bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationships between storage protein composition, protein content, growing season and flour quality of bread wheat
Authors: Færgestad Ellen Mosleth , Solheim Flæte Nina E , Magnus Ellen Merethe , Hollung Kristin , Martens Harald , Uhlen Anne‐Kjersti
Published: May 2004

The storage protein composition from the Glu‐1, Glu‐3 and Gli‐1 loci encoding high and low molecular weight glutenin subunits (HMW‐GS and LMW‐GS) and gliadins, respectively, was determined on 30 wheat (T aestivum L) genotypes from three growing seasons. The gliadins and the LMW‐GS were identified as gliadin/LMW‐GS pairs. All samples were analysed by two one‐dimensional electrophoretic techniques, and selected samples were also subjected to two‐dimensional electrophoretic separation. Different statistical/data‐analytical techniques were evaluated in the study of how the presence or absence of the protein alleles, the protein content and the growing seasons are related to flour quality. The year of growth had a large impact on mixograph peak time. When predicting mixograph peak time from the presence or absence of significant proteins and the year of growth, 70% of the variability in mixograph peak time could be explained, whereas only 49% of the variability could be explained when the year of growth was deleted from the model. Protein had no effect on mixograph peak time as expected, and the well‐known positive effect of HMW‐GS 5 + 10, and the negative effects of 2 + 12 and 6 + 8 was observed. Furthermore, some of the gliadin/LMW‐GS combinations influenced mixograph peak time significantly. The gliadin/LMW‐GS at the combined Gli‐A1, Glu‐A3 loci b; 𝒻 was positively related to mixograph peak time, whereas 𝒻; 𝒻 and a;a was negatively related. Although the LMW‐GS component 𝒻 of the alleles b; 𝒻 and 𝒻; 𝒻 alleles appear similar on one‐dimensional gels, two‐dimensional separation of selected samples may suggest that the 𝒻 components in these alleles are different proteins. Cross‐validated partial least squares regression combined with empirical uncertainty estimates (jack‐knifing) of the parameters estimated in the model, gave similar results to ANOVA in identifying quality related protein alleles. The applicability of the multivariate approach in proteomics is, however, much wider. Copyright © 2004 Society of Chemical Industry
Influence of genotype and protein content on the baking quality of hearth bread
Authors: Uhlen Anne‐Kjersti , Sahlstrøm Stefan , Magnus Ellen M , Færgestad Ellen M , Dieseth Jon A , Ringlund Kåre
Published: May 2004

The relationships between protein content and quality and the loaf characteristics of hearth breads made from the respective flours were investigated for 20 wheat genotypes, comprising both commercially grown cultivars and advanced breeding lines, grown in 1997 and 1998. In both years the average protein content was increased by increasing the level of nitrogenous fertiliser application, this effect being more pronounced in 1997 than in 1998. Grain samples were milled and evaluated for endosperm hardness, protein content, SDS sedimentation volume, mixing properties and baking quality determined by a hearth bread baking test using optimal mixing and fixed proving time. When producing hearth bread, both the form (height/width) ratio and loaf volume are critical external characteristics. Genotype differences, mainly due to differences in protein quality, were more important for both form ratio and loaf volume than differences in protein content. The dough development time of the Farinograph, operated at high speed (126 rev min−1), was the best indicator of heart bread baking quality, giving high positive correlations to both the volume (r = 0.89) and form ratio of the loaf (r = 0.90). Climatic differences between the two years, in particular temperature during ripening, also affected the quality parameters studied. Copyright © 2004 Society of Chemical Industry
Concurrent cystic mediastinal lymphangioma and paratesticular rhabdomyosarcoma
Authors: Emir Suna , Uǧur Handan , Koloǧlu Meltem , Erekul Selim , Ünal Emel , Taçyildiz Nurdan , Yavuz Gülsan
Published: May 2004

The current report describes a 23‐month‐old boy with paratesticular rhabdomyosarcoma who was incidentally diagnosed as also having an isolated mediastinal cystic lymphangioma. The association of childhood rhabdomyosarcoma with various congenital anomalies and genetic alterations such as p53 mutations have been well known. However, mediastinal cystic lymphangioma has not been reported among the congenital anomalies diagnosed in rhabdomyosarcoma. Both rhabdomyosarcoma and lymphangioma originate from mesenchymal cells. This association may be coincidental or may point to a common genetic and/or developmental disorder of the mesencymal tissue. © 2004 Wiley‐Liss, Inc.
Trypanocidal activity of chemical constituents from Lychnophora salicifolia Mart
Authors: Jordão Christiane Oliveira , Vichnewski Walter , de Souza Glória Emília Petto , Albuquerque Sérgio , Lopes João Luis Callegari
Published: May 2004

The ethyl acetate extract from leaves plus inﬂorescences of L. salicifolia showed signiﬁcant trypanocidal activity against trypomastigote forms of T. cruzi, which was due to the ﬂavonoid quercetin‐7,3′,4′‐trimethyl ether and the sesquiterpenoid lychnopholic acid. Despite the use of some species of Lychnophora in folk medicine for pain relief, crude extracts from L. salicifolia Mart. showed no antinociceptive activity in the mouse writhing test. Copyright © 2004 John Wiley & Sons, Ltd.
Effects of betaine, coumarin and flavonoids on mucin release from cultured hamster tracheal surface epithelial cells
Authors: Lee Choong Jae , Lee Jae Heun , Seok Jeong Ho , Hur Gang Min , Park Ji sun , Bae Sohyun , Lim Jong Ho , Park Yang Chun
Published: May 2004

