bluuebunny/crossref_metadata_2025 huggingface dataset:
Five-hour half-life of mouse liver gap-junction protein.
Authors: Fallon R F , Goodenough D A
Published: May 2004

The half-life of a gap-junction polypeptide band migrating at 21,000 Mr on SDS polyacrylamide gels isolated from mouse liver is measured to be 5 h. Two low-molecular wight bands, probably related to the 21,000 Mr material by proteolysis, have measured half-lives of 4.6 and 5.2 h. Gap junctions are labeled in vivo using the 14C-bicarbonate labeling procedure, followed by quantitative fluorography.
Sizing of protein A-colloidal gold probes for immunoelectron microscopy.
Authors: Slot J W , Geuze H J
Published: May 2004

Gold particles in colloidal solutions often vary considerably in size. The finest sols (diameter less than 15 nm), especially, are very heterogeneous, as is indicated by coefficients of variance (CV) of 25-35%. We have complexed staphylococcal protein A with gold particles (PA/Au) and then fractionated the preparations by glycerol or sucrose gradient centrifugation into very homogeneous subfractions. In this way, PA/Au probes of almost any size between 4.5 and 15 nm could be prepared. The variation of the gold particles in these fractions resulted in CV's between 9 and 16%. The reactivity of the PA/Au complex was not affected by the gradient procedure, as was shown by single- and double-labeling immunocytochemistry of ultrathin cryosections of rat pancreatic tissue.
Calcium regulates the commitment of murine erythroleukemia cells to terminal erythroid differentiation.
Authors: Bridges K , Levenson R , Housman D , Cantley L
Published: May 2004

An alteration in the rate of calcium transport appears to be the rate-limiting event for the commitment of murine erythroleukemia (MEL) cells to initiate a program of terminal erythroid differentiation. The dimethyl sulfoxide (DMSO)-induced commitment of MEL cells to erythroid differentiation can be inhibited by treatment of cells with the calcium-chelating agent EGTA. Upon removal of EGTA, cells initiate commitment without the 12-h lag normally observed after treatment with DMSO alone. Treatment of cells with DMSO in the presence of calcium ionophore A23187 causes cells to initiate commitment from time zero with no lag. These results suggest that the lag is the time required for DMSO to alter the calcium transport properties of the cell.
Specification of cell morphology by endogenous determinants.
Authors: Solomon F
Published: May 2004

We are studying the mechanisms by which cells elaborate their differentiated morphologies. Here we discuss one aspect of this issue: the specification of the detailed shape of individual cells. We describe an experimental system in which endogenous determinants of morphology are expressed. These determinants originally were detected in the morphological relationships between sister neuroblastoma cells. Approaches to analyzing these relationships are presented. The properties and behavior of the endogenous determinants have been partially characterized by further experiments, which are also described. The significance and the prospects for further analysis of our findings are discussed.
Cytoplasmic vacuolation of mouse peritoneal macrophages and the uptake into lysosomes of weakly basic substances.
Authors: Ohkuma S , Poole B
Published: May 2004

With few exceptions, weakly basic compounds that are sufficiently lipophilic in their neutral forms and sufficiently hydrophilic in their protonated forms accumulate in lysosomes. When the concentration within the lysosomes becomes sufficiently high, osmotic swelling occurs. The cells than take on a vacuolated appearance. The concentrations at which different weak bases cause lysosomal vacuolation vary over almost three orders of magnitude. For any particular weak base, it is the concentration of the neutral form that determines the extent of uptake and the degree of vacuolation. Chloroquine is anomalous in that concentrations greater than approximately 30 microM cause less uptake and less vacuolation than do lower concentrations.
Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages.
Authors: Poole B , Ohkuma S
Published: May 2004

The spectral characteristics of dextran, labeled with fluorescein, depend upon pH. We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions. The pH of the medium has no effect on the intralysosomal pH. Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH. However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form. Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH. This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A. The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.
Galactose-specific recognition system of mammalian liver: receptor distribution on the hepatocyte cell surface.
Authors: Wall D A , Hubbard A L
Published: May 2004

