bluuebunny/crossref_metadata_2025 huggingface dataset:
Estrogen Activation of Cyclic Adenosine 5′-Monophosphate Response Element-Mediated Transcription Requires the Extracellularly Regulated Kinase/Mitogen-Activated Protein Kinase Pathway
Authors: Wade Christian B. , Dorsa Daniel M.
Published: February 2003

The ability of estrogen to rapidly initiate a variety of signal transduction cascades is increasingly recognized as playing an important role in a number of tissue-specific transcriptional actions of the hormone. In vivo, estrogen rapidly elicits phosphorylation of cAMP response element-binding protein (CREB). We have previously shown that both ERα and ERβ are capable of activating the MAPK pathway in response to a low dose of 17β-estradiol. In the present study, the ability of estrogen to act through both ERα and ERβ to increase CREB phosphorylation was evaluated in an immortalized hippocampal cell line stably expressing either receptor. Estrogen treatment promoted rapid CREB phosphorylation, reaching a maximum by 15 min. This activation is completely blocked by the antiestrogen ICI 182,780, suggesting an estrogen receptor-dependent mechanism. The addition of the mitogen/ERK kinase-1 inhibitor, PD98059, also blocked the ability of estrogen to signal to CREB phosphorylation. Estrogen also caused an increase in p90Rsk activity, a critical mediator of MAPK effects. Surprisingly, blockade of the protein kinase A pathway in cells treated with estrogen did not affect estrogen-mediated CREB phosphorylation. Thus, MAPK and p90Rsk appear to be the primary mediators of estrogen-induced gene transcription through ERα and ERβ.
In Vivo Dimerization of Types 1, 2, and 3 Iodothyronine Selenodeiodinases
Authors: Curcio-Morelli Cyntia , Gereben Balazs , Zavacki Ann Marie , Kim Brian W. , Huang Stephen , Harney John W. , Larsen P. Reed , Bianco Antonio C.
Published: February 2003

The goal of the present investigation was to test the hypothesis that types 1, 2, and 3 iodothyronine selenodeiodinases (D1, D2, and D3) can form homodimers. The strategy included transient coexpression of wild-type (wt) deiodinases (target), and FLAG-tagged alanine or cysteine mutants (bait) in human embryonic kidney epithelial cells. SDS-PAGE of the immunoprecipitation pellet of 75Se-labeled cell lysates using anti-FLAG antibody revealed bands of the correct sizes for the respective wt enzymes, which corresponded to approximately 2–5% of the total deiodinase protein in the cell lysate. Western blot analysis with anti-FLAG antibody of lysates of cells transiently expressing individual FLAG-tagged-cysteine deiodinases revealed specific monomeric bands for each deiodinase and additional minor bands of relative molecular mass (Mr) of 55,000 for D1, Mr 62,000 for D2, and Mr 65,000 for D3, which were eliminated by 100 mm dithiothreitol at 100 C. Anti-FLAG antibody immunodepleted 10% of D1 and 38% of D2 activity from lysates of cells coexpressing inactive FLAG-tagged Ala mutants and the respective wt enzymes (D1 or D2) but failed to immunodeplete wtD3 activity. D1 or D2 activities were present in these respective pellets. We conclude 1) that overexpressed selenodeiodinases can homodimerize probably through disulfide bridges; and 2) at least for D1 and D2, monomeric forms are catalytically active, demonstrating that only one wt monomer partner is required for catalytic activity of these two deiodinases.
Identification of a New Growth Hormone Family Protein, Somatolactin-Like Protein, in the Rainbow Trout (Oncorhyncus mykiss) Pituitary Gland
Authors: Yang Bih-Ying , Chen Thomas T.
Published: February 2003

A cDNA encoding a new GH family protein expressed in the rainbow trout (Oncorhyncus mykiss) pituitary gland was identified. Because the deduced amino acid sequence of this cDNA shares 56% homology with rainbow trout (rt) somatolactin (rtSL), we named it as rtSL-like protein (rtSLP). Comparison of the amino acid sequences of rtGH, rainbow trout prolactin (rtPRL), and rtSLP revealed that rtSLP shares 26% and 21% identity with that of rtGH and rtPRL, respectively. Trout SLP contains not only the known GH family protein conserved domains but also all of the four cysteine residues that are responsible for the formation of two disulfide linkages in GH, PRL, and SL. Immunoblot analysis revealed that rtSLP is secreted from the pituitary gland once it is synthesized. Unlike rtSL, rtSLP mRNA was readily detectable in pituitary glands of 2-yr fish (30–34 cm in length) but not in embryos or 1-yr fish (17–25 cm in length). Furthermore, the level of rtSLP mRNA in male fish is 2-fold higher than that in female fish. These results suggest that rtSLP might play a role in regulating the reproductive maturation in rainbow trout.
Stem cells, niches and cadherins: a view fromDrosophila
Authors: González-Reyes Acaimo
Published: February 2003

Stem cells are essential for the correct development and homeostasis of adult organisms, as well as having obvious potential therapeutic importance. Analysis of the biology of stem cells and their regulatory microenvironment in adult organs has, however, been hindered by the rarity of these cells in mature tissues and by the lack of positive markers for them. The ovary of the Drosophila melanogaster female is a stem cell niche in which such analyses can be performed. The stromal cells of the microenvironment act as a regulatory centre to control the proliferation and differentiation of the germline stem cells, using several signalling molecules, among them the protein DPP – a Drosophila homologue of the human bone morphogenetic proteins BMP2 and BMP4. Recent work shows that DE-cadherin-mediated adhesion is used for the initial recruitment and posterior anchoring of the germline-derived stem cells in their niche.
Megalin and the neurodevelopmental biology of sonic hedgehog and retinol
Authors: McCarthy Robert A. , Argraves W. Scott
Published: February 2003