Betaine, coumarin, hesperidin and kaempferol are the components derived from Lycium chinense, Angelicae decursiva, Poncirus trifoliata and Polygonatum odoratum, respectively. These plants have been used for the treatment of respiratory diseases in oriental medicine and their respective components were reported to have various biological effects. In this study, we investigated whether these natural products affect mucin release from cultured hamster tracheal surface epithelial cells and compared the possible activities of these agents with the inhibitory action on mucin release by poly‐L‐lysine and the stimulatory action by adenosine triphosphate. Conﬂuent primary hamster tracheal surface epithelial cells were metabolically radiolabeled using 3H‐glucosamine for 24 h and treated for 30 min in the presence of varying concentrations of each agent to assess the effects on 3H‐mucin release. The results were as follows: (i) Coumarin and kaempferol did not affect mucin release signiﬁcantly; (ii) Betaine and hesperidin increased mucin release at the highest concentration; (iii) Poly‐L‐lysine inhibited and adenosine triphosphate increased mucin release. We conclude that betaine and hesperidin can increase mucin release by direct acting on airway mucin‐secreting cells and suggest these agents be further studied for the possible use as mild expectorants during the treatment of chronic airway diseases. Copyright © 2004 John Wiley & Sons, Ltd.
Wealth versus political power: the Russian case
Authors: Shlapentokh Vladimir
Published: May 2004

The interplay between the state and the super rich has been a subject of intense debate since the time of Aristotle, who introduced the notion of oligarchs and the oligarchic regime as the ruling of a few rich people. The ideologically loaded debates about the role of wealthy people in society can be found in each country in the contemporary world. In recent times, the fact that Silvio Berlusconi is both Italy’s prime minister and the richest person in the country (who has almost complete control over Italian TV) has aroused intense debates about the impact of big business on politics (Stille, 2003). It is not surprising that the case of the Italian prime minister draws attention in Russia, where he is often compared to both the Russian president as well as to the oligarchs (Remnik, 2003). A dramatic struggle between Russian President Vladimir Putin, and the oligarch Mikhail Khodorkovsky, whose wealth was estimated before his arrest at eight-billion dollars,1 unfolded in the summer and fall of 2003. This conflict will likely be recounted in any future textbook that discusses the relationship between political power and big money.
East-Central Europe’s new security concern: foreign land ownership
Authors: Tesser Lynn M.
Published: May 2004

While foreign land ownership has become a post-Cold War security concern for East-Central Europe, it has been neglected in security studies focused on more traditional topics. This article offers a comparative analysis of: (1) the post-1989 development of policy towards land sales to non-citizens, (2) why foreign land ownership has been the most controversial in Hungary and especially Poland, and (3) why foreign ownership can be a useful tool for nationalists. Mining the land issue ultimately strikes a deeper tension between foreign-driven pressures to liberalize land markets within a pan-European free market and the still-strong belief that states should control land sales for the good of the nation.
Postscript to the Special Issue on Gossip
Authors: Bloom Paul
Published: May 2004

It is tempting to ask about the origins and functions of gossip, but this temptation should be resisted. From a psychological perspective, gossip is likely to be an arbitrary and unnatural category. The articles in this issue demonstrate how scientifically rich the study of gossip is, but this is not because gossip is a natural kind; it is instead because it is a domain where the most interesting aspects of mental life are laid bare.
Lessons from border cell migration in the
Drosophila
ovary: A role for myosin VI in dissemination of human ovarian cancer
Authors: Yoshida Hiroyuki , Cheng Wenjun , Hung Jamie , Montell Denise , Geisbrecht Erika , Rosen Daniel , Liu Jinsong , Naora Honami
Published: May 2004

Dissemination of ovarian cancer is a major clinical challenge and is poorly understood at the molecular level due to a lack of suitable experimental models. During normal development of the
Drosophila
ovary, a dynamic process called border cell migration occurs that resembles the migratory behavior of human ovarian cancer cells. In this study, we found that myosin VI, a motor protein that regulates border cell migration, is abundantly expressed in high-grade ovarian carcinomas but not in normal ovary and ovarian cancers that behave indolently. Inhibiting myosin VI expression in high-grade ovarian carcinoma cells impeded cell spreading and migration
in vitro
. Optical imaging and histopathologic studies revealed that inhibiting myosin VI expression reduces tumor dissemination in nude mice. Therefore, using genetic analysis of border cell migration in
Drosophila
is a powerful approach to identify novel molecules that promote ovarian cancer dissemination and represent potential therapeutic targets.
Auxin regulation of cytokinin biosynthesis in
Arabidopsis thaliana
: A factor of potential importance for auxin–cytokinin-regulated development
Authors: Nordström Anders , Tarkowski Petr , Tarkowska Danuse , Norbaek Rikke , Åstot Crister , Dolezal Karel , Sandberg Göran
Published: May 2004

