bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction.
Authors: Ceccarelli B , Hurlbut W P
Published: May 2004

Frog cutaneous pectoris muscles were treated with low doses of crude black widow spider venom (BWSV) or purified alpha-latrotoxin, and neuromuscular transmission, quantal secretion, changes in ultrastructure and uptake of horseradish peroxidase (HRP) were studied. When these agents were applied to muscles bathed in a Ca2+-free solution with 1 mM EGTA and 4 mM Mg2+, the rate of quantal secretion rose to high levels but quickly subsided; neuromuscular transmission was totally and irreversibly blocked within 1 h. The terminals became swollen and were depleted of vesicles; HRP was not taken up. When BWSV was applied to other muscles bathed in a solution with 1.8 mM Ca2+ and 4 mM Mg2+, the rate of secretion rose to high levels and then declined to intermediate levels that were sustained throughot the hour of exposure. Neuromuscular transmission was blocked in fewer than 50% of these fibers. The ultrastructure of these terminals was normal and they contained large numbers of synaptic vesicles. If HRP had been present, most of the synaptic vesicles were labeled with reaction product. These observations suggest that Ca2+ plays an important role in endocytosis at the frog neuromuscular junction.
Regulated expression of the prolactin gene in rat pituitary tumor cells.
Authors: Brennessel B A , Biswas D K
Published: May 2004

Prolactin (PRL) gene expression in three strains of GH cells (rat pituitary tumor cells) has been quantitated by measurement of: (a) intracellular and extracellular PRL, (b) cytoplasmic translatable PRL-specific mRNA (mRNAPRL), and (c) molecular hybridization of cytoplasmic poly(A) RNA to cDNAPRL (DNA complementary to mRNAPRL). Three GH cell lines utilized in this investigation were a PRL-producing (PRL+) strain, GH4C1, a PRL nonproducing 5-bromo-deoxyuridine resistnat (PRL- BrdUrdr) strain, F1BGH12C1, and a new strain, 928-9b, derived by fusion of PRL+ cells with a nuclear monolayer of the PRL-, BrdUrdr GH cell strain. PRL production is a characteristic of 928-9b cells, but the level of PRL production (2-4 micrograms/mg protein/24 h) is much lower than that of the PRL+ strain, GH4C1 (15-25 micrograms/mg protein/24 h). Levels of cytoplasmic translatable mRNAPRL and cytoplasmic PRL-RNA sequences quantitated with a cDNAPRL probe were also much lower in 928-9b as compared to the PRL+ parent. PRL-RNA sequences could not be detected in the PRL- strain. Thyrotopin-releasing hormone (TRH) stimulates PRL synthesis about threefold and inhibit a growth hormone (GH) synthesis 72% in the PRL+ strain. TRH has no effect on the synthesis of either PRL or GH in the 928-9b strain, although TRH receptors could be detected in these cells. Stimulation of PRL synthesis in the PRL+ strain by TRH could be correlated with increases in levels of cytoplasmic translatable mRNAPRL and increases in cytoplasmic PRL-RNA sequences. These results demonstrate that the graded expression of the PRL gene at the basal level, and in response to TRH, is caused by the regulated production of specific mRNA, i.e., mRNAPRL in these three GH cell strains.
Uptake and utilization of mRNA by myogenic cells in culture.
Authors: Mroczkowski B , Dym H P , Siegel E J , Heywood S M
Published: May 2004

Primary chick myoblast cultures demonstrate the ability to take up exogenously supplied polyadenylated RNA and express the encoded information in a specific manner. This expression is shown to exhibit tissue specificity. Analysis of creatine kinase activity monitored at various times of incubation in the presence of either polyadenylated or nonpolyadenylated RNA indicates that only the poly(A)+ mRNA is capable of being actively translated. Radioactively labled poly(A)+ mRNA is taken up by the cell cultures in a time-dependent manner and subsequently shown to be associated with polysomes. This association with polysomes does not occur in the presence of puromycin and is unaffected by actinomycin D. Thus, nonspecific interaction with polysomes and induction of new RNA synthesis are ruled out and the association of the exogenously supplied poly(A)+ mRNA with polysomes is indicative of its translation in the recipient cells. When heterologous mRNA (globin) is supplied to the myoblasts, it is also taken up and properly translated. In addition, exogenously supplied myosin heavy chain mRNA is found associated with polysomes consisting of 4-10 ribosomes in myoblast cell cultures while in myotubes it is associated with very large polysomes, thus reflecting the different translational efficiencies that this message exhibits at two very different stages of myogenesis. The results indicate that muscle cell cultures can serve as an in vitro system to study translational controls and their roles in development.
Calmodulin confers calcium sensitivity on ciliary dynein ATPase.
Authors: Blum J J , Hayes A , Jamieson G A , Vanaman T C
Published: May 2004

