stimuli in the osseus environment and are involved in the large fluxes of ions and protons that necessarily occur during bone formation and resorption and thus play an important role in intracellular ion homeostasis in osteoblasts.
REGULATION OF EXTRACELLULAR MATRIX PROTEINS BY TRANSFORMING GROWTH FACTOR β1IN CULTURED PULMONARY ENDOTHELIAL CELLS
Authors: Shanker Gouri , Olson Dan , Bone Roger , Sawhney Rajinder
Published: September 2002

Transforming growth factor beta‐1 (TGF‐β1), which is present in lung tissue, has been suggested to play a role in modulating vascular cell function in vivo. The action of TGF‐β1in vivo, especially at the local site of application to connective tissue, is anabolic and leads to pulmonary fibrosis and angiogenesis, strongly indicating that TGF‐β may have practical applications in repair of tissue injury caused by burns, trauma, or surgery. In the present study, we have used cultured bovine pulmonary artery endothelial (BPAE) cells as a model system. Expression of various proteins, including SPARC (secreted protein acidic and rich in cysteines), type IV procollagen and fibronectin (FN) was examined by radiolabeling the cells with [3H]proline, immunoprecipitation with specific antibodies, and Northern blot analyses by using specific cDNA probes. Cultured cells were labeled with [3H]proline for 24h in either the absence or in the presence of TGF‐β1(0–20ng/ml). Incorporation of radioactivity was observed in a concentration‐dependent manner, maximal at 5ng/ml. Northern blot hybridization demonstrated that TGF‐β1(5ng/ml) treatment of BPAE cells caused an increase in steady‐state levels of mRNA for SPARC (75%) and FN, thus, indicating a positive modulation. To determine whether TGF‐β1has an effect on cell proliferation in the cultures, the incorporation of [3H]thymidine into cellular nuclei was used to measure DNA replication; 5ng/ml had an inhibitory effect (42%) on cell proliferation. Protein production by TGF‐β1, surprisingly, showed decrease in SPARC levels (42%) and in contrast increased levels of FN (86%) and type IV procollagen (334%). Our results indicate that on exposure to TGF‐β1, cultured BPAE cells differentially change expression of extracellular matrix proteins which may be important in remodeling of tissue and healing at sites of injury.
POSSIBLE INVOLVEMENT OF A CHLORIDE—BICARBONATE EXCHANGER IN APOPTOSIS OF ENDOTHELIAL CELLS AND CARDIOMYOCYTES
Authors: Fujita Hiroshi , Ishizaki Yasuki , Yanagisawa Atsuo , Morita Ikuo , Murota Sei‐Itsu , Ishikawa Kyozo
Published: September 2002

We examined the role of ion movement in staurosporine‐induced apoptosis of vascular endothelial cells. Cultured vascular endothelial cells from bovine carotid arteries were used. Apoptosis was determined by propidium iodide assay. Treatment of the endothelial cells with staurosporine (10nmol/l‐1μmol/l) for 6h induced nuclear fragmentation in a dose‐dependent manner. Staurosporine (1μmol/l) elicited apoptosis in 70.5±1.5% of cells. Concomitant treatment of endothelial cells with 1mmol/l of 4,4‐diisothiocyanatostilbene‐2,2‐disulfonic acid (DIDS), a chloride—bicarbonate exchange blocker, completely inhibited staurosporine‐induced apoptosis. Other ion transporter inhibitors such as dimethyl amiloride and anthracene‐9 carboxylic acid were less effective inhibitors of staurosporine‐induced apoptosis of endothelial cells. DIDS prevented staurosporine‐induced apoptosis of endothelial cells as well as cardiomyocytes. Next, we determined whether chloride ions or bicarbonate are involved in apoptosis. Incubation with a chloride ion removal buffer did not inhibit staurosporine‐induced apoptosis of endothelial cells. However, endothelial cell apoptosis was completely suppressed by an inhibitor of caspase, benzyloxycarbonyl‐Asp‐CH2‐O(C)O‐dichlorobenzene (zD‐dcb, 50μmol/l). Staurosporine (1μmol/l) increased the intracellular pH of endothelial cells, and DIDS (1mmol/l), but not a caspase inhibitor, inhibited this increase in pH caused by staurosporine. Our findings suggest that endothelial cell apoptosis induced by staurosporine may be associated with the Cl−and bicarbonate (HCO−3) ions. Thus, Cl−efflux from cells or HCO−3 influx to cells (which increases pH) may play an important role in signal transduction leading events such as activation of caspase in staurosporine‐induced apoptosis.
GLYCOLIPID GLYCOSYLTRANSFERASE ACTIVITIES DURING EARLY DEVELOPMENT OF XENOPUS: EFFECT OF RETINOIC ACID
Authors: Rossi Federica , Gornati Rosalba , Rizzo Angela M. , Venturini Loretta , Bernardini Giovanni , Berra Bruno
Published: September 2002