One of the most long-lived models in plant science is the belief that the long-distance transport and ratio of two plant hormones, auxin and cytokinin, at the site of action control major developmental events such as apical dominance. We have used
in vivo
deuterium labeling and mass spectrometry to investigate the dynamics of homeostatic cross talk between the two plant hormones. Interestingly, auxin mediates a very rapid negative control of the cytokinin pool by mainly suppressing the biosynthesis via the isopentenyladenosine-5′-monophosphate-independent pathway. In contrast, the effect of cytokinin overproduction on the entire auxin pool in the plant was slower, indicating that this most likely is mediated through altered development. In addition, we were able to confirm that the lateral root meristems are likely to be the main sites of isopentenyladenosine-5′-monophosphate-dependent cytokinin synthesis, and that the aerial tissue of the plant surprisingly also was a significant source of cytokinin biosynthesis. Our demonstration of shoot-localized synthesis, together with data demonstrating that auxin imposes a very rapid regulation of cytokinin biosynthesis, illustrates that the two hormones can interact also on the metabolic level in controlling plant development, and that the aerial part of the plant has the capacity to synthesize its own cytokinin independent of long-range transport from the root system.
Effect of Fidarestat and α‐Lipoic Acid on Diabetes‐Induced Epineurial Arteriole Vascular Dysfunction
Authors: Yorek M. A. , Coppey L. J. , Gellett J. S. , Davidson E. P. , Lund D. D.
Published: May 2004

In the present study, the authors examined whether treating streptozotocin‐induced diabetic rats with the combination of α‐lipoic acid and fidarestat, an aldose reductase inhibitor, can promote the formation of dihydrolipoic acid in diabetic animals and thereby enhance the efficacy of α‐lipoic acid as monotherapy toward preventing diabetic vascular and neural dysfunction.Treating diabetic rats with the combination of 0.25% α‐lipoic acid (in the diet) and fidarestat (3 mg/kg body weight) prevented the diabetesinduced slowing of motor nerve conduction velocity and endoneurial blood flow. This therapy also significantly improved acetylcholine‐mediated vasodilation in epineurial arterioles of the sciatic nerve compared to nontreated diabetic rats. Treating diabetic rats with 0.25% α‐lipoic acid and fidarestat (3 mg/kg body weight) was equally or more effective in preventing vascular and neural dysfunction than was monotherapy of diabetic rats with higher doses of α‐lipoic acid or fidarestat. Treating diabetic rats with the combination of 0.25% α‐lipoic acid and fidarestat (3 mg/kg body weight) significantly improved several markers of oxidative stress and increased the serum levels of both α‐lipoic acid and dihydrolipoic acid. These studies suggest that combination therapy consisting of α‐lipoic acid and fidarestat may be more efficacious in preventing diabetes‐induced vascular and neural dysfunction in peripheral tissue compared to monotherapy, which requires higher doses to be equally effective. The effect of this combination therapy may in part be due to the increased production and/or level of dihydrolipoic acid.
Tissue-specific expression of distinct spectrin and ankyrin transcripts in erythroid and nonerythroid cells.
Authors: Moon R T , Ngai J , Wold B J , Lazarides E
Published: May 2004

cDNA probes for three components of the erythroid membrane skeleton, alpha spectrin, beta spectrin, and ankyrin, were obtained by using monospecific antibodies to screen a lambda gt11 expression vector library containing cDNA prepared from chicken erythroid poly(A)+ RNA. Each cDNA appears to hybridize to one gene type in the chicken genome. Qualitatively distinct RNA species in myogenic and erythroid cells are detected for beta spectrin and ankyrin, while alpha spectrin exists as a single species of transcript in all tissues examined. This tissue-specific expression of RNAs is regulated quantitatively during myogenesis in vitro, since all three accumulate only upon myoblast fusion. Furthermore, RNAs for two of the three genes do not accumulate to detectable levels in chicken embryo fibroblasts, demonstrating that their accumulation can be noncoordinate. These observations suggest that independent gene regulation and tissue-specific production of heterogeneous transcripts from the beta spectrin and ankyrin genes underlie the formation of distinct membrane skeletons in erythroid and muscle cells.
Junctions between lens fiber cells are labeled with a monoclonal antibody shown to be specific for MP26.
Authors: Sas D F , Sas M J , Johnson K R , Menko A S , Johnson R G
Published: May 2004