Extraction of demembranated cilia of Tetrahymena by Tris-EDTA (denoted by the suffix E) yields 14S-E and 30S-E dyneins with ATPase activities that are slightly increased by Ca++. This effect is moderately potentiated when bovine brain calmodulin is added to the assay mixture. Extraction with 0.5 M KCl (denoted by the suffix K) yeilds a 14S-K dynein with a low basal ATPase activity in the presence of Ca++. Subsequent addition of calmodulin causes marked activation (up to 10-fold) of ATPase activity. Although 14S-K and 14S-E dyneins have Ca++-dependent ATPase activities that differ markedly in the degree of activation, the concentration of calmodulin required for half-maximal saturation is similar for both, approximately 0.1 microM. Both 30S-K and 30S-E dyneins, however, require approximately 0.7 microM bovine brain calmodulin to reach half-maximal activation of their Ca++-dependent ATPase activities. Tetrahymena calmodulin is as effective as bovine brain calmodulin in activating 30S dynein , but may be slightly less effective than the brain calmodulin in activating 14S dynein. Rabbit skeletal muscle troponin C also activates the Ca++-dependent ATPase activity of 30S dynein and, to a lesser extent, that of 14S dynein, but in both cases is less effective than calmodulin. The interaction of calmodulin with dynein that results in ATPase activation is largely complete in less than 1 min, and is prevented by the presence of low concentrations of ATP. Adenylyl imidodiphosphate can partially prevent activation of dynein ATPase by calmodulin plus Ca++, but at much higher concentrations than required for prevention by ATP. beta, gamma-methyl-adenosine triphosphate appears not to prevent this activation. The presence of Ca++-dependent calmodulin-binding sites on 14S and 30S dyneins was demonstrated by the Ca++-dependent retention of the dyneins on a calmodulin-Sepharose-4B column. Gel electrophoresis of 14S dynein that had been purified by the affinity-chromatography procedure showed that presence of two major and one minor high molecular weight components. Similar analysis of 30S dynein purified by this procedure also revealed on major and one minor high molecular weight components that were different from the major components of 14S dynein. Ca++-dependent binding sites for calmodulin were shown to be present on axonemes that had been extracted twice with Tris-EDTA or with 0.5 M KCl by the use of 35S-labeled Tetrahymena calmodulin. It is concluded that the 14S and 30S dyneins of Tetrahymena contain Ca++-dependent binding sites for calmodulin and the calmodulin mediates the Ca++-regulation of the dynein ATPases of Tetrahymena cilia.
Intracellular distribution of low molecular weight RNA species in HeLa cells.
Authors: Gurney T , Eliceiri G L
Published: May 2004

Intracellular distributions of the low molecular weight RNA species of HeLa cells were determined by a nonaqueous method of cell fractionation, in which lyophilized cells were homogenized and centrifuged in anhydrous glycerol. The nonaqueous method was used to avoid artifactual extraction of weakly bound nuclear RNA during cell fractionation. We found that the mature small RNA species K, A, C, and D were almost entirely (greater than 95%) nuclear, and that mature 4S tRNA was partially (5-10%) nuclear. Our results gave higher nuclear content of the mature species K, A, C, and 4S than was shown previously with conventional aqueous cell fractionation. The nonaqueous method also gave higher nuclear proportions of some short-lived precursors to mature small RNAs. We found that approximately one-half of recently synthesized pre-4S RNA and more than one-half of recently synthesized 5S RNA were nuclear, whereas these species had been thought to be cytoplasmic from previous work. The species C' and D', precursors to the stable nuclear species C and D respectively, were found to be partially nuclear, also in contrast to earlier work. The stable cytoplasmic species L (oncornavirus 7S RNA) was found to be mostly nuclear shortly after synthesis.
Association of a protease with polytene chromosomes of Drosophila melanogaster.
Authors: Cavagnaro J , Pierce D A , Lucchesi J C , Chae C B
Published: May 2004

Incubation of Drosophila salivary glands with radioactive diisopropyl fluorophosphate results in the uniform labeling of polytene chromosomes. Extensive labeling is seen only when chromosome squashes are prepared by a formaldehyde fixation procedure and not by standard acetic acid techniques. The labeling is inhibited in the presence of tosylphenylalanine chloromethyl ketone and phenylmethane sulfonylfluoride but not by tosyllysine chloromethyl ketone, suggesting that a chymotrypsin-like serine protease is associated with the chromosomes. Protease inhibitors show no apparent effect on heat-shock specific puffing.
Continuous culture of rat C6 glioma in serum-free medium.
Authors: Wolfe R A , Sato G H , McClure D B
Published: May 2004

In this communication we describe serum-free culture conditions for the serial propagation of the C6 glioma cell line. The growth rate, saturation density, and morphology of these cells are equivalent to those of their serum-grown counterparts when cultured in a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham's medium F-12 supplemented with trace elements, insulin, transferrin, fibroblast growth factor, linoleic acid complexed to fatty acid-free bovine serum albumin, and a serum-spreading factor (SSF) partially purified from human plasma. The requirement for SSF in the medium can be satisfied by preincubating the tissue culture dishes with SSF. Tissue culture dishes sequentially pretreated with poly-D-lysine and purified cold insouluble globulin will also substitute for this requirement. The fatty acid-free bovine serum albumin/linoleic acid complex increases the growth rate of these cells but has no appreciable effect on their morphology, saturation density, or ability to grow with repeated subculture. The growth stimulation caused by this complex appears to be dependent on the fatty acid, as the fatty acid-free bovine serum albumin alone has no effect on the growth rate. Linoleic acid is cytotoxic in the absence of bovine serum albumin, and the fatty acid-free bovine serum albumin prevents this toxicity. Other fatty acids including oleic, arachidonic, and palmitic only partially substitute for the growth-promoting effect of linoleic acid.
Membrane assembly in retinal photoreceptors I. Freeze-fracture analysis of cytoplasmic vesicles in relationship to disc assembly.
Authors: Besharse J C , Pfenninger K H
Published: May 2004