An isolated perfused liver system was used to study the distribution of asialoglycoprotein (ASGP) binding sites on rat hepatocyte cell surfaces. The number of surface receptors was quantitated by monitoring clearance of 125I-labeled ligands from the perfusate medium under two conditions that blocked their internalization: low temperature (less than 5 degrees C) or brief formaldehyde fixation. The cell surface distribution of binding sites was visualized in the electron microscope with either asialoorosomucoid covalently coupled to horseradish peroxidase (ASOR-HRP) or lactosaminated ferritin (Lac-Fer), both of which were bound with similar kinetics and to similar extents as ASOR itself. At low temperature or after prefixation, ASGP binding sites were present over much of the sinusoidal cell surface, but were concentrated most heavily over coated pits. Quantitation of ligand distribution at 4 degrees C with Lac-Fer gave an approximately 70-fold greater density of ferritin particles over coated membrane than over uncoated regions. We obtained no evidence for gradual movement of ASGP receptors into or out of coated pits within the time-course of our experiments. Finally, the number and distribution of cell surface binding sites was unaffected by previous exposure to ASOR or by inhibition of endocytic vesicle-lysosome fusion and ASOR degradation at 16 degrees C.
Involucrin synthesis and tissue assembly by keratinocytes in natural and cultured human epithelia.
Authors: Banks-Schlegel S , Green H
Published: May 2004

Different stratified squamous epithelia, whether they bear a stratum corneum or not, are shown by immunofluorescence to possess the precursor protein of the cross-linked envelope that is characteristic of epidermal s. corneum. This protein, involucrin, is not present in the deepest epithelial cells but appears in the course of their outward migration. The boundary at which involucrin first appears can sometimes by correlated with a visible boundary between zones of large and small cells. Cultured keratinocytes, derived from all stratified squamous epithelia (epidermal, corneal, conjuctival, esophageal, lingual, and vaginal), form colonies that grow together to form a stratified epithelium. The cells of the basal layer are nearly always free of detectable involucrin, but, in contrast to the natural epithelium, this protein usually makes its appearance in the cells immediately above the basal layer. When a cultured epithelium derived from epidermal keratinocytes is detached and applied as a graft to animals, the cells flatten and the distinctness of the basal layer is at first reduced; but with time the organization of the epithelium becomes more characteristic of epidermis. Cell size and shape become more orderly along the cell migration pathway, and involucrin first appears at some distance from the basal layer, instead of in immediately suprabasal cells, as in the cultured epithelium. The progeny of dissociated and cultured keratinocytes are therefore able, when grafted, to reassemble an epidermis in which the timing of specific gene expression is restored to that of the original tissue.
Preferential phosphorylation of the 150,000 molecular weight component of neurofilaments by a cyclic AMP-dependent, microtubule-associated protein kinase.
Authors: Leterrier J F , Liem R K , Shelanski M L
Published: May 2004

Highly purified preparations of bovine brain and rabbit nerve root neurofilaments were found to be lacking in protein kinase activity when either histone FIIA or the neurofilaments themselves were used as acceptors. There was no augmentation of activity in the presence of cyclic AMP. Addition of microtubule proteins prepared by cycles of assembly and disassembly resulted in phosphorylation of histone, phosphorylation of tubulin and the microtubule-associated proteins, and phosphorylation of neurofilament subunits. The phosphorylation of neurofilaments was predominantly in the 150,000-dalton species and was completely cyclic AMP dependent.
Intracellular Na+:K+ ratios in human cancer cells as revealed by energy dispersive x-ray microanalysis.
Authors: Nagy I Z , Lustyik G , Nagy V Z , Zarándi B , Bertoni-Freddari C
Published: May 2004