A monoclonal antibody (mcAb) that recognizes an intracellular domain of the major lens membrane protein in both chicken and bovine lenses is described. Mice were immunized with chicken lens fiber cell membranes that had been washed with 7 M urea. Hybridomas were screened by means of enzyme-linked immunosorbent assays and the molecular specificities of the mcAbs were determined using electrophoretic transfer procedures, "Westerns." One of these mcAbs, an IgG designated B2, reacted with a single band of 28,000 Mr from the chicken embryo lens (MP28) and the analogous 26,000 Mr protein in the bovine lens (MP26). Monoclonal B2 was shown to be specific for these proteins, since (a) heating in SDS caused MP26 to aggregate and reduced B2 binding to the protein band at an Mr of 26,000 in Western transfer analysis; (b) apparent dimers were bound by B2 in Western transfers; (c) soluble protein fractions from the lens contained no detectable B2 antigens; and (d) a cyanogen bromide fragment of MP26 was bound by B2. Studies with several proteases indicated that the antigenic site for B2 resides on a 2-kd, protease-sensitive region at the C-terminal end of MP26 and MP28. Evidence for B2 binding on the cytoplasmic side of the membrane comes from labeling studies done at the ultrastructural level. These studies, utilizing indirect methods with peroxidase and colloidal gold markers, clearly demonstrated that B2 labels two types of junctional profiles. In our calf lens membrane preparations after tannic acid staining, the predominant type (80%) measured 16-18 nn thick, with the second type measuring only 12-14 nm. Chick embryo lens cells that had differentiated in vitro and formed groups of lens fiber-like cells (termed lentoids), fluoresced brightly only when they had been permeabilized before labeling with B2 and a fluorochrome-conjugated antibody. This binding was concentrated at the plasma membranes of cells within the lentoids, even outside areas of cell-cell contact. Surrounding epithelioid cells were not stained. Solubilized lens cultures, examined by Westerns, displayed a single immunoreactive band, which co-migrated with MP28.
Membrane-related specializations associated with acetylcholine receptor aggregates induced by electric fields.
Authors: Luther P W , Peng H B
Published: May 2004

The localization of membrane-associated specializations (basal lamina and cytoplasmic density) at sites of acetylcholine receptor (AChR) aggregation is consistent with an involvement of these structures in receptor stabilization. We investigated the occurrence of these specializations in association with AChR aggregates that develop at the cathode-facing edge of Xenopus muscle cells during exposure to a DC electric field. The cultures were labeled with a fluorescent conjugate of alpha-bungarotoxin and the receptor distribution on selected cells was determined before and after exposure to the field. In thin sections taken from the same cells, the cathode-facing edge was characterized by plaques of basal lamina and cytoplasmic density co-extensive with sarcolemma of increased density. In sections cut in a plane similar to the fluorescence image, it was possible to demonstrate that the specializations were concentrated at areas of field-induced AChR aggregation, and at receptor clusters existing on control cells. This finding further indicates that these structures participate in AChR stabilization, and that the mechanisms involved in AChR aggregation that result from field exposure and nerve contact may be similar.
The rat liver mitochondrial DNA-protein complex: displaced single strands of replicative intermediates are protein coated.
Authors: Van Tuyle G C , Pavco P A
Published: May 2004

Mitochondrial DNA (mtDNA)-protein complexes were released from the organelles by sodium dodecyl sulfate-lysis and purified by Phenyl-Sepharose CL-4B chromatography. The mitochondrial DNA-binding protein P16 was the only detectable protein in the complex. Treatment of the complex with proteinase K, or subtilisin, revealed the presence of a protease-insensitive, submolecular domain (Mr approximately equal to 6,000) that retained the capacity to bind tenaciously to the DNA. Analysis of chemically fixed complexes by CsCl isopycnic gradient centrifugation showed that P16 was bound to a large subpopulation of mtDNA enriched in displacement loops (D-loops). Based upon the effective buoyant density of the complex in CsCl gradients and the molecular weights of P16 and mtDNA, it was estimated that a mean of 49 P16 molecules were bound per mtDNA. For this measurement, the variation in hydration of protein and DNA at different CsCl concentrations was ignored. Analysis of restriction endonuclease-digested complexes by glass fiber filters that bind only protein-associated DNA resulted in the retention of a single fragment regardless of the enzyme, or enzymes, used. In each case, the retained fragment was the D-loop-containing fragment. With direct electron microscopy, the protein was readily visualized on the displaced single strand portions of D-loops and expanding D-loops. The nucleoprotein fibers were approximately 12 nm in diameter without correcting for the thickness of tungsten coating and roughly 1/3 the length of the double strand segment of the corresponding D-loop structure. In addition, occasional molecules with the characteristics of gapped circles were seen exhibiting a nucleoprotein fibril, presumably containing the single strand gap segment, linking the ends of double strand DNA. P16 was not seen on the double strand portions in any of the complexes.
Cyclic AMP effects on cell-to-cell junctional membrane permeability during adipocyte differentiation of 3T3-L1 fibroblasts.
Authors: Azarnia R , Russell T R
Published: May 2004

Mouse 3T3-L1 fibroblast cells, also know as preadipocytes, differentiate in vitro into adipocytes when treated with promoting agents and acquire numerous properties characteristic of mature fat cells. We studied junctional cell-to-cell communication by measuring the incidence of electrical coupling and transfer of carboxy-fluorescein among these cells. When 3T3-L1 cells were induced to differentiate into adipocytes, they lost virtually all cell-cell communication. Preadipocytes that remained nondifferentiated after the treatment maintained normal communication. Loss of communication in the adipocytes invariably coincided with appearance of lipid droplets and not with other phenotypic changes. In the differentiating cells, loss of cell-to-cell communication and lipid accumulation was prevented if dibutyryl cyclic AMP and caffeine were present in the culture medium. Addition of dibutyryl cyclic AMP and caffeine to already differentiated adipocytes resulted in loss of lipid and simultaneously improved junctional permeability. The results demonstrate that in the in vitro 3T3-L1 cell system, (a) cell-to-cell communication and lipid synthesis are intimately related during the adipose conversion and (b) cAMP affects the expression of the two phenotypes.
Bidirectional organelle transport can occur in cell processes that contain single microtubules.
Authors: Koonce M P , Schliwa M
Published: May 2004