To study precursor-product relationships between cytoplasmic membranes of the inner segment of photoreceptors and the continually renewed outer disc membrane, we have compared the density and size distribution of intramembrane particles (IMP) in various membrane compartments of freeze-fractured photoreceptor inner and outer segments. Both rod and cone outer segments of Xenopus laevis are characterized by a relatively uniform distribution of approximately 4,400-4,700 IMP/micron2 in P-face (PF) leaflets of disc membranes. A similar distribution of IMP is found in the outer segment plasma membrane, the ciliary plasma membrane, and in the plasma membrane of the inner segment in the immediate periciliary region. In each case the size distribution of IMP can be characterized as unimodal with a mean diameter of approximately 10 nm. PF leaflets of endoplasmic reticulum, Golgi complex, and vesicles near the cilium have IMP with a size distribution like that in the cilium and outer segment, but with an average density of approximately 2,000/micron2. In contrast, IMP are smaller in average size (approximately 7.5 nm) in PF leaflets of inner segment plasma membrane, exclusive of the periciliary rgion. The similarity of size distribution of IMP in inner segment cytoplasmic membranes and those within the plasmalemma of the cilium and outer segment suggest a precursor-product relationship between the two systems. The structure of the vesicle-rich periciliary region and the segregation of IMP with different size distributions in this region suggest that components destined for incorporation into the outer segment exist as preformed membrane packages (vesicles) which fuse with the inner segment plasma membrane in the periciliary region. Subsequently, membrane components may be transferred to forming discs of the outer segment via the ciliary plasma membrane.
Primary sequence of ovomucoid messenger RNA as determined from cloned complementary DNA.
Authors: Catterall J F , Stein J P , Kristo P , Means A R , O'Malley B W
Published: May 2004

Ovomucoid messenger RNA (mRNAom) comprises approximately 8% of the total mRNA in the estrogen-stimulated oviduct. The recombinant plasmid pOM100 contained DNA complementary to the 3' end of mRNAom. DNA complementary to the 5' end of mRNAom was obtained from a partially purified preparation of mRNAom by polymerization by reverse transcriptase in the presence of a restriction fragment primer from pOM100. The complementary DNA mixture was amplified by molecular cloning using poly dG/dC tailing to form recombinant bacterial plasmids. Recombinant plasmids containing ovomucoid DNA sequences were selected by in situ hybridization to 32P-labeled pOM100 fragments. The longest plasmid containing ovomucoid DNA sequences was designated pOM502. The complete DNA sequence of both pOM100 and pOM502 was determined. The two plasmids appear to contain sequences complementary to the entire length of mRNAom. The nucleic acid sequence agrees with the known amino acid sequences for both ovomucoid and its N-terminal signal peptide. Highly homologous sequences occur in two regions that coincide with structural domains of the protein. Comparison of the sequence of mRNAom with that for other eucaryotic mRNAs allowed identification of possible functional regions in the mRNA molecule.
Structural diversity of occluding junctions in the low-resistance chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus).
Authors: Ernst S A , Dodson W C , Karnaky K J
Published: May 2004

The structural features of the chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus) were examined by thin-section and freeze-fracture electron microscopy, with particular emphasis on the morphological appearance of occluding junctions. This epithelium is a flat sheet consisting predominantly of groups of mitochondriarich chloride cells with their apices associated to form apical crypts. These multicellular groups are interspersed in an otherwise continuous pavement cell epithelial lining. The epithelium may be mounted in Ussing-type chambers, which allow ready access to mucosal and serosal solutions and measurement of electrocal properties. The mean short-circuit current, potential difference (mucosal-side negative), and DC resistance for 19 opercular epithelia were, respectively, 120.0 +/- 18.2 microA/cm2, 12.3 +/- 1.7 mV, and 132.5 +/- 26.4 omega cm2. Short-circuit current, a direct measure of Cl- transport, was inhibited by ouabain (5 micron) when introduced on the serosal side, but not when applied to the mucosal side alone. Autoradiographic analysis of [3H]-ouabain-binding sites demonstrated that Na+,K+-ATPase was localized exclusively to basolateral membranes of chloride cells; pavement cells were unlabeled. Occluding junctions between adjacent chloride cells were remarkably shallow (20-25 nm), consisting of two parallel and juxtaposed junctional strands. Junctional interactions between pavement cells or between pavement cells and chloride cells were considerably more elaborate, extending 0.3-0.5 micron in depth and consisting of five or more interlocking junctional strands. Chloride cells at the lateral margins of crypts make simple junctional contacts with neighboring chloride cells and extensive junctions with contiguous pavement cells. Accordingly, in this heterogeneous epithelium, only junctions between Na+,K+-ATPase-rich chloride cells are shallow. Apical crypts may serve, therefore, as focal areas of high cation conductivity across the junctional route. This view is consistent with the electrical data showing that transmural resistance across the opercular eptihelium is low, and with recent studies demonstrating that transepithelial Na+ fluxes are passive. The simplicity of these junctions parallels that described recently for secretory cells of avian salt gland (Riddle and Ernst, 1979, J. Membr. Biol., 45:21-35) and elasmobranch rectal gland (Ernst et al., 1979, J. Cell Biol., 83:(2, Pt. 2):83 a[Abstr.]) and lends morphological support to the concept that paracellular ion permeation plays a central role in ouabain-sensitive transepithelial NaCl secretion.
Identification and characterization of a membrane component essential for the translocation of nascent proteins across the membrane of the endoplasmic reticulum.
Authors: Meyer D I , Dobberstein B
Published: May 2004