Megalin is a receptor expressed by embryonic epithelia that mediates endocytosis of numerous ligands, including sonic hedgehog (Shh) and retinol,the precursor to retinoic acid (RA). The importance of Shh and RA signaling in neurodevelopment, combined with the fact that megalin-deficient mice show profound neurodevelopmental abnormalities, has raised questions as to the possible role of megalin in Shh and RA signaling. Several mechanisms could explain how megalin influences Shh and RA signaling in the context of neurodevelopment. These include the involvement of megalin in the transport of Shh and retinol within neuroepithelia, as well as direct signal transduction as a response to binding of Shh and retinol to megalin.
Alterations in the Arf6-regulated plasma membrane endosomal recycling pathway in cells overexpressing the tetraspan protein Gas3/PMP22
Authors: Chies Romina , Nobbio Lucilla , Edomi Paolo , Schenone Angelo , Schneider Claudio , Brancolini Claudio
Published: February 2003

Growth arrest specific 3 (Gas3)/peripheral myelin protein 22 (PMP22) is a component of the compact peripheral nerve myelin, and mutations affecting gas3/PMP22 gene are responsible for a group of peripheral neuropathies in humans. We have performed in vivo imaging in order to investigate in detail the phenotype induced by Gas3/PMP22 overexpression in cultured cells. Here we show that Gas3/PMP22 triggers the accumulation of vacuoles, before the induction of cell death or of changes in cell spreading. Overexpressed Gas3/PMP22 accumulates into two distinct types of intracellular membrane compartments. Gas3/PMP2 accumulates within late endosomes close to the juxtanuclear region, whereas in the proximity of the cell periphery, it induces the formation of actin/phosphatidylinositol (4,5)-bisphosphate(PIP2)-positive large vacuoles. Gas3/PMP22-induced vacuoles do not contain transferrin receptor, but instead they trap membrane proteins that normally traffic through the ADP-ribosylation factor 6 (Arf6) endosomal compartment. Arf6 and Arf6-Q67L co-localize with Gas3/PMP22 in these vacuoles,and the dominant negative mutant of Arf6, T27N, blocks the appearance of vacuoles in response to Gas3/PMP22, but not its accumulation in the late endosomes. Finally a point mutant of Gas3/PMP22 responsible for the Charcot-Marie-Tooth 1A disease is unable to trigger the accumulation of PIP2-positive vacuoles. Altogether these results suggest that increased Gas3/PMP22 levels can alter membrane traffic of the Arf6 plasma-membrane–endosomal recycling pathway and show that, similarly to other tetraspan proteins, Gas3/PMP22 can accumulate in the late endosomes.
Role of the Vtc proteins in V-ATPase stability and membrane trafficking
Authors: Müller Oliver , Neumann Heinz , Bayer Martin J. , Mayer Andreas
Published: February 2003

Vtc proteins have genetic and physical relations with the vacuolar H+-ATPase (V-ATPase), influence vacuolar H+ uptake and,like the V-ATPase V0 sectors, are important factors in vacuolar membrane fusion. Vacuoles from vtc1Δ and vtc4Δmutants had slightly reduced H+-uptake activity. These defects could be separated from Vtc function in vacuole fusion, demonstrating that Vtc proteins have a direct role in membrane fusion. We analyzed their involvement in other membrane trafficking steps and in VATPase dynamics. Deletion of VTC genes did not impede endocytic trafficking to the vacuole. However, ER to Golgi trafficking and further transport to the vacuole was delayed in Δvtc3 cells. In accordance with that,Δ vtc3 cells showed a reduced growth rate. Vtc mutations did not interfere with regulated assembly and disassembly of the V-ATPase, but they affected the number of peripheral V1 subunits associated with the vacuoles. Δvtc3 vacuoles carried significantly more V1 subunits, whereas Δvtc1, Δvtc2 andΔ vtc4 had significantly less. The proteolytic sensitivity of the V0 subunit Vph1p was different in Δvtc and wild-type cells in vivo, corroborating the physical interaction of Vtc proteins with the V-ATPase observed in vitro. We suggest that Vtc proteins affect the conformation of V0. They might thereby influence the stability of the VATPase holoenzyme and support the function of its V0 sector in vacuolar membrane fusion.
Retrograde flow rate is increased in growth cones from myosin IIB knockout mice
Authors: Brown Michael E. , Bridgman Paul C.
Published: February 2003