Intracellular organelle transport was studied in a new model system, the giant freshwater ameba Reticulomyxa. The ameba extends a large reticulate network of cytoplasmic strands in which various phase-dense organelles can be seen to move at a rate of up to 25 microns/s. This combined light and high voltage electron microscopic study shows that organelles move bidirectionally in even the finest network strands that contain only a single microtubule. In terms of microtubule-associated intracellular transport, this observation defines a minimum set of conditions necessary for such movement. The implications of this finding for possible models of force generation are discussed.
Changes in the distribution of a spectrin-like protein during development of the preimplantation mouse embryo.
Authors: Sobel J S , Alliegro M A
Published: May 2004

The mouse blastocyst expresses a 240,000-mol-wt polypeptide that cross-reacts with antibody to avian erythrocyte alpha-spectrin. Immunofluorescence localization showed striking changes in the distribution of the putative embryonic spectrin during preimplantation and early postimplantation development. There was no detectable spectrin in either the unfertilized or fertilized egg. The first positive reaction was observed in the early 2-cell stage when a bright band of fluorescence delimited the region of cell-cell contact. The blastomeres subsequently developed continuous cortical layers of spectrin and this distribution was maintained throughout the cleavage stages. A significant reduction in fluorescence intensity occurred before implantation in the apical region of the mural trophoblast and the trophoblast outgrowths developed linear arrays of spectrin spots that were oriented in the direction of spreading. In contrast to the alterations that take place in the periphery of the embryo, spectrin was consistently present in the cortical cytoplasm underlying regions of contact between the blastomeres and between cells of the inner cell mass. The results suggest a possible role for spectrin in cell-cell interactions during early development.
Human erythrocyte myosin: identification and purification.
Authors: Fowler V M , Davis J Q , Bennett V
Published: May 2004

Human erythrocytes contain an Mr 200,000 polypeptide that cross-reacts specifically with affinity-purified antibodies to the Mr 200,000 heavy chain of human platelet myosin. Immunofluorescence staining of formaldehyde-fixed erythrocytes demonstrated that the immunoreactive myosin polypeptide is present in all cells and is localized in a punctate pattern throughout the cell. Between 20-40% of the immunoreactive myosin polypeptide remained associated with the membranes after hemolysis and preparation of ghosts, suggesting that it may be bound to the membrane cytoskeleton as well as being present in the cytosol. The immunoreactive myosin polypeptide was purified from the hemolysate to approximately 85% purity by DEAE-cellulose chromatography followed by gel filtration on Sephacryl S-400. The purified protein is an authentic vertebrate myosin with two globular heads at the end of a rod-like tail approximately 150-nm long, as visualized by rotary shadowing of individual molecules, and with two light chains (Mr 25,000 and 19,500) in association with the Mr 200,000 heavy chain. Peptide maps of the Mr 200,000 heavy chains of erythrocyte and platelet myosin were seen to be nearly identical, but the proteins are distinct since the platelet myosin light chains migrate differently on SDS gels (Mr 20,000 and 17,000). The erythrocyte myosin formed bipolar filaments 0.3-0.4-micron long at physiological salt concentrations and exhibited a characteristic pattern of myosin ATPase activities with EDTA, Ca++, and Mg++-ATPase activities in 0.5 M KCl of 0.38, 0.48, and less than 0.01 mumol/min per mg. The Mg++-ATPase activity of erythrocyte myosin in 0.06 M KCl (less than 0.01 mumol/min per mg) was not stimulated by the addition of rabbit muscle F-actin. The erythrocyte myosin was present in about 6,000 copies per cell, in a ratio of 80 actin monomers for every myosin molecule, which is an amount comparable to actin/myosin ratios in other nonmuscle cells. The erythrocyte myosin could function together with tropomyosin on the erythrocyte membrane (Fowler, V.M., and V. Bennett, 1984, J. Biol. Chem., 259:5978-5989) in an actomyosin contractile apparatus responsible for ATP-dependent changes in erythrocyte shape.
Isolation of a cell-surface receptor for chick neural retina adherons.
Authors: Schubert D , LaCorbiere M
Published: May 2004

Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion. In solution they increase the rate of cell-cell aggregation. Cell-cell and adheron-cell adhesions of cultured retina cells are selectively inhibited by heparan-sulfate glycosaminoglycan, but not by chondroitin sulfate or hyaluronic acid, suggesting that a heparan-sulfate proteoglycan may be involved in the adhesion process. We isolated a heparan-sulfate proteoglycan from the growth-conditioned medium of neural retina cells, and prepared an antiserum against it. Monovalent Fab' fragments of these antibodies completely inhibited cell-adheron adhesion, and partially blocked spontaneous cell-cell aggregation. An antigenically and structurally similar heparan-sulfate proteoglycan was isolated from the cell surface. This proteoglycan bound directly to adherons, and when absorbed to plastic, stimulated cell-substratum adhesion. These data suggest that a heparan-sulfate proteoglycan on the surface of chick neural retina cells acted as a receptor for adhesion-mediating glycoprotein complexes (adherons).
Analysis of pre- and postsynaptic factors of the serotonin system in rabbit retina.
Authors: Mitchell C K , Redburn D A
Published: May 2004