When rough microsomes are subjected to limited proteolysis and high salt, a soluble fraction can be separated from the membrane. Neither fraction alone is capable of vectorially translocating nascent peptides. When the soluble extract is recombined with the residual membrane fraction, translocating activity is restored. Standard biochemical techniques were used to identify and characterize the active component derived by treating rough microsomes with elastase and high salt. The active factor is a peptide fragment with an apparent molecular weight of 60,000. It represents the cytoplasmic domain of a larger membrane protein. The fragment is basic and has at least one accessible sulfhydryl group. These characteristics facilitated its purification and identification as a membrane component required for translocation of nascent peptides across microsomal membranes.
Organization of spindle microtubules in Ochromonas danica.
Authors: Tippit D H , Pillus L , Pickett-Heaps J
Published: May 2004

The entire framework of microtubules (MTs) in the mitotic apparatus of Ochromonas danica is reconstructed (except at the spindle poles) from transverse serial sections. Eleven spindles were sectioned and used for numerical data, but only four were reconstructed: a metaphase, an early anaphase, a late anaphase, and telophase. Four major classes of MTs are observed: (a) free MTs (MTs not attached to either pole); (b) interdigitated MTs (MTs attached to one pole which laterally associate with MTs from the opposite pole); (c) polar MTs (MTs attached to one pole); (d) kinetochore MTs (kMTs). Pole-to-pole MTs are rare and may be caused by tracking errors. During anaphase, the kMTs, free MTs, and polar MTs shorten until most disappear, while interdigitated MTs lengthen. In the four reconstructed spindles, the number of MTs decreases between early anaphase and telophase from 881 to 285, while their average length increases from 1.66 to 4.98 micron. The total length of all the MTs in the spindle (placed end to end) remains at 1.42 +/- 0.04 mm between these stages. At late anaphase and telophase the spindle is comprised mainly of groups of interdigitated MTs. Such MTs from opposite poles form a region of overlap in the middle of the spindle. During spindle elongation (separation of the poles), the length of the overlap region does not decrease. These results are compatible with theories that suggest that MTs directly provide the force that elongates the spindle, either by MT polymerization alone or by MT sliding with concomitant MT polymerization.
Characterization of the turning response of dorsal root neurites toward nerve growth factor.
Authors: Gundersen R W , Barrett J N
Published: May 2004

This study reports that chick dorsal root ganglion neurites undergo a rapid (20 min) reorientation of their direction of growth in response to nerve growth factor (NGF) concentration gradients in vitro. Dorsal root ganglia from chick embryos were explanted onto a collagen-poly-L-lysine substrate. After 24-48 h in culture, NGF gradients were applied to individual growth cones via a micropipette containing 50 biological units NGF/ml. The growth cones turned and grew toward these NGF sources. This turning response was not caused by the trophic effects of NGF on neurite initiation, survival, or growth rate. Dorsal root neurites also grew toward sources of mono- and dibutyryl cyclic adenosine monophosphate (dB cAMP), cyclic guanosine monophosphate (cGMP), and elevated calcium in the presence of the calcium ionophore A23187. These results are consistent with the hypothesis that intracellular levels of cAMP and /or cGMP and calcium may play a role in the turning response of dorsal root neurites toward NGF, but do not establish a causal relationship between the mechanisms of action of NGF, cyclic nucleotides and calcium. Total growth cone adherence to the substrate was measured using a timed microjet of perfusion medium. NGF increased the adherence of growth cones to the substrate, but caffeine and dB cAMP which also elicit the positive turning response, decreased growth cone adherence. Calcium, which did not elicit the positive turning response, produced a greater growth cone adherence to the substrate than that observed with NGF. Although these results do not rule out a role of adhesion changes in axonal turning to NGF, they show that a general increase in adherence does not correlate well with the rapid turning response observed in this study.
Permanently proliferating rat vascular smooth muscle cell with maintained expression of smooth muscle characteristics, including actin of the vascular smooth muscle type.
Authors: Franke W W , Schmid E , Vandekerckhove J , Weber K
Published: May 2004