Growth cones of myosin-IIB-knockout mice have reduced outgrowth rates and traction force. There is a close relationship between traction force,retrograde flow and forward advance of growth cones. All three activities appear to be at least partially myosin dependent. Therefore, we have now tested for differences in retrograde flow rates between growth cones from myosin-IIB-knockout mice and their normal littermates. By placing nerve-growth-factor-coated silica beads on the surface of growth cones with laser tweezers, or by tracking GFP-myosin IIA spots, we found that the retrograde flow rate was increased more than two fold in the knockout growth cones compared with the wild type. These data suggest that both myosin IIA and IIB normally contribute to retrograde flow and the properties of the flow are strongly influenced by myosin IIB because of its location and abundance. However, in the absence of myosin IIB, myosin IIA takes over this function. The change in retrograde flow rate may reflect the difference in functional properties of these two myosins. Knockout growth cones also exhibited reduced stability of lamellipodia, possibly as a partial consequence of this increased retrograde flow rate. In addition, microtubules penetrated a shorter distance into filopodia, which suggests that the increase in flow rate may adversely affect the microtubule-dependent maturation of filopodia. Taken together these data support the idea that the forward advance of the growth cone is myosin II dependent and involves multiple myosin II isoforms.
Combination System of Fluorine-containing Phenoxy-imine Ti Complex and Chain Transfer Agent: A New Methodology for Multiple Production of Monodisperse Polymers
Authors: Mitani Makoto , Mohri Jun-ichi , Furuyama Rieko , Ishii Seiichi , Fujita Terunori
Published: February 2003

Multiple production of Zn-terminated polyethylene with narrow molecular weight distribution was attempted using the combination of fluorine-containing phenoxy-imine Ti complexes and Et2Zn. One of the two complexes promotes living polymerization in the presence of ethylene and Et2Zn and, moreover, undergoes chain transfer with Et2Zn in the absence of ethylene, resulting in a rare example of multiple production of nearly monodisperse Zn-terminated polyethylenes which are valuable intermediates for functional polymers and block copolymers.
Preparation of Hyperbranched Aromatic Poly(ethersulfone)s Possessing Sulfonic Acid Terminal Groups for Polymer Electrolyte
Authors: Takeuchi Masaki , Jikei Mitsutoshi , Kakimoto Masa-aki
Published: February 2003

Hyperbranched poly(ethersulfone) (HPES) having terminal sulfonic acid group was synthesized from 2,6-bis(p-sodium sulfophenoxy)benzonitrile. These films of polymer blend of HPES and polyvinyl alcohol (PVA) exhibited the proton transportation ability.
Effect of Addition of Pt, La2O3, Nd2O3 and ZrO2 to PdO/Al2O3 on Catalytic Combustion of Methane
Authors: Ozawa Yasushi , Tochihara Yoshihisa , Watanabe Ayako , Nagai Masatoshi , Omi Shinzo
Published: February 2003

The addition of Pt prolongs the lifetime of the PdO/Al2O3 catalyst during CH4 combustion at 1073 K. Furthermore, the addition of ZrO2, La2O3 and Nd2O3 greatly extends the lifetime of the Pt–PdO/Al2O3 catalyst. This is due not to the transformation of PdO to Pd–Pt but to the prevention of the growth of PdO particles during the reaction.
Ceric(IV) Ammonium Nitrate: A Novel Reagent for the Synthesis of Homoallyl Alcohols
Authors: Yadav J S , Reddy B V S , Krishna A D , Sadasiv K , Chary Ch Janardhana
Published: February 2003

A rapid and highly efficient method has been developed for the allylation of aldehydes with allyltributylstannane using a catalytic amount of ceric ammonium nitrate in acetonitrile under mild and neutral conditions to afford the corresponding homoallyl alcohols in excellent yields with high chemoselectivity. Allylation of ketones has also been achieved with tetraallytin under similar reaction conditions.
Studies on Synthesis of MMH and Rheological Properties of Concentrated MMH Dispersions
Authors: Guo Peizhi , Sun Dejun , Zhang Jie , Zhang Chunguang
Published: February 2003

A positively charged mixed metal hydroxides (MMH) was synthesized by a non-steady method. The chemical composition and morphology of the positive MMH nanoparticles were studied by ICP, Mastersizer and TEM. Preliminary rheological measurements indicated that when adding NaCl to exhaustively deionized MMH dispersion, the yield stress, G′ and G″ first decreased to a minimum, and then rose again.
Electropolymerization of Bithienyl-appended Cerium(III) Triple Decker Porphyrin Complex
Authors: Yamashita Kousei , Ikeda Masato , Takeuchi Masayuki , Shinkai Seiji
Published: February 2003

A cerium(III) triple decker porphyrin complex bearing bithienyl substituents (1) at the peripheral meso-positions was synthesized. Electrochemical polymerization of the triple decker porphyrin gave a unique polymer film on the ITO electrode surface without decomposition.
The refining influence of ethics committees on animal experimentation in Sweden
Authors: Hagelin Joakim , Hau Jann , Carlsson Hans-Erik
Published: February 2003

Mandatory scrutiny of projects by animal ethics committees was introduced in Sweden in 1979. The present study investigated the minutes of meetings held between 1989 and 2000 at which consideration of applications for experimental work in animals resulted in requests for modification ( n = 3607). 18.1% of the applications received were approved only after modifications. The majority of the changes requested may be classified as 'Refinement'. The most common requests were for improvement of project design, euthanasia method and housing and husbandry. There was a relative increase in modifications requested by the committees related to anaesthesia, choice of licensed supervisor and the need for licenses or informed consent from animal owners during the period investigated. There was a relative decrease in modifications related to euthanasia, housing and husbandry, and general endpoint assertions.