Intranuclear sodium, potassium, and chloride contents were measured by energy-dispersive x-ray microanalysis in freeze-fractured, freeze-dried, bulk-tumor samples taken from 10 patients suffering from invasive urogenital cancers. Human biopsies were carried out during the first diagnostic interventions before any cytostatic treatment had been applied. Pathohistological diagnosis established the malignancy in each case. The cancers were classified in three types: keratinizing, transitional cell, and hypernephroid carcinoma. More than 250 cell nuclei were measured from each type of cancer. The results were compared with those obtained in intact human urothelium taken from patients having no malignant processes. Proximal and distal tubular epithelial cell nuclei representing the origin of human hypernephroid cancer were also measured in rat kidney because corresponding healthy human material cannot be obtained. The analyses revealed, in all three types of cancer cells, that the average intranuclear sodium content increased more than three-fold, the potassium content decreased 32, 16, and 13%, respectively; meanwhile the chloride content increased, but to a lesser extent than did the sodium. The intranuclear Na+:K+ ratios were more than five-fold higher in the cancer cells on the average, and their distribution histograms were much broader than in the normal human urothelium and in the tubular cell nuclei of the rat kidney. The results obtained fit well with the theory of Cone, C. D., Jr. 1971. J. Theor. Biol. 30: 151-181 according to which the sustained depolarization of the cell membrane may be of mitogenic effect.
Cytoplasmic actin in postsynaptic structures at the neuromuscular junction.
Authors: Hall Z W , Lubit B W , Schwartz J H
Published: May 2004

We used an antibody prepared against Aplysia (mollusc) body-wall actin that specifically reacts with certain forms of cytoplasmic actin in mammalian cells to probe for the presence of actin at the neuromuscular junction. Immunocytochemical studies showed that actin or an actinlike molecule is concentrated at neuromuscular junctions of normal and denervated adult rat muscle fibers. Actin is present at the neuromuscular junctions of fibers of developing diaphragm muscles as early as embryonic day 18, well before postsynaptic folds are formed. These results suggest that cytoplasmic actin may play a role in the clustering or stabilization of acetylcholine receptors at the neuromuscular junction.
Detection of actin-binding proteins in human platelets by 125I-actin overlay of polyacrylamide gels.
Authors: Snabes M C , Boyd A E , Bryan J
Published: May 2004

Actin-binding proteins have been identified in human platelets with a gel-overlay technique that uses 125I-G-actin. Platelet proteins were separated on SDS polyacrylamide gels using the buffer system of Laemmli (1970, Nature [Lond.] 227:680-685). The proteins were fixed in the gels with methanol-acetic acid, the SDS was washed out, and the proteins were renatured. The gels were incubated with 125I-G-actin from rabbit skeletal muscle that was radiolabeled with 125I according to the method of Bolton and Hunter (1973, Biochem. J. 133:529-538) and has been shown to retain biological activity. After nonspecifically bound radioactivity was washed out, gels were dried and processed for autoradiography. The 125I-G-actin binds to several proteins in human platelets, platelet extracts, and the particulate fraction. Control experiments demonstrate that the 125I-G-actin can be displaced by use of increasing amounts of unlabeled actin, that the binding is stable to 0.6 M NaCl, and that preheating the 125I-G-actin to 90 degrees C for 3 min eliminates all binding. Prominent 125I-G-actin-binding activities were present at Mr 90,000 and 40,000. The binding to the 90,000 Mr protein appears to be at least partially Ca++ sensitive, whereas the binding to the 40,000 Mr protein does not. 125I-G-actin bound to proteins in the SDS gels can be fixed in situ and compared directly with the stained gel. This technique should prove generally useful in identification and purification of some actin-binding proteins from cells and tissues.
Biochemical studies of the excitable membrane of Paramecium tetraurelia VI. Endogenous protein substrates for in vitro and in vivo phosphorylation in cilia and ciliary membranes.
Authors: Lewis R M , Nelson D L
Published: May 2004