Cells of an established clonal line (RVF-SMC) derived from rat vena cava are described by light and electron microscope methods and biochemical analysis of the major proteins. The cells are flat, and they moderately elongate and form monolayers. They are characterized by prominent cables of microfilaments bundles decoratable with antibodies to actin and alpha-actinin. These bundles contain numerous densely stained bodies and are often flanked by typical rows of surface caveolae and vesicles. The cells are rich in intermediate-sized filaments of the vimentin type but do not show detectable amounts of desmin and cytokeratin filaments. Isoelectric focusing and protein chemical studies have revealed actin heterogeneity. In addition to the two cytoplasmic actins, beta and gamma, common to proliferating cells, two smooth muscle-type actins (an acidic alpha-like and a gamma-like) are found. The major (alpha-type) vascular smooth muscle actin accounts for 28% of the total cellular actin. No skeletal muscle or cardiac muscle actin has been detected. The synthesis of large amounts of actin and vimentin and the presence of at least three actins, including alpha-like actin, have also been demonstrated by in vitro translation of isolated poly(A)+ mRNAs. This is, to our knowledge, the first case of expression of smooth muscle-type actin in a permanently growing cell. We conclude that permanent cell growth and proliferation is compatible with the maintained expression of several characteristic cell features of the differentiated vascular smooth muscle cell including the formation of smooth muscle-type actin.
Tyrosine aminotransferase sensitivity to bromodeoxyuridine during restricted intervals of S phase in hepatoma cells.
Authors: O'Brien J C
Published: May 2004

Synchronized hepatoma tissue culture (HTC) cells, accumulated at the G1/S boundary with aminopterin, were released into S phase with either thymidine or 5-bromodeoxyuridine (BUdR). Tyrosine aminotransferase (TAT) activity was found to be unaffected by BUdR over the initial 3 h of S phase, but then to rapidly decline to a new basal level of 40% of control by 9 h. There was no corresponding response in the activities of alcohol dehydrogenase, malate dehydrogenase, acid phosphatase, and alkaline phosphatase, or in the rate of protein and RNA synthesis. If BUdR incorporation was restricted to limited periods of S phase, TAT was found to be maximally suppressed by incorporation into the initial 40% of the DNA. Incorporation of the analogue into the latter 60% of DNA synthesized during S phase had no effect on TAT. This is the first report that the effect of BUdR on TAT in HTC cells is associated with incorporation of the analog into DNA synthesized during a specific interval of S phase.
Fibronectin localization in the rat glomerulus.
Authors: Courtoy P J , Kanwar Y S , Hynes R O , Farquhar M G
Published: May 2004

Fibronectin (FN) has been localized in the rat glomerulus using indirect immunolabeling. It was demonstrated in frozen sections by immunofluorescence, in sections of fixed kidneys by both peroxidase and ferritin-labeled antibodies, and in isolated glomerular basement membranes (GBM) with ferritin-labeled antibodies. Complementary and convergent results were obtained with these approaches. FN was most abundant in the mesangial matrix where it was especially concentrated at the interface between the endothelial and mesangial cells. In the peripheral capillary loop, FN was also detected in the laminae rarae (interna and externa) of the GBM--i.e., between the endothelial and epithelial cells, respectively, and the GBM. These findings indicate that FN is an important constituent of the glomerulus, and they are compatible with the assumption that, in the glomerulus, as in cultured cells, FN is involved in cell-to-cell (mesangial-mesangial, mesangial-endothelial) and cell-to-substrate (mesangial cell-mesangial matrix, epithelium-GBM, endothelium-GBM) attachment.
Linkage in cultured Chinese hamster cells of two genes, emtB and leuS, involved in protein synthesis and isolation of cell lines with mutations in three linked genes.
Authors: Wasmuth J J , Chu L Y
Published: May 2004

We have determined via segregation analyses from appropriate hybrids that two genes involved in protein synthesis, one encoding for a ribosomal protein (emtB) and one encoding for leucyl-tRNA synthetase (leuS), cosegregate at a very high frequency and are linked in both Chinese hamster ovary and lung cells. In contrast, the emtA locus, defined by a second complementation group of emetine-resistant mutants which also have alterations affecting protein synthesis and probably the ribosome, is not linked to leuS. In addition, we have determined that a third gene, one that can be altered to give rise to chromate resistance, is syntenic with emtB and leuS. We have selected cell lines with mutations in each of these three linked genes and have shown that the three loci cosegregate at a high frequency. Because the mutations in these three linked genes provide easily distinguishable phenotypes, these cell lines should provide a powerful tool for examining several important questions concerning mitotic recombination in somatic cells.
Gap junction dynamics: reversible effects of divalent cations.
Authors: Peracchia C , Peracchia L L
Published: May 2004

Reversible changes in gap junction structure similar to those previously seen to parallel electrical uncoupling (9, 33, 34) are produced by treating with Ca++ or Mg++ gap junctions isolated in EDTA from calf lens fibers. The changes, characterized primarily by a switch from disordered to crystalline particle packings, occur at a [Ca++] of 5 x 10(-7) M or higher and a [Mg++] of 1 x 10(-3) M or higher and can be reversed by exposing the junctions to Ca++- and Mg++-free EGTA solutions. Similar changes are obtained in junctions of rat stomach epithelia incubated at 37 degrees C in well-oxygenated Tyrode's solutions containing a Ca++ ionophore (A23187). Deep etching experiments on isolated lens junctions show that the true cytoplasmic surface of the junctions (PS face) is mostly bare, suggesting that the particles may not be connected to cytoskeletal elements. A hypothesis is proposed suggesting a mechanism of particle aggregation and channel narrowing based on neutralization of negative charges by divalent cations or H+.
Organization of the photosystem II centers and their associated antennae in the thylakoid membranes: a comparative ultrastructural, biochemical, and biophysical study of Chlamydomonas wild type and mutants lacking in photosystem II reaction centers.
Authors: Wollman F A , Olive J , Bennoun P , Recouvreur M
Published: May 2004