[3H]Serotonin is accumulated by a specific set of amacrine cells in the rabbit retina. These cells also accumulate the neurotoxin, 5,7-dihydroxytryptamine, and show signs of necrosis within 4 h of in vivo exposure to the drug. Biochemical analysis of [3H]serotonin uptake reveal a sodium- and temperature-dependent, high affinity uptake system with a Km of 0.94 microM and Vmax of 1.08 pmol/mg protein/min. [3H]Tryptophan is also accumulated in rabbit retinal homogenates by a high affinity process. Accumulated [3H]serotonin is released in response to potassium-induced depolarization of intact, isolated retinas. In vitro binding studies of rabbit retinal homogenate membranes demonstrate specific sets of binding sites with characteristics of the postsynaptic serotonin receptor. These data strongly suggest that rabbit retina contains virtually all of the molecular components required for a functional serotonergic neurotransmitter system. The only significant difference between the serotonin system in rabbit retina and that in the well-established serotonin transmitter systems in nonmammalin retinas and in brains of most species is the relatively low concentration of endogenous serotonin in rabbit retinas, as demonstrated by high-performance liquid chromatography, histofluorescence, or immunocytochemistry.
Neuronal regulation of astroglial morphology and proliferation in vitro.
Authors: Hatten M E
Published: May 2004

To analyze the interdependence of neurons and astroglia during central nervous system development, a rapid method for purifying early postnatal cerebellar neurons and astroglia, and recombining them in vitro, has been developed. The influence of neurons on astroglial shape and proliferation has been evaluated with an in vitro model system previously used to describe the role of cerebellar astroglia in neuronal migration and positioning (Hatten, M. E., and R. K. H. Liem, 1981, J. Cell Biol., 90:622-630; and Hatten, M. E., R. K. H. Liem, and C. A. Mason, 1984, J. Cell Biol., 98:193-204. Cerebellar tissue harvested from C57Bl/6J mouse cerebellum on the third or fourth day postnatal was dissociated into a single cell suspension with trypsin, and enriched glial and neuronal fractions were separated with a step gradient of Percoll. Highly purified astroglial and neuronal fractions resulted from subsequently preplanting the cells on a polylysine-coated culture surface. In the absence of neurons, astroglia, identified by staining with antisera raised against purified glial filament protein, assumed a flattened shape and proliferated rapidly. In the absence of astroglia, cerebellar neurons, identified by staining with antisera raised against the nerve growth factor-inducible large external (NILE) glycoprotein and by electron microscopy, formed cellular reaggregates, had markedly impaired neurite outgrowth, and survived poorly. When purified neurons and isolated astroglia were recombined, astroglial proliferation slowed markedly and the flattened shape expressed in the absence of neurons transformed into highly elongated profiles that resembled embryonic forms of cerebellar astroglia. After longer periods (48-72 h) in the presence of neurons, astroglia had "Bergmann-like" or "astrocyte-like" shapes and neurons commonly associated with them. These results suggest that neurons influence the differentiation of astroglia.
Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules.
Authors: Resh M D , Erikson R L
Published: May 2004

Antiserum to the Rous sarcoma virus (RSV)-transforming protein, pp60v-src, was produced in rabbits immunized with p60 expressed in Escherichia coli. alpha p60 serum immunoprecipitated quantitatively more pp60v-src than did tumor-bearing rabbit (TBR) sera. When RSV-transformed cell lysates were preadsorbed with TBR serum, the remaining lysate contained additional pp60v-src, which was recognized only by reimmunoprecipitation with alpha p60 serum and not by TBR serum. In subcellular fractions of RSV-infected chicken embryo fibroblasts (RSV-CEFs) and field vole cells probed with TBR serum, the majority of the pp60v-src was associated with the plasma membrane-enriched P100 fraction. However, alpha p60 serum revealed equal distribution of pp60v-src and its kinase activity between the P1 (nuclear) and P100 fractions. The same results were obtained for pp60c-src in uninfected CEFs. On discontinuous sucrose gradients nearly 50% of the P1-pp60v-src sedimented with nuclei, in fractions where no plasma membrane was detected. Indirect immunofluorescence microscopy of RSV-CEFs with alpha p60 serum revealed a distinct pattern of perinuclear fluorescence, in addition to staining at the cell periphery. Thus the use of a highly specific antibody reveals that enzymatically active pp60v-src and pp60c-src molecules are present in other intracellular structures, probably juxtareticular nuclear membranes, in addition to the plasma membrane in normal, uninfected, and wild-type RSV-infected cells.
Endothelial fenestral diaphragms: a quick-freeze, deep-etch study.
Authors: Bearer E L , Orci L
Published: May 2004