Retinoic acid (RA) plays an important role in differentiation stage in which it also influences glycoconjugate metabolism. Previous work in our laboratory has shown that treatment with RA modifies glycolipid synthesis and distribution in total Xenopus embryos during development. In this study we have investigated the activity of the following anabolic enzymes involved in glycolipid biosynthesis: sialyltransferase‐1 (SAT‐1), GM3(β1,4)‐N‐acetylgalactosaminyltransferase (GalNAcT‐1) and LacCer(β1,3)N‐acetylglucosaminyltransferase (GlcNAcT‐1). These enzymes are located at the branching point of lactosylceramide (Lc2) metabolism. Enzyme activities were assayed after treatment with different doses of RA added exogenously to the medium during the first 7 days of Xenopus embryo development. Our results show that RA activates GlcNAcT‐1, the enzyme that drives Lc2to the glycolipids of the lacto‐series, and SAT‐1 that inserts Lc2in the ganglio‐series pathway. These data support our previous analysis of glycolipid pattern in Xenopus embryos after RA treatment (Rizzo et al., 1995;Cell Biol Int19: 895–901) indicating a possible correlation between the distribution of glycolipids and the enzymes involved in their metabolism.
Ha‐ ras‐TRANSFORMATION ALTERS THE METABOLISM OF PHOSPHATIDYLETHANOLAMINE AND PHOSPHATIDYLCHOLINE IN NIH 3T3 FIBROBLASTS
Authors: Momchilova A. , Markovska T. , Pankov R.
Published: September 2002

Cultured NIH 3T3 fibroblasts were employed to investigate the changes in the phospholipid metabolism induced by Ha‐ ras transformation. All phospholipid fractions were reduced in ras ‐transformed fibroblasts except phosphatidylethanolamine (PE). The incorporation of labeled choline and ethanolamine into phosphatidylcholine (PC), PE and their corresponding metabolites were elevated in a similar manner in the transformed cells. The enhanced uptake of choline and ethanolamine correlated with the activation of choline kinase and ethanolamine kinase. Similarly, the uptake of arachidonic, oleic and palmitic acids by PC and PE was higher in ras ‐cells. Acyl‐CoA synthetases, which esterify fatty acid before their incorporation into lysophospholipids, were also activated. However, both CTP:phosphocholine‐cytidylyltransferase and CTP:phosphoethanolamine‐chytidyltransferase were inhibited in the transformed cells. This fact, taken together with the observed activation of choline‐ and ethanolamine kinases, led to accumulation of phosphocholine and phosphoethanolamine, which have been presumed to participate in the processes of tumor development. PC biosynthesis seemed to be carried out through the CDP‐choline pathway, which was stimulated in the oncogenic cells, whereas PE was more likely, a product of phosphatidylserine decarboxylation rather than the CDP‐ethanolamine pathway.
DIFFERENCES IN RAT RBC CYTOSOL INDUCED AFTER IN VIVO PHENYLHYDRAZINE TREATMENT
Authors: Djurkovic Aleksandra , Zaric Jelena , Lušic Marina , Glišin Vladimir , Popovic Zvezdana
Published: September 2002

Analysis of rat red blood cell (RBC) cytosol protein fraction after in vivo phenylhydrazine treatment revealed increased amounts of 68‐kDa protein. This protein is present in trace amounts in normal rat RBC cytosol. We also present data that 68‐kDa protein from RBC cytosol has an identical isoelectric point, partial proteolytic map and immunological determinants as a protein of the same molecular mass from rat exosomes. These data indicate that 68‐kDa protein is normally present in rat RBC cytosol, is exported via exosomes during reticulocyte maturation, and is increased in induced haemolytic anaemia.
INHIBITION OF PROLIFERATION OF CONTAMINATING FIBROBLASTS BY D‐VALINE IN CULTURES OF SMOOTH MUSCLE CELLS FROM HUMAN MYOMETRIUM
Authors: Hongpaisan Jarin
Published: September 2002