The results suggest that the work of the committees may be perceived as an ongoing process, since several of the applications for which modification was requested were projects that had been approved on a previous occasion but were now up for renewal. In order to have maximal influence on the refinement of scientific protocols it is important that the scientists in the committees are continuously updated on developments in laboratory animal science.
Lymph node topography of the head and neck in New Zealand White rabbits
Authors: Dunne Anja-A. , Plehn S. , Schulz S. , Levermann A. , Ramaswamy A. , Lippert B. M. , Werner J. A.
Published: February 2003

Investigations of the lymphogenic metastatic spread of VX2 carcinomas in New Zealand White rabbits require an exact knowledge of the topography of cervical and facial lymph nodes. The topography of neck lymph nodes was evaluated from 16 rabbits macroscopically, histologically and by lymphographic investigations, and the possibility of their surgical removal (neck dissection) was examined. The upper aerodigestive tract and the ear of New Zealand White rabbits drain via four consistent groups of 12–18 lymph nodes. Except for the paratracheal lymph node, they are all easily accessible to surgery. The data presented in this study encourage the use of induced VX2 carcinomas in New Zealand White rabbits as an animal model to study the lymphogenic metastatic spread of squamous cell carcinomas of the head and neck. Such investigations could lead to an improvement of surgical and pharmaceutical treatment of this tumour entity.
The effect of body weight on the degree of blood velocity profile skewness in the aortic annulus in domestic pigs
Authors: Matre K. , Kvitting P. , Zhou Y. Q. , Færestrand S.
Published: February 2003

We investigated the blood velocity profile in the aortic annulus (AA) in two groups of domestic pigs using epicardial Doppler echocardiography. The velocity profile skewness in terms of max/mean TVI (the ratio of maximal to cross-sectional mean time-velocity integral along the diameter) was 1.107 ± 0.01 in the small pigs ( n = 10; body weight: 24.6 ± 0.8 kg) and 1.216 ± 0.026 in the large pigs ( n = 8; body weight: 50.6 ± 2.5 kg) ( P = 0.002). The velocity profile in the AA is more skewed in large animals than in small animals and the skewness in the larger animals is similar to that in normal adult humans. This study shows the importance of choosing animals of sufficient size if flow method investigations are to be performed. This is particularly important for ultrasound Doppler investigations based on a limited sample of velocities across the flow channel.
Plant actin filament and microtubule interactions during anaphase‐telophase transition: effects of antagonist drugs
Authors: SCHMIT Anne‐Catherine , LAMBERT Anne‐Marie
Published: February 2003

F‐actin and microtubule co‐distribution and interaction were studied during anaphase‐telophase. Rapid and drastic changes in the cytoskeleton during these particular stages were studied in isolated plant endosperm cells of the blood lily. These wall‐free cells can be considered as natural dividing protoplasts. As identified previously, an F‐actin cytoskeletal network characterized the plant cortex and formed an elastic cage around the spindle, remaining throughout interphase, mitosis and cytokinesis. Actin was specifically labeled by fluorescent phalloidin and/or monoclonal antibodies. Gold‐labeled secondary antibodies were used for ultrastructural observations and silver‐enhancement was applied for video‐enhanced microscopy. Microtubule and microfilament dynamics and interaction were studied using drug antagonists to actin (cytochalasins B, D) and to tubulin (colchicine). This permitted precise correlations to be made between chromosome movement inhibition and alteration in the actin/tubulin cytoskeleton. During anaphase chromosome migration, the cortical actin network was stretched along the microtubular spindle, while it remained homogeneous when anaphase was inhibited by colchicine. Cytochalasins did not inhibit chromosome movement but altered actin distribution. A new population of actin filaments appeared at the equator in late anaphase before the microtubular phragmoplast was formed and contributed to cell plate formation. Our conclusion is that F‐actin‐microtubule interaction may contribute to the regulatory mechanism of plant cytokinesis.
Flagellar wave reversal in the kinetoplastid flagellate Crithidia oncopelti
Authors: Surgue Phillip , Hirons Michael R. , Adam Juliet U. , Holwill Michael E.J.
Published: February 2003

Summary— Living Crithidia oncopelti cells swim through their environment by means of tip‐to‐base waves on their single flagellum. The cells are able to re‐orient themselves by using a short burst of asymmetrical base‐to‐tip waves. All points on a flagellum are capable of initiating waves. Placing a population of cells in a medium of high viscosity initially produces a large number of organisms beating in the reverse mode. An individual cell has a random “switching” behaviour. Viscosity affects the frequency of forward and reverse waves in different ways. The concentration of free Ca++ ions determines the direction of wave propagation in reactivated axonemes. Calmodulin may play a role in mediating the Ca++ dependence of wave direction.
Biology of African trypanosomes in the tsetse fly
Authors: Vickerman Keith , Tetley Laurence , Hendry Kay A.K. , Turner C. Michael R.
Published: February 2003

African trypanosomes present several features of interest to cell biologists. These include: a repressible single mitochondrion with a large mass of mitochondrial DNA, the kinetoplast; a special organelle, the glycosome, which houses the enzymes of the glycolytic chain; a surface coat of variable glycoprotein which enables the parasite to evade the mammalian host's immune response; and a unique flagellum‐to‐host attachment mechanism associated with novel cytoskeletal elements. Trypanosome development during the life cycle involves cyclical activation and repression of genes controlling these activities. Understanding the complexity of parasite development in the tsetse fly vector is especially challenging but may help to suggest new methods for the control of trypanosomiasis.
Expression and function of erythrocyte‐associated surface antigens in malaria
Authors: Hommel Marcel , Semoff Sam
Published: February 2003