The endogenous protein kinases of isolated Paramecium tetraurelia cilia phosphorylated approximately 30 ciliary polypeptides in vitro. Labeling with [gamma-32P]ATP was not proportional to the amount of each protein in cilia; some minor polypeptides (e.g., 67,000 and 180,000 mol wt) were more heavily labeled than some major polypeptides. Certain of the endogenous substrates for protein kinase were localized in the ciliary membrane (130,000, 86,000, 67,000, and 45,000 mol wt); others were found in axonemes or in both fractions. With cilia from bacterized cultures in the undefined Cerophyl medium, the labeling of specific endogenous phosphate acceptors was altered by pH, cyclic AMP, and cyclic GMP, but the labeling pattern was not affected by the presence of Na+ or K+ (15 mM), Ba++ (5 mM), Ca++ (10(-5) or 10(-4) M), or EGTA. Very similar results were obtained with cilia from cells grown axenically in a semidefined medium; the molecular weights and the extent of phosphorylation of the phosphopolypeptides were comparable to those of cilia from bacterized Cerophyl cultures, although no significant cyclic nucleotide effects were observed in the axenic cilia. Most of the phosphopolypeptides labeled in vitro also turned over rapidly in vitro. The phosphoprotein phosphatase responsible for turnover was partially inhibited by 5 mM NaF. The pattern of ciliary polypeptides labeled in vivo was similar to that observed in the in vitro experiments, although the relative intensities of labeling differed. Six behavioral mutants of Paramecium, known to have defects in the excitable membrane that regulates the ciliary beat, showed normal patterns of ciliary protein phosphorylation in vitro, with and without added cyclic nucleotides, at both pH 6.0 and pH 8.0. The mutants also had apparently normal phosphoprotein phosphatase. The Paranoiac A mutant, however, showed a reduction in cyclic GMP-stimulated protein kinase activity.
Isolation and characterization of actin and actin-binding protein from human platelets.
Authors: Rosenberg S , Stracher A , Lucas R C
Published: May 2004

Human blood platelets, which are highly motile cells essential for the maintenance of hemostasis, contain large quantities of actin and other contractile proteins. We have previously introduced a method (Lucas, R. C., T. C. Detwiler, and A. Stracher, J. Cell Biol., 1976, 70(2, Pt. 2):259 a) for the quantitative recovery of the platelets' cytoskeleton using a solution containing 1% Triton X-100 and 10 mM EGTA. This cytoskeleton contains most of the platelets' actin, actin-binding protein (ABP, subunit molecular weight = 260,000), and a 105,000-dalton protein. Negative staining of this Triton-insoluble residue on an EM grid shows it to consist of branched cables of actin filaments aligned in parallel. When this cytoskeletal structure is dissolved in high-salt solutions, the actin and ABP dissociate and can subsequently be separated. Here we will present simple and rapid methods for the individual purifications of platelet actin and platelet ABP. When purified actin and ABP are recombined in vitro, they are shown to be both necessary and sufficient for the reformation of the cytoskeletal complex. The reformed structure is visualized as a complex array of fibers, which at the EM level are seen to be bundles of actin filaments. The reformation of the cytoskeleton requires only that the actin be in the filamentous form--no accessory proteins, chelating agents, divalent cations, or energy sources are necessary. In vivo, however, the state of assembly of the platelets' cytoskeleton appears to be under the control of the intracellular concentration of free calcium. Under conditions where proteolysis is inhibited and EGTA is omitted from the Triton-solubilization step, no cytoskeleton can be isolated. The ability of Ca+2 to control the assembly and disassembly of the platelets' cytoskeleton provides a mechanism for cytoskeletal involvement in shape change and pseudopod formation during platelet activation.
Alterations of new methylated phospholipid synthesis in the plasma membranes of macrophages exposed to chemoattractants.
Authors: Pike M C , Snyderman R
Published: May 2004