The route by which water, solutes, and macromolecules traverse the endothelial cell has long been a subject of study for both physiologists and cell biologists. Recent physiologic studies describe a slit-shaped pore (5.1-5.7-nm wide) as the communicating channel, although no channel of such dimensions has been visible in electron microscopic preparations. That this channel should be found within the fenestral diaphragm has long been suggested. In this report, by the aid of a new technique in tissue processing, we are able to demonstrate a possible morphologic correlate within the fenestral diaphragm of fenestrated capillaries. Quick-freezing and deep-etching of whole tissue blocks allows the sublimation of water from the endothelial pores, thus leaving the channels through the diaphragms empty and readily replicated with a platinum-carbon shadow. The structure of the diaphragm was revealed thus to be composed of radial fibrils of 7 nm in diameter, interweaving in a central mesh, and creating by their geometric distribution, wedge-shaped channels around the periphery of the pore. The average channel had a maximum arc length of 5.46 nm. Fenestrated endothelia from various tissues, including endocrine and exocrine pancreas, adrenal cortex, and kidney peritubular capillaries, displayed the same diaphragmatic structure, whereas continuous capillaries in muscle had no such diaphragm. Photographic augmentation of electron micrographs of etched replicas displayed marked enhancement at n = 8, confirming an octagonal symmetry of the fenestral diaphragm. Finally, cationic ferritin, clearly visible as a marker after etching, heavily bound to the flowerlike structure within the fenestral pore. We conclude that the fenestral diaphragm contains the structure responsible for fenestrated capillary permeability and that the communicating channel has the shape of a wedge.
Reversal of the posttranslational modification on Chlamydomonas flagellar alpha-tubulin occurs during flagellar resorption.
Authors: L'Hernault S W , Rosenbaum J L
Published: May 2004

We previously have shown that a posttranslational modification of alpha-tubulin takes place in the flagellum during Chlamydomonas flagellar assembly (L'Hernault, S. W., and J. L. Rosenbaum, 1983, J. Cell Biol., 97:258-263). In this report, we show that the posttranslationally modified alpha-3 tubulin is changed back to its unmodified alpha-1 precursor form during the microtubular disassembly that takes place during flagellar resorption. These data indicate that the addition and removal of a posttranslational modification on alpha-tubulin might be a control step in the assembly and disassembly of flagella.
Maize plastid photogenes: mapping and photoregulation of transcript levels during light-induced development.
Authors: Rodermel S R , Bogorad L
Published: May 2004

Positively photoregulated regions that show increased transcript levels upon illumination of dark-grown seedlings are scattered over approximately 19% of the maize plastid chromosome. Some photogenes, i.e., genes within these regions, are transcribed individually, whereas others that are transcribed as polycistronic mRNAs appear to be functionally organized into operons. Multiple light-induced transcripts are complementary to most photogenes; these mRNAs are not present in equimolar amounts during plastid photomorphogenesis, but particular transcripts predominate at specific stages of development. Most, but not all, photogene RNA pools reach a maximum size (after either 10, 20, or 44 h of illumination) and then fall to approximately preillumination levels. These data and other considerations argue that photogene expression control is fundamentally transcriptional and that there is more than one expression class. Transcripts of the maize plastid gene for the large subunit of ribulose bisphosphate carboxylase reach a maximum by 20 h of illumination; transcripts of the nuclear gene for the small subunit of this enzyme continue to accumulate and fall considerably later. These data suggest that the level of transcription of the latter gene in the nucleus may be regulated by events in the chloroplast.
Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor.
Authors: Kato Y , Gospodarowicz D
Published: May 2004

We examined the effect of fibroblast growth factor (FGF) on proteoglycan synthesis by rabbit costal chondrocyte cultures maintained on plastic tissue culture dishes. Low density rabbit costal chondrocyte cultures grown in the absence of FGF gave rise at confluency to a heterogeneous cell population composed of fibroblastic cells and poorly differentiated chondrocytes. When similar cultures were grown in the presence of FGF, the confluent cultures organized into a homogenous cartilage-like tissue composed of rounded cells surrounded by a refractile matrix. The cell ultrastructure and that of the pericellular matrix were similar to those seen in vivo. The expression of the cartilage phenotype in confluent chondrocyte cultures grown from the sparse stage in the presence vs. absence of FGF was reflected by a fivefold increase in the rate of incorporation of [35S]sulfate into proteoglycans. These FGF effects were only observed when FGF was present during the cell logarithmic growth phase, but not when it was added after chondrocyte cultures became confluent. High molecular weight, chondroitin sulfate proteoglycans synthesized by confluent chondrocyte cultures grown in the presence of FGF were slightly larger in size than that produced by confluent cultures grown in the absence of FGF. The major sulfated glycosaminoglycans associated with low molecular weight proteoglycan in FGF-exposed cultures were chondroitin sulfate, while in cultures not exposed to FGF they were chondroitin sulfate and dermatan sulfate. Regardless of whether or not cells were grown in the presence or absence of FGF, the 6S/4S disaccharide ratio of chondroitin sulfate chains associated with high and low molecular weight proteoglycans synthesized by confluent cultures was the same. These results provide evidence that when low density chondrocyte cultures maintained on plastic tissue culture dishes are grown in the presence of FGF, it results in a stimulation of the expression and stabilization of the chondrocyte phenotype once cultures become confluent.
MAP3: characterization of a novel microtubule-associated protein.
Authors: Huber G , Alaimo-Beuret D , Matus A
Published: May 2004