We investigated the ultrastructure of thylakoid membranes that lacked either some or all of their Photosystem II centers in the F34SU3 and F34 mutants of Chlamydomonas reinhardtii. We obtained the following results: (a) There are no particles of the 160-A size class on the EF faces of the thylakoids in the absence of Photosystem II centers (as in F34); the F34SU3 contains 50% of the wild-type number of PSII centers and EF particles. (b) The density of the particles on the PF faces of the thylakoids is higher in the mutants than in the wild type. (c) The fluorescence analysis shows that the organization of the pigments is the same regardless of whether 50% of the PSII centers are temporarily inactivated (by preilluminating the wild type) or are actually missing from the thylakoid membrane (F34SU3). Our results, therefore, support a model in which: (a) each 160-A EF particle has only one PSII center surrounded by light-harvesting complexes and (b) part of the PSH antenna is associated with 80-A PF particles in both of the mutants and the wild type.
Experimental modulation of occluding junctions in a cultured transporting epithelium.
Authors: Martinez-Palomo A , Meza I , Beaty G , Cereijido M
Published: May 2004

The experimental opening and resealing of occluding junctions in monolayers of cultured MDCK cells (epithelioid of renal origin) was explored by measuring changes in the electrical resistance across the monolayer and by freeze-fracture electron microscopy. As in natural epithelia, the function of occluding junctions as permeability barriers specifically depends on extracellular Ca++ concentration and fails if this ion is replaced by Mg++ or Ba++. The removal of Ca++ and the addition of EGTA to the bathing medium opened the junctions and reduced the transepithelial resistance. Resealing was achieved within 10-15 min by restoring Ca++. Quantitative freeze-fracture electron microscopy showed that junctional opening, caused by lack of Ca++, was accompanied by simplification of the pattern of the membrane strands of the occluding junction without disassembly or displacement of the junctional components. Resealing of the cellular contacts involved the gradual return to a normal junctional pattern estimated as the average number of strands constituting the junction. The occluding junctions were also opened by the addition of the ionophore A23187, suggesting that the sealing of the contacts requires high Ca++ on the extracellular side and low Ca++ concentration of the cytoplasmic compartment. The opening process could be blocked by low temperature (7.5 degrees C). Resealing did not depend on serum factors and did not require protein synthesis; therefore, it seems to be caused by reassembly of preexisting membrane junctional components. The restoration of the junctions occurred simultaneously with the establishment of ion-selective channels; the Na+/Cl- and the cation/cation selectivity were recovered with the same time-course as the electrical resistance. The role of the cytoskeleton in the process of junctional reassembly is reported in the companion article.
Isolation and characterization of Chinese hamster ovary cell variants defective in adhesion to fibronectin-coated collagen.
Authors: Harper P A , Juliano R L
Published: May 2004

Variant clones of Chinese hamster ovary (CHO) cells were selected for reduced adhesion to serum-coated tissue culture plates. These clones also displayed reduced adhesion to substrata composed of collagen layers coated with bovine serum or with fibronectin (cold-insoluble globulin). Wild-type (WT) and adhesion variant (ADv) cells grew at comparable rates in suspension culture, but the adhesion variants could not be grown in monolayer culture because of their inability to attach to the substratum. The adhesion deficit in these cells was not corrected by raising the concentration of divalent cations or of serum to levels 10-fold greater than those normally utilized in cell culture. However, both WT and ADv clones could adhere, spread, and attain a normal CHO morphology on substrata coated with concanavalin A or poly-L-lysine. In addition, the adhesion variants could attach to substrata coated with "footpad" material (substratum-attached material) derived from monolayers of human diploid fibroblasts or WT CHO cells. These observations suggest that the variant clones may have a cell surface defect that prevents them from utilizing exogeneous fibronectin as an adhesion-promoting ligand; however the variants seem to have normal cytoskeletal and metabolic capacities that allow them to attach and spread on substrata coated with alternative ligands. These variants should be extremely useful in studying the molecular basis of cell adhesion.
Visualization of nucleosomes in thin sections by stereo electron microscopy.
Authors: Olins A L , Olins D E , Zentgraf H , Franke W W
Published: May 2004

Nucleosomes (approximately diameter) were clearly visualized in thin sections (approximately 0.1 micrometer thick) of isolated chicken erythrocytes. The cells were lysed and fixed in low ionic strength buffers that maintained the chromatin as dispersed filaments and prevented the reformation of supranucleosomal structures. Stereo electron micrographs at high magnification demonstrate the stability of nucleosome structure in the dispersed chromatin state during fixation, dehydration, and embedding.
Primary structure of diphtheria toxin fragment B: structural similarities with lipid-binding domains.
Authors: Lambotte P , Falmagne P , Capiau C , Zanen J , Ruysschaert J M , Dirkx J
Published: May 2004