Chemotactic factors have been shown to inhibit the methylation of phosphatidylethanolamine in macrophages without affecting total phospholipid synthesis. It would thus be anticipated that newly synthesized membranes of macrophages exposed to chemoattractants would have an increased ratio of phosphatidylethanolamine to its methylated derivatives. These ratios were measured directly in newly synthesized phospholipids of plasma membranes isolated from guinea pig peritoneal macrophages. The phosphatidylethanolamine: methylated phospholipid ratio in such plasma membranes was increased by 53 to 111% upon exposure of the cells to chemotactic factors. This increase was due to decreased synthesis of methylated phospholipids and not to altered formation of phosphatidylethanolamine or activation of phospholipases. Methylated phospholipid ratios were also studied in the leading front lamellipodia isolated from macrophages migrating under chemotactic and nonchemotactic conditions. The phosphatidylethanolamine:methylated phospholipid ratios were increased up to fourfold in lamellipodia of macrophages migrating towards chemotactic agents when compared to those from cells migrating randomly. Biophysical changes in the plasma membrane produced by an increase in the ratio of phosphatidylethanolamine:methylated phospholipids as a result of exposure of cells to chemoattractants may be required for sustained directed migration.
Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin
Authors: Rossomando EF , Jahngen EG , Varnum B , Soll DR
Published: May 2004

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium.

Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation.

Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium.

In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity.

After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated.

These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.
Sperm morphogenesis in wild-type and fertilization-defective mutants of Caenorhabditis elegans.
Authors: Ward S , Argon Y , Nelson G A
Published: May 2004

Taking advantage of conditions that allow spermatogenesis in vitro, the timing and sequence of morphological changes leading from the primary spermatocyte to the spermatozoon is described by light and electron microscopy. Together with previous studies, this allows a detailed description of the nuclear, cytoplasmic, and membrane changes occurring during spermatozoan morphogenesis. By comparison with wild type, abnormalities in spermatogenesis leading to aberrant infertile spermatozoa are found in six fertilization-defective (fer) mutants. In fer-1 mutant males, spermatids appear normal, but during spermiogenesis membranous organelles (MO) fail to fuse with the sperm plasma membrane and a short, though motile. pseudopod is formed. In fer-2, fer-3, and fer-4 mutants, spermatids accumulate 48-nm tubules around their nuclei where the centriole and an RNA containing perinuclear halo would normally be. In all three mutants, spermatids still activate to spermatozoa with normal fusion of their MOs, but the pseudopods formed are aberrant in most fer-2 and fer-4 spermatozoa and in some fer-3 spermatozoa. In fer-5 mutant males, spermatozoa do not form. Instead, defective spermatids with crystalline inclusions and abnormal internal laminar membranes accumulate. In fer-6 mutant males, only a few spermatozoa form and these have defective pseudopods. These spermatozoa retain their fibrous bodies, a structure which normally disassembles in the spermatid. The time of appearance of developmental abnormalities in all of these mutants correlates with the temperature-sensitive periods for development of infertility. The observation that each of these mutants has a different and discreet set of morphological defects, a structure which normally disassembles in the spermatid. The time of appearance of developmental abnormalities in all of these mutants correlates with the temperature-sensitive periods for development of infertility. The observation that each of these mutants has a different and discreet set of morphological defects, a structure which normally disassembles in the spermatid. The time of appearance of developmental abnormalities in all of these mutants correlates with the temperature-sensitive periods for development of infertility. The observation that each of these mutants has a different and discreet set of morphological defects shows that the strict sequence of morphogenetic events that occurs during wild-type spermatogenesis cannot arise because each event is dependent on previous events. Instead, spermatozoa, like bacteriophages, must be formed by multiple independent pathways of morphogenesis.
Evidence for the sorting of endocytic vesicle contents during the receptor-mediated transport of IgG across the newborn rat intestine.
Authors: Abrahamson D R , Rodewald R
Published: May 2004