Using monoclonal antibodies we have characterized a brain protein that copurifies with microtubules. We identify it as a microtubule-associated protein (MAP) by the following criteria: it copolymerizes with tubulin through repeated cycles of microtubule assembly in vitro; it is not associated with any brain subcellular fraction other than microtubules; in double-label immunofluorescence experiments antibodies against this protein stain the same fibrous elements in cultured cells as are stained by antitubulin; and this fibrous staining pattern is dispersed when cytoplasmic microtubules are disrupted by colchicine. Because it is distinct from previously described MAPs we designate this novel species MAP3. The MAP3 protein consists of a closely spaced pair of polypeptides on SDS gels, Mr 180,000, which are present in both glial (glioma C6) and neuronal (neuroblastoma B104) cell lines. In brain the MAP3 antigen is present in both neurons and glia. In nerve cells its distribution is strikingly restricted: anti-MAP3 staining is detectable only in neurofilament-rich axons. It is not, however, a component of isolated brain intermediate filaments.
Assembled and unassembled pools of clathrin: a quantitative study using an enzyme immunoassay.
Authors: Goud B , Huet C , Louvard D
Published: May 2004

Using polyclonal antibodies raised against clathrin, we have developed an enzyme-linked immunoassay that can specifically measure the quantity of clathrin in crude cell extracts. We found that the quantity (weight percent of total protein) of clathrin was similar in cell types that exhibit large differences in their levels of endocytosis and exocytosis (lymphoid cells, 0.11%; liver cells, 0.07%, fibroblasts, 0.18%; myeloma cells, 0.16%). However, the quantity of clathrin was found to be significantly higher in brain cortex (0.75%). Cellular clathrin was separated by high-speed centrifugation into two fractions: an unassembled form present in high-speed supernatants and an assembled form (clathrin coats) present in the pellets. We show that the fraction of clathrin in the unassembled state varies considerably depending on the cell type studied (14% in brain cortex to 70% in lymphocytes). Our data support the view that the amount of clathrin (relative to total cell protein) in eucaryotic cells is not related to the extent of receptor-mediated endocytosis and intracellular membrane traffic. However, the fraction of assembled clathrin seems to be higher in endocytically and/or exocytically active cells.
Fc receptor-mediated binding and endocytosis by human mononuclear phagocytes: monomeric IgG is not endocytosed by U937 cells and monocytes.
Authors: Jones D H , Nusbacher J , Anderson C L
Published: May 2004

Fc receptor-mediated endocytosis of monomeric IgG1 by human mononuclear phagocytes was evaluated under conditions where aggregated IgG and insulin readily undergo receptor-mediated internalization. U937 cells or normal human peripheral blood monocytes were incubated at 37 degrees C in the absence of free radioligand after having first bound 125I-IgG1 at 0 degrees C. To determine the amount of cell-associated IgG1 internalized after varying periods of 37 degrees C incubation, surface-bound IgG1 was removed by sequential exposure of cells at 0 degrees C to a nonspecific proteinase for 1 h and to acetic acid at pH 3.2 for 3 min. The failure to develop a proteinase- and acid-resistant fraction, similar to that seen over time at 37 degrees C in parallel experiments with 125I-insulin and 125I-aggregated IgG, and the lack of degradation of the IgG1 released into the medium from the same cells over time show that these cells do not endocytose and degrade monomeric IgG by an Fc receptor-specific mechanism and suggest that constitutive recycling without degradation is unlikely to be occurring. These data fulfill one prediction of the hypothesis that receptor-receptor interaction triggers Fc receptor-mediated endocytosis.
Novel peripheral blood-derived human cell lines with properties of megakaryocytes.
Authors: Morgan D A , Brodsky I
Published: May 2004

For 18 mo, we derived 18 cell lines from 11 donors with various clinical profiles ranging from normal to leukemic. Suspension cultures were initiated with 1 X 10(6) mononuclear blood cells/ml of nutrient medium containing 10% human serum and 10% lectin-stimulated human lymphocyte conditioned medium. The cultures were monitored weekly by morphological analyses of Wright-Giemsa-stained cell preparations. All successful cultures showed a significant decline in viability during the first 3-4 wk with rate "lymphoid" cells observed in mitosis. Within the next 2 wk, the proliferating cells gave rise to a rapidly expanding population of mononuclear cells. As the cultures expanded, cell morphology became heterogeneous with respect to cell size and nuclear ploidy, with an accumulation of giant multinuclear cells that were suggestive of megakarocytes. Even though the cells did not have the classical morphology of mature platelet-forming megakaryocytes, 90% of the cells within a cell line were positive by direct or indirect immunofluorescence for the platelet membrane glycoproteins IIb and IIIa; for surface markers HLA-Dr and B2-microglobulin; for intracellular platelet-derived growth factor and platelet factor IV; and for membrane affinity or binding with serum platelet-derived growth factor and platelet factor IV. These results suggest that a blood precursor cell, most likely a primitive megakaryoblast, was isolated from the peripheral blood and was provided with an optimal culture environment for sustained growth. These cells did not mature to a more differentiated stage, perhaps owing to regulatory factor deficiencies in this in vitro system. The remarkable frequency of obtaining cell lines with megakaryocyte properties from normal peripheral blood and the capacity of some normal donors to repeatedly yield these cell lines make this cell culture system indeed unique by being selective for putative megakaryocyte precursors.