Two different lipid-associating domains have been identified in the B fragment of diphtheria toxin using automated Edman degradation of its cyanogen bromide peptides, secondary structure prediction analysis, and comparisons with known phospholipid-interacting proteins. The first domain is located in the highly hydrophilic (polarity index [PI] = 61.0%) 9.00-dalton N-terminal region of fragment B. This region shows primary and predicted secondary structures dramatically similar to those found for the phospholipid headgroup-binding domains of human apolipoprotein A1 (surface lipid-associating domain). The second domain is located in the highly hydrophobic (PI = 32.4%) middle region of fragment B. Its structure resembles that found for the membranous domain of intrinsic membrane proteins (transverse lipid-associating domain). In contrast, the hydrophilic C-terminal 8,000-dalton region of fragment B (PI = 53.8%) does not show structural similarity with lipid-associating domains.
The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement.
Authors: Draper R K , Simon M I
Published: May 2004

Lysosomotropic amines, such as ammonium chloride, are known to protect cells from the cytotoxic effects of diphtheria toxin. These drugs are believed to inhibit the transport of the toxin from a receptor at the cell exterior into the cytoplasm where a fragment of the toxin arrests protein synthesis. We studied the effects of lysosomotropic agents on the cytotoxic process to better understand how the toxin enters the cytoplasm. The cytotoxic effects of diphtheria toxin were not inhibited by antitoxin when cells were preincubated at 37 degrees C with toxin and ammonium chloride, exposed to antitoxin at 4 degrees C, washed to relieve the ammonium chloride inhibition, and finally warmed to 37 degrees C. The antigenic determinants of the toxin were, therefore, either altered or sheltered. It is likely that the combination of ammonium chloride and a low temperature trapped the toxin in an intracellular vesicle from which the toxin could proceed to the cytoplasm. Because lysosomotropic amines raise the pH within acidic intracellular vesicles, such as lysosomes, they could trap the toxin within such a vesicle if an acidic environment were necessary for the toxin to penetrate into the cytoplasm. We simulated acidic conditions which the toxin might encounter by exposing cells with toxin bound to their surface to acidic medium. We then measured the effects of lysosomotropic amines on the activity of the toxin to see if the acidic environment substituted for the function normally inhibited by the drugs. The drugs no longer protected the cells. This suggests that exposing the toxin to an acidic environment, such as that found within lysosomes, is an important step in the penetration of diphtheria toxin into the cytoplasm.
Rat hepatocytes bind to synthetic galactoside surfaces via a patch of asialoglycoprotein receptors.
Authors: Weigel P H
Published: May 2004

The binding of rat hepatocytes to flat polyacrylamide surfaces containing galactose is sugar-specific, requires Ca+2, and occurs only above a critical concentration of sugar in the substratum [Weigel et al., 1979, J. Biol. Chem., 254, 10,830). Binding is completely inhibited by asialo-orosomucoid but not by orosomucoid or asialo-agalacto-orosomucoid, suggesting that cell binding is mediated by asialoglycoprotein receptors. Asialo-orosomucoid was labeled with fluorescein isothiocyanate and used as a direct fluorescent probe to monitor the distribution of cell surface asialoglycoprotein receptors before and after hepatocyte binding to galactoside or control substrata. Cells bound at 37 degrees C were de-adhered at 4 degrees C using the Ca+2 chelator EGTA. The released cells were then stained with fluorescein-asialo-orosomucoid, fixed, washed, and examined by fluorescence microscopy. On freshly isolated cells before binding, the distribution of asialoglycoprotein receptors appears diffuse and nonclustered. However, more than half of the cells released intact from a galactoside surface had a single large (4 micrometer2) fluorescent patch. The receptor patch cannot be detected on cells while they are bound to a galactoside surface but rather only on released cells, indicating that the cell-substratum junction is the site of the receptor patch. No asialoglycoprotein receptor patches (less than or equal to 1%) were observed on cells that were incubated on, but did not bind to, an underivatized polyacrylamide surface or to a surface with a galactose concentration below the critical concentration for binding. Furthermore, no receptor patches were present on cells that had bound to and were subsequently released from substrata that did not contain galactose, including glass, tissue culture plastic, nontissue culture plastic, and collagen. The distribution of asialoglycoprotein receptors is preserved at 4 degrees C because at 37 degrees C the patches disappear with a half-life of approximately 2.6 min. The results directly demonstrate that a large cluster of asialoglycoprotein receptors mediates the binding of rat hepatocytes to a galactoside surface.
Antibodies to neurofilament, glial filament, and fibroblast intermediate filament proteins bind to different cell types of the nervous system.
Authors: Yen S H , Fields K L
Published: May 2004