As the malaria parasite develops within the erythrocyte, a series of molecules are produced, which find their way first accross the parasitophorous vacuole membrane, then through the system of membranous clefts in the cytoplasm of the infected cell, to end up associated with the erythrocyte membrane. The domains of the erythrocyte‐associated malaria antigens which are exposed at the cell surface are readily recognised by the host's immune system and represent important targets in the early stages of acquired immunity to malaria. The malaria parasite, in turn, appears to have developed some very effective mechanisms of escaping this immune response, including sequestration and antigenic variation. This paper reviews recent findings in the field of erythrocyte‐associated malarial antigens and discusses these findings in the context of disease severity and malaria immunity.
Plasmodium vivax malaria: parasite biology defines potential targets for vaccine development
Authors: David Peter H. , del Portillo Hernando A. , Mendis Kamini N.
Published: February 2003

Although progress in the development of an antimalarial vaccine has been mostly obtained through the study of P. falciparum, significant advances have recently been made in the study of P. vivax, the other major human malarial parasite. Antigens which have been shown to be important in P. falciparum have been characterized and in some cases cloned in P. vivax. Other studies have examined some of the more specific biological characteristics of P. vivax. Among these are studies on components present in caveolae‐vesicle complexes of the infected erythrocyte, on the occurrence of delayed hepatic development leading to relapse, or on the Duffy erythrocyte antigen as a key receptor for parasite invasion. Although progress has been made in the short‐term in vitro maintenance of P. vivax, the inability to maintain the parasite in continuous culture led to the investigation of wild parasite populations in patients; occurrence of extensive antigenic and karyotype polymorphism was detected in this way, as was a double‐blocking and enhancing activity of human antibodies on parasite development in the vector. The association of monoclonal antibodies with DNA recombinant technology allowed the characterization of a number of P. vivax antigens to be made. Among these, an antigen shared between sexual and asexual stages was shown to constitute a target for transmission‐blocking immunity. The cloning of an antigen involved in transmission‐blocking immunity, along with that of the surface antigen of the sporozite (CSP) and of a major surface antigen of the invasive merozoite (PV200) constitutes a significant step towards the development of a multivalent recombinant vaccine against P. vivax.
Cell cycle‐related proteins: a flow cytofluorometric study in human tumors
Authors: DANOVA Marco , RICCARDI Alberto , GIORDANO Monica , GIRINO Margherita , MAZZINI Giuliano , DEZZA Laura , ASCARI Edoardo
Published: February 2003

We used 2‐parameter flow cytometry (FCM) to investigate the relationship between the cell cycle phases and 3 proteins whose expression is known to increase in proliferating cells: the surface antigen transferrin receptor Trf‐r), the “cyclin” (a proliferating cell nuclear antigen, PCNA), and the nuclear antigen recognized by the monoclonal antibody (MoAb) Ki‐67. FITC‐labeled antibodies against Trf‐r, PCNA, and the Ki‐67‐reactive antigen, as well as propidium iodide‐DNA distribution, were simultaneously measured on human leukemia HL‐60 and K562, and breast carcinoma MCF‐7 cell lines and on fresh human leukemic and glioblastoma cells. The 70% ethanol fixation for Trf‐r and PCNA and the 95% acetone fixation for Ki‐67 plus permeabilization (with 0.1% and 1% Triton X100, respectively, for the surface and the nuclear antigens) produced cell suspensions with negligible cell clumping, high‐quality DNA profiles, and bright specific immunofluorescent staining. The investigated proteins have different relationships with the proliferative state of the cell. Trf‐r is expressed mainly at the transition from G0/G1 to S‐phase. PCNA expression is prominent in late G1 and through S‐phase and decreases in G2‐M. The Ki‐67‐reactive antigen is widely distributed in G1, S, and G2‐M phases. Knowledge regarding the relationships between proliferation‐associated antigens and cell cycle phase in normal and neoplastic cells could improve our understanding of the mechanisms underlying growth regulation and neoplastic transformation. Bivariate FCM is an easy method for obtaining these data from large numbers of cells.
Image processing of electron micrographs of human alpha2‐macroglobulin half‐molecules induced by Cd2+
Authors: THOMAS Daniel , FLIFLA Mohamed J. , ESCOFFIER Brigitte , BARRAY Martine , DELAIN Etienne
Published: February 2003

Human alpha2‐macroglobulin is a tetrameric plasma inhibitor of proteinases. Its dissociation by Cd2+ gives functional dimers. Electron microscopy of negatively stained dimers shows their round‐ended cylindrical shape with furrows delimiting 3 main stain‐excluding domains. Image processing of electron micrographs shows the existence of 2 main orientations of the dimers on the carbon support film. The dimer is composed of 2 curved monomers linked in a central domain, and related by a 90° rotation. Taking into account the known primary structure of alpha2‐macroglobulin and the linkage of the 2 constitutive monomers by 2 disulfide bonds, the molecular organization of the dimer is discussed, extended to the tetrameric molecule and compared to the published models of human alpha2‐macroglobulin.
Assembly of a protein sharing epitopes with sperm dynein heavy chains into meiotic spindle in the prometaphase starfish oocyte
Authors: OGAWA Kazuo , YOKOTA Etsuo , SHIRAI Hiroko
Published: February 2003