Replacement of L‐valine with D‐valine in a standard culture medium can selectively inhibit fibroblast proliferation. The aim of the present study was to investigate whether human myometrial cells cultured with D‐valine instead of L‐valine can survive and express their characteristics. Cultured cells (95–98%) maintain expression of the intermediate filament desmin, which is the specific marker for mature muscle cells. By transmission electron microscopy, the cells showed the general morphology of smooth muscle cells in culture. Oxytocin in serum‐free culture medium at 37°C (5min) caused a concentration‐dependent increase in cellular Na and total Ca, and a decrease in K content as determined by X‐ray microanalysis. The percentage of cells cultured with D‐valine responding to oxytocin stimulation was larger than that of cells cultured with L‐valine, suggesting less contamination of smooth muscle cells by fibroblasts in the presence of D‐valine. As shown by measurements with fura‐2, D‐valine‐cultured cells retained the characteristic increase in intracellular free Ca2+ions after oxytocin stimulation.
TISSUES FORMED DURING DISTRACTION OSTEOGENESIS IN THE RABBIT ARE DETERMINED BY THE DISTRACTION RATE: LOCALIZATION OF THE CELLS THAT EXPRESS THE mRNAs AND THE DISTRIBUTION OF TYPES I AND II COLLAGENS
Authors: Li G. , Virdi A. S. , Ashhurst Doreen E. , Simpson A.H.R.W. , Triffitt J. T.
Published: September 2002

An experimental model of leg lengthening was used to study the morphology of, the collagenous proteins present, and the collagen genes expressed in the regenerating tissue following 20% lengthening at four different distraction rates. At a distraction rate of 0.3mm/day (8 weeks distraction), the regenerate consists of intramembranous bone and localized areas of fibrocartilage. At rates of 0.7 (4 weeks) and 1.3mm/day (2 weeks), the bone that grows from the cut ends of the cortical bone is separated by fibrous tissue and cartilage is present. At 2.7mm/day (1 week), only fibrous tissue and sparse bone are present. Type I collagen is present in the matrices around the cells expressing its mRNA and similarly, type II collagen is located around the chondrocytes. Type I collagen mRNA is expressed predominantly by the fibroblasts in the fibrous tissue, the bone surface cells and to a reduced extent by the osteocytes. Type II collagen mRNA is expressed by chondrocytes. The results suggest that osteoblasts and chondrocytes within the regenerate originate from the same pool of progenitor cells, and the differentiation of these cells and the expression of types I and II collagen genes are altered by different rates of distraction. These observations suggest that the optimal rate of distraction in the model is 0.7mm/day.
INHIBITION OF CELL DIVISION BUT NOT NUCLEAR DIVISION BY 1‐ O ‐OCTADECYL‐2‐ O ‐METHYL‐ Sn ‐GLYCERO‐3‐PHOSPHOCHOLINE
Authors: Pushkareva Marina Y. , Janoff Andrew S. , Mayhew Eric
Published: September 2002

1‐ O ‐Octadecyl‐2‐ O ‐methyl‐glycero‐3‐phosphocholine (ET‐18‐OCH3) selectively inhibits the growth of cancer cells. Here we show that in some cell types ET‐18‐OCH3and liposome‐associated ET‐18‐OCH3inhibit cell division without concurrent inhibition of nuclear division, leading to multinucleate cell formation, and cell death through apoptosis. Cell cycle analysis revealed that ET‐18‐OCH3‐treated U‐937 cells continued to move through the cell cycle, but many cells were not able to divide and instead accumulated as tetraploid cells or octaploid cells in the G0/G1 phase of the cell cycle. Inhibition of cytokinesis has been shown to be paralleled by activation of U‐937 cells, including upregulation of some cell‐surface markers, acquisition of phagocytic activity, and secretion of tumor necrosis factor (TNF)‐α (Pushkareva et al., 2000). Furthermore, treatment of cells with ET‐18‐OCH3results in the accumulation of apoptotic cells in time‐ and dose‐dependent manner. It is possible that inhibition of cytokinesis may be related to cytoskeletal effects.
INHIBITION OF CYTOKINESIS BY ASBESTOS AND SYNTHETIC FIBRES
Authors: Jensen Cynthia G. , Watson Maureen
Published: September 2002

Using high‐resolution timelapse microscopy, we have followed individual phagocytized fibres through the later stages of division in MeT‐5A human mesothelial cells and LLC‐MK2monkey epithelial cells. The fibres used were crocidolite and chrysotile asbestos, fibrous glass (MMVF), and refractory ceramic fibres (RCF). Long fibres (15–80μm) trapped within the cleavage furrow can partially or completely block cytokinesis. Cells proceed in one of three ways: (1) eventual completion of cytokinesis; (2) incomplete cytokinesis, resulting in two cells joined by a fibre‐containing intercellular channel; or (3) failure of cytokinesis, resulting in a binucleate or trinucleate cell. Two factors associated with fibre‐induced bi/trinucleation are: (1) an initial association between the fibre and the forming daughter nuclei, which is sometimes lost over time, and (2) disintegration of the midbody. The studies suggest that delay of cytokinesis by interzonal fibres can result in bi/trinucleation through the loss of midbody/intercellular bridge proteins that are required for completion of cytokinesis.