Antisera were raised to the 210,000-dalton and the 49,000-dalton proteins of a fraction enriched in intermediate (10 nm) filaments from human brain. Proteins of the filament preparation were separated by SDS-polyacrylamide gel electrophoresis and used for immunization and subsequent analysis of the reactions of the sera by rocket immunoelectrophoresis. Anti-210,000-dalton serum precipitated proteins of molecular weights 210,000, 160,000, and 68,000, and, thus, reacted with all the neurofilament triplet components. Anti-49,000-dalton serum did not react with the triplet proteins but precipitated the 49,000-dalton protein. By immunofluorescence on tissue sections, anti-210,000-dalton serum bound to neuronal axons in sciatic nerve and cerebellum. In dissociated cell cultures, rat dorsal root ganglion cells and their processes bound the serum, whereas nonneuronal cells did not. Some cultured cerebellar neurons were also positive, whereas astrocytes were not. At the ultrastructural level, anti-210,000-dalton serum bound to intermediate filaments inside axonal processes. Anti-49,000-dalton serum bound to astrocytes in sections of the cerebellum, and cultured astrocytes had filaments that stained, whereas other cell types did not. In sciatic nerve sections, elements stained with this serum, but cultured cells from newborn sciatic nerve were negative. An antiserum against the 58,000-dalton protein of the cytoskeleton of NIL-8 fibroblasts strongly stained sciatic nerve sections, binding to Schwann cells but not to axons or to myelin. In cerebellar sections, astrocytes were positive, as were blood vessels and cells in the pia. In cell cultures, anti-58,000-dalton serum stained filaments inside Schwann cells, fibroblasts, and astrocytes, but neurons were negative. Cells in the cultures and tissue sections of the nervous system failed to react with antiserum to the 58,000-dalton protein of skin intermediate filaments. In these studies, astrocytes in vivo and in culture were the only cells which had antigens related to two classes of intermediate filaments.
Studies on cell adhesion and recognition. III. The occurrence of α-mannosidase at the fibroblast cell surface, and its possible role in cell recognition
Authors: Rauvala H , Hakomori S
Published: May 2004

The occurrence of α-mannosidase activity at the surface of hamster embryo (NIL) fibroblasts is indicated by the following findings: (a) When NIL cells were incubated on the glass surfaces on which ovalbumin glycopeptides were covalently linked, a rapid release of free mannose from ovalbumin glycopeptides was observed as evidenced by analysis on gas chromatography/mass spectrometry. (b) Cell suspensions as well as intact cell monolayers hydrolyzed rapidly p-nitrophenyl-α-D-mannoside, and the time-course of the hydrolytic cleavage was linear from the moment of mixing of the substrate with the cells. The hydrolysis of the nitrophenyl glycosides of β-D-mannose, α-D-galactose, β-D-galactose, α-L-fucose, β-D-glucose, β-D-N-acetylgalactosamine and β-D-N-acetylglucosamine was negligible or more than ten times lower as compared with the hydolysis of α-D-mannoside. (c) No released or secreted activity of mannosidase could be detected under the conditions used. (d) Studies using known proportions of broken cells in the incubation mixture indicated that more than 90 percent of the mannosidase activity measured was attributable to intact cells and not to broken cells or cell fragments. (e) Hydrolysis of p-nitrophenyl-α-D-mannoside by cell monolayers was inhibited, in the order of decreasing inhibitory activity, by yeast mannan, ovalbumin, α-1,4-L-mannonolactone, α-methylmannoside, and mannose-6-phosphate. High inhibitory activity of the mannan polysaccharide and of ovalbumin favored the presence of the mannosidase activity at the cell surface, as these substrates may not penetrate rapidly into the cells.

The following findings indicated that the cell surface mannosidase is mediating the cell adhesion based on the recognition of high-mannose-type glycopeptide: (a) Ovalbumin- coated plastic surfaces strongly promoted attachment and spreading of NIL fibroblasts, whereas the same ovalbumin coat did not promote attachment and spreading of some other cell types (BALB/c 3T3 fibroblasts and freshly prepared rat liver cells). (b) Digestion of ovalbumin with α-mannosidase greatly reduced the adhesion-mediating activity. (c) Cell adhesion to ovalbumin-coated surfaces was strongly inhibited by mannose tetrasaccharides, moderately by α-1,4-L-mannonolactone, and weakly by α- methylmannoside and mannose-6-phosphate. This order of the inhibitory activity for cell attachment is the same as that for the inhibition of mannosidic hydrolysis. The interpretation that the cell surface mannosidase is able to mediate cell adhesion is in agreement with previous studies suggesting that polyvalent glycosidase surfaces can promote cell adhesion to a degree similar to that caused by fibronectin and several lectins by interacting with their cell surface substrate site (the accompanying papers of this series).
The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N-acetylmannosamine. II. Observations in tissues other than liver.
Authors: Bennett G , Kan F W , O'Shaughnessy D
Published: May 2004

Biochemical evidence from the preceding paper indicated that [3H]N-acetylmannosamine may be used as a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids) in radioautographs of rat liver and duodenum. In order to study the site of incorporation of this label in cell types of various tissues, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of 8 mCi of [3H]N-acetylmannosamine and sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N-acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Light microscope radioautographic analysis revealed that in a great variety of cell types the label was initially localized to the Golgi region. Electron microscope radioautographic analysis of duodenal villous columnar and goblet cells, pancreatic acinar cells and Paneth cells, from rats and mice sacrificed 10 min after injection, showed that the silver grains were localized over Golgi saccules (and adjacent secretion granules). In kidney proximal and distal tubule cells reaction was initially localized to the Golgi apparatus in some areas of the kidney cortex whereas in other areas it was more diffuse. In all cells, the proportion of silver grains over the Golgi apparatus decreased with time after injection while an increasing number of grains appeared over secretion products in secretory cells or over the plasma membrane in other cell types. Lysosomes also became increasingly labeled at later time intervals. The above results suggest that in most cell types sialic acid residues are incorporated into glycoproteins (and perhaps glycolipids), primarily in the Golgi apparatus. With time, these newly synthesized molecules migrate to secretion products, to the plasma membrane, or to the lysosomes.