Starfish oocyte meiosis provides a good system for studying the mechanism for prometaphase chromosome movement. Since a protein sharing epitopes with sperm dynein might be a force generator for mitosis, the contribution of such a protein was assessed in this movement. Specific antibodies to heavy chains (HCs) and intermediate chains (ICs) of dynein subunits were affinity‐purified from whole antidynein serum. We confirmed that the oocytes contain several polypeptides identical to sperm dynein subunits. The anti‐HCs binding to in situ antigen was examined in the oocytes permeabilized with detergent at appropriate stages of maturation with special reference to tubulin and chromosomes, and the meiotic apparatus‐establishing process was described in terms of a force generator (oocyte dynein). Before resumption of maturation, dynein HCs were particularly associated with prophase chromosomes within the germinal vesicle (GV). After GV breakdown, there was a striking local accumulation of dynein HCs in the “fading GV” (nuclear matrix). When chromosomes were pulled toward the central area between 2 asters, dynein was accumulated at first at the presumptive equator and then moved to the poles, showing uneven localization on the meuotic spindle.
Cilia‐lacking respiratory cells in ciliary aplasia
Authors: de SANTI Maria Margherita , GARDI Concetta , BARLOCCO Gabriele , CANCIANI Mario , MASTELLA Gianni , LUNGARELLA Giuseppe
Published: February 2003

This report describes the ultrastructural alterations observed in the nasal and bronchial mucosa of an 11‐yr‐old male suffering from immotile cilia syndrome (ICS).

The morphological features observed in this patient are consistent with a ciliary aplasia. In fact, ciliated cells appeared to be replaced by columnar cells lacking cilia and basal bodies, and bearing on their surface cilium‐like projections without any internal axonemal structure.

In spite of the absence of basal bodies, centrioles, and kinocilia, these cells unexpectedly showed mature striated roots and centriolar precursor material scattered throughout the apical cytoplasm.

These data suggest that control over basal body assembly is distinct from control over striated root formation. The presence of the above‐reported structures in cells otherwise presenting many morphological features of normal ciliated cells is discussed on the basis of current knowledge of respiratory cilia biogenesis.
Purification and biological properties of vasculotropin, a new angiogenic cytokine
Authors: Favard Catherine , Moukadiri Hafida , Dorey Christiane , Praloran Vincent , Plouët Jean
Published: February 2003

Summary— Anglogenesis is a key step in organ development and remodeling during embryogenesis or tissue regeneration. Some pathological events such as tumor growth or diabetic retinopathy also lead to angiogenesis formation. Several molecules have already been identified as promoting anglogenesis in vivo. Whether their bioactivity is mediated by other angiogenic growth factors or not is still unclear. We identified and purified recently a new anglogenic growth factor. Its unique specificity for vascular endothelial cells led us to provisionally name it vasculotropin (VAS). We describe the biochemical properties of VAS and its biological functions. Structural data showed that VAS is related to the SIS family. In vivo VAS was recognized as an inducer of angiogenesis and vascular permeability. In vitro, despite a moderate action on proliferation, VAS strongly stimulates the cell migration. The screening of the presence of cellular receptors and VAS production showed that the cells which bind VAS do not synthesize it, whereas the cells which synthesize VAS do not bind it. Thus, VAS seems to act through a paracrine pathway. We also present data suggesting that VAS has a lymphokine activity.
Mammalian cell lines can be efficiently established in vitro upon expression of the SV40 large T antigen driven by a promoter sequence derived from the human vimentin gene
Authors: Schwartz Bertrand , Vicart Patrick , Delouis Claude , Paulin Denise
Published: February 2003

Summary— The aim of this study was to investigate a new method to enhance the efficiency to create mammalian cell lines. Cell immortalization was achieved by intranuclear microinjection of a recombinant DNA construct composed of a constitutive promoter controlling the genes encoding immortalizing proteins; the sequences coding for the large T and small t antigens were fused downstream of regulatory elements from the vimentin gene, the activation of which characterizes the vast majority of cells growing in vitro. Data show that the efficiency of the immortalizing procedures using the SV40 early genes could be enhanced by the control elements derived from the human vimentin (HuVim) 5′ sequences that contained nucleotides −878 to +93 from the CAP site. This HuVim 830−T/t recombinant was used to create cell lines from numerous primary cultures of different origins: rabbit, porcine and human endothelial cells, rabbit and bovine epithelial cells. A set of large T‐expressing cells was derived, and these cells retained characteristics of differentiated cells: binding of Ulex europaeus lectin and synthesis of Factor VIII for human endothelial cells; network of cytokeratin for bovine oviductal cells and rabbit mammary cells.
Co‐expression of the proto‐oncogene FOS (c‐fos) and an embryonic interferon (ovine trophoblastin) by sheep conceptuses during implantation
Authors: Xavier Françoise , Guillomot Michel , Charlier Madia , Martal Jacques , Gaye Pierre
Published: February 2003

Summary— Expression of the c‐fos proto‐oncogene by ovine conceptuses was analyzed by Northern and slot blots and indirect immunohistofluorescence in relation to the expression of the embryonic interferon‐α (oTP) during implantation. c‐fos was expressed initially in the trophoblast, and then in the allantois, when this tissue began to develop (day 17). In the embryonic tissues, the c‐fos proto‐oncogene was weakly expressed up to day 22 and increased thereafter. In the trophoblast, the expression of c‐fos proto‐oncogene was transient, occurring when the oTP gene was transcribed at a maximal level at the beginning of implantation (days 14–15), and decreased thereafter, following the pattern of oTP gene expression. This decline is due essentially to the arrest of c‐fos and oTP gene expression by the trophoblastic cells which established cellular contacts with the uterine epithelium during the implantation process.
Ultrastructural features of soybean somatic cells at the beginning of an organogenic process: toward a new concept*
Authors: Fournier Dominique , Bonnelle Christine , Tourte Yves
Published: February 2003

Summary— Histological and ultrastructural observations of soybean protoplast‐derived calli allowed establishment of the very early cellular reorganization that initiates a proliferating process at the beginning of regeneration. These cytological events appeared more clearly in an inductive medium containing 2.4 D. The cells that participate in the somatic regeneration are characterized by special features that chiefly affect the nucleus and mitochondria: an increase of nucleo‐cytoplasmic exchange by means of deep invaginations of the nuclear envelope, the occurrence of specific attachments of chromatin dense masses at the bottom of these invaginations and the disappearance of some mitochondria while a great number of them proliferates around the nucleus. All these events are comparable to the cytological reorganization affecting the female gametic lineage that generally occurs during the female gametophytic phase in sexual plant reproduction.
The Balbiani ring 6 induction in Chironomus
Authors: Botella Luisa M , Edström Jan‐Erik
Published: February 2003

Summary— Balbiani ring (BR) genes in polytene chromosomes of Chironomus salivary glands code for secretory proteins of the sp‐I family, 106D. They are used by the aquatic larva to spin a housing and feeding tube. The expression of the BR gene family undergoes correlated changes depending on the environment. In the presence of certain sugars, ethanol or glycerol the normally most active BR2 regresses and its products disappear. There is a parallel induction of a new BR, BR6, and a new sp‐I protein. The change seems to represent an adaptive response to phosphate depletion in the larval haemolymph produced by the inducing agents. The BR2 (and BR1) products are heavily phosphorylated and the BR6 product non‐phosphorylated. One of the BR2‐coded sp‐I proteins is cleaved off close to the C‐terminus and material with properties expected of the resulting polypeptide can be recovered in the nuclei, accumulating in the BR. This might represent a feed‐back signal from translation to transcription.
Protein folding in vitro and in the cellular environment
Authors: Yon Jeannine M , Betton Jean‐Michel
Published: February 2003

Summary— The main concepts concerning protein folding have been developed from in vitro refolding studies. They state that the folding of a polypeptide chain is a spontaneous process depending only on the amino‐acid sequence in a given environment. It is thermodynamically controlled and driven by the hydrophobic effect. Consequently, it has been accepted that the in vitro refolding process is a valuable model to understand the mechanisms involved during the folding of a nascent polypeptide chain in the cell. Although it does not invalidate the main rules deduced from the in vitro studies, the discovery of molecular chaperones has led to a re‐evaluation of this last point. Indeed, in cells molecular chaperones are able to mediate the folding of polypeptide chains and the assembly of subunits in oligomeric proteins. The possible mechanisms by which these folding helpers act are discussed in the light of the data available in the literature. The folding process is assissted in the cell in different ways, preventing premature folding of the polypeptide chain and suppressing the incorrectly folded species and aggregates. Molecular chaperones bind to incompletely folded proteins in a conformation which suggests that the latter are in the “molten globule” state. However, very little is known about the recognition process.
Mitochondrial biogenesis in rabbit articular chondrocytes transferred to culture
Authors: Mignotte Françoise , Champagne Anne‐Marie , Froger‐Gaillard Béatrice , Benel Laurent , Gueride Monique , Adolphe Monique , Mounolou Jean‐Claude
Published: February 2003

Summary— The effect of the switch to aerobic growth conditions was examined in rabbit articular chondrocytes transferred to culture. Spectroscopic analysis of the cytochromes of the respiratory chain shows that only cytochrome b is present in chondrocytes from cartilage, cytochromes c, c1 and a·a3 being undetectable as compared with the typical spectrum found in a primary cell culture on day 4. Steady state levels of RNA transcripts of nuclear (cytochrome c) and mitochondrial genes (cytochrome b and cytochrome oxidase subunits II and III) involved in the oxidative metabolism were determined relative to the RNA transcripts of the nuclear gene for glyceraldehyde phosphate dehydrogenase involved in the glycolytic pathway and to mitochondrial ribosomal RNAs. Chondrocytes transferred to culture showed a general increase in the levels of all transcripts, but the effect on mitochondrial transcripts was much greater (× 20) than the effect on nuclear transcripts (× 3–4). These results show the absence of a coordinate regulation of the expression of mitochondrial and nuclear genes coding for components of the respiratory chain. The increase in mitochondrial DNA triggered by culture conditions does not appear to be sufficient to account for the enhanced transcription. Concomitant with these mitochondrial changes, the level of transcripts for the collagen II gene involved in the differentiation function decreases dramatically (3% of the control on day 3).
Substance P and calbindin D‐28k‐immunoreactivity in primary sensory neurons of chick embryos: differential neuronal birthdates and transient co‐localization
Authors: Duc Christophe , Barakat‐Walter Ibtissam , Droz Bernard
Published: February 2003

Summary— During the ontogenesis of dorsal root ganglia (DRG), the immunoreactivity to substance P (SP) and calbindin D‐28k (CaBP) appears in chickens at embryonic day 5 (E5) and E10 respectively. To establish the birthdates of primary sensory neurons expressing SP or CaBP, chick embryos were given repetitive intra‐amniotic injections of [HH]‐thymidine. The neuroblasts giving rise to SP‐expressing neurons were labeled up to E6 while those generating CaBP‐immunoreactive neurons stopped to incorporate [3H]‐thymidine before E5.5. This finding indicates that neurons exhibiting distinct phenotypes may originate from neuroblasts which arrest to proliferate at close but distinct stages of development. To determine whether SP and CaBP are co‐expressed or not in DRG neurons, chick embryos at E12, E18, and chickens two weeks after hatching were perfused and fixed to detect simultaneously SP‐ and CaBP‐immunoreactivity in DRG sections. The results showed that SP and CaBP were transiently co‐expressed by a subset of neurons at E12. Later, however, the SP‐immunoreactivity was gradually lost by these ganglion cells, so that the SP‐ and CaBP‐immunoreaction defined two distinct neuronal subpopulations after hatching. In conclusion, most CaBP‐immunoreactive DRG cells derive from a subset of neurons in which SP and CaBP are transiently co‐localized.
Immunocytochemical localization of the DNA‐binding protein HCc during the cell cycle of the histone‐less dinoflagellate protoctista Crypthecodinium cohnii B
Authors: Géraud Marie‐Line , Sala‐Rovira Montserrat , Herzog Michel , Soyer‐Gobillard Marie‐Odile
Published: February 2003

Summary— The major basic nuclear protein HCc (previously named Histone‐like) of the dinoflagellate Crypthecodinium cohnii B was immunolocalized in light and electron microscopy using an affinity‐purified polyclonal antibody. Complementary conventional and cryo‐techniques were used to study the distribution of the DNA‐binding protein in interphase cells and to follow its behaviour throughout the mitotic cycle. In non‐dividing cells, the HCc protein was found to be located on extra‐chromosomal loops and chromosomal nucleofilaments dispersed in the nucleoplasm. In mitotic cells, from prophase to early telophase, it was homogeneously distributed in the (whole) dividing chromosomes. HCc protein was also detected in two compartments of all the permanently observable nucleoli: the nucleolar organizing region and the fibrillo‐granular region. In this paper we discuss the hypothetical roles, structural and/or functional, of this DNA‐binding protein, which is specific to dinoflagellates, the only eukaryotes whose chromatin is devoid of histones and nucleosomes.
Cellular interactions and tubulin detyrosination in fibroblastic and epithelial cells
Authors: Bré Marie‐Hélèn , Pepperkok Rainer , Kreis Thomas E. , Karsenti Eric
Published: February 2003

Summary— In mammalian cells most microtubules are enriched in tyrosinated α‐tubulin (tyr‐tubulin). Other subclasses of microtubules are present in variable amounts and some are enriched in detyrosinated α‐tubulin (glu‐tubulin). We examined the effect of cell‐cell interactions on the level of glu‐tubulin in microtubules. This was studied by quantitative immunofluorescence using antibodies against tyr‐ and glu‐tubulin. We found that in cells which have established cell‐cell contacts, the ratio of glu‐/tyr‐tubulin is higher than in isolated cells. We also examined the effect of cell‐cell interactions on the glu‐/tyr‐tubulin ratio by using the antibody blocking method of Schulze and Kirschner [42]. Microtubules containing mainly tyr‐tubulin had been blocked first by a polyclonal antibody against tyr‐tubulin and several layers of secondary antibodies. The unblocked microtubules were then labeled by a monoclonal antibody against α‐tubulin. Since the coating efficiency of microtubules by the anti‐tyr tubulin depends on the amount of tyr‐tubulin in each microtubule, this procedure allows the visualization of microtubules enriched or depleted in tyr‐tubulin in specific domains of each cell. Microtubules were more extensively blocked in subconfluent than in confluent cells and preferentially at the periphery of the cytoplasm. In cells present at the margin of an artificial wound produced in a confluent monolayer, the amount of blocked microtubules increased slowly with time (between 2 and 4 h). These results are consistent with the hypothesis that cell‐cell contacts lead to increased tubulin detyrosination both in fibroblastic and epithelial cells.
Dynamic image analysis applied to the study of ciliary beat on cultured ciliated epithelial cells from rabbit trachea
Authors: Romet Sylvie , Schoevaert Damien , Marano Francelyne
Published: February 2003

Summary— We have developed an automated image analysis method to study the ciliary beat frequency of ciliated cells of the primary culture from rabbit trachea. The ciliated outgrowth image is digitized and the variation in optical density is automatically calculated for each selected area of interest. 32 measurements of ciliary beat frequency are, in this way, calculated simultaneously in 6 min. With this reliable device, some studies on baseline frequency of control culture have been carried out. There was no variation in the mean frequencies of ciliated cells of the primary culture of different tracheas in our culture conditions. Moreover, the values of ciliary beat frequency at the starting point of the outgrowth were similar to those at the periphery of the outgrowth. There is nevertheless a slow decrease in frequencies versus the duration of culture. We have also established that the frequency of ciliary beat of some cells fluctuates in a periodic pattern whereas the majority of the ciliated population beat in a stable way. The image analysis process allows us to perform a cartography of frequencies on the video display. It also allows us to have access to the frequency of one cilium. Our method therefore seems to be reliable and furthermore simple in the evaluation of the potential effect of inhaled toxic compounds on ciliated cells of mammalian respiratory tract.