Fc receptors on the luminal membranes of intestinal epithelial cells in the neonatal rat mediate the vesicular transfer of functionally intact IgG from the intestinal lumen to the circulation. In addition, there is a low level of nonselective protein uptake, but in this case transfer does not occur. To determine whether a specialized class of endocytic vesicles could account for the selective transfer of IgG, mixtures of IgG conjugated to ferritin (IgG-Ft) and unconjugated horseradish peroxidase (HRP) were injected together into the proximal intestine of 10-d-old rats, and the cellular distribution of these two different tracers was determined by electron microscopy. Virtually all apical endocytic vesicles contained both tracers, indicating simultaneous uptake of both proteins within the same vesicle. However, only IgG-Ft bound to the apical plasma membrane, appeared within coated vesicles at the lateral cell surface, and was released from cells. HRP did not bind to the luminal membrane and was not transferred across cells but was confined to apical lysosomes as identified by acid phosphatase and aryl sulfatase activities. To test the possibility that the binding of IgG to its receptor stimulated endocytosis, HRP was used as a fluid volume tracer, and the amount of HRP taken up by cells in the presence and absence of IgG was measured morphologically and biochemically. The results demonstrate that endocytosis in these cells is constitutive and occurs at the same level in the absence of IgG. The evidence presented indicates that the principal selective mechanism for IgG transfer is the binding of IgG to its receptor during endocytosis. Continued binding to vesicle membranes appears to be required for successful transfer because unbound proteins are removed from the transport pathway before exocytosis. These results favor the proposal that IgG is transferred across cells as an IgG-receptor complex.
Induction of polarity in mouse 8-cell blastomeres: specificity, geometry, and stability.
Authors: Johnson M H , Ziomek C A
Published: May 2004

We studied the cellular mechanisms underlying the induction of polarity in individual blastomeres of the 8-cell mouse embryo. The ability to induce polarity is lacking in the membranes of unfertilized and newly fertilized mouse eggs, then develops during the 2-cell stage, and is present in membranes of cells from 4-, 8-, and 16-cell stages. The axis of polarity takes 3-5 h to become established and thereafter appears to be stable. Multiple cell contacts affect the orientation of the axis of polarity, and no polarity develops in cells which are totally surrounded. Polarized cells show evidence of an limited capacity for slight adjustments in their position relative to other cells. The implications of these results for the mechanisms by which a blastocyst is generated are discussed briefly.
Response of basal epithelial cell surface and Cytoskeleton to solubilized extracellular matrix molecules.
Authors: Sugrue S P , Hay E D
Published: May 2004

Corneal epithelium removed from underlying extracellular matrix (ECM) extends numerous cytoplasmic processes (blebs) from the formerly smooth basal surface. If blebbing epithelia are grown on collagen gels or lens capsules in vitro, the basal surface flattens and takes on the smooth contour typical of epithelium in contact with basal lamina in situ. This study examines the effect of soluble extracellular matrix components on the basal surface. Corneal epithelia from 9- to 11-d-old chick embryos were isolated with trypsin-collagenase or ethylenediamine tetraacetic acid, then placed on Millipore filters (Millipore Corp., Bedford, Mass.), and cultured at the medium-air interface. Media were prepared with no serum, with 10% of calf serum, or with serum from which plasma fibronectin was removed. Epithelia grown on filters in this medium continue to bleb for the duration of the experiments (12-14 h). If soluble collagen, laminin, or fibronectin is added to the medium, however, blebs are withdrawn and by 2-6 h the basal surface is flat. Epithelia grown on filters in the presence of albumin, IgG, or glycosaminoglycans continue to bleb. Epithelia cultured on solid substrata, such as glass, also continue to bleb if ECM is absent from the medium. The basal cell cortex in situ contains a compact cortical mat of filaments that decorate with S-1 myosin subfragments; some, if not all, of these filaments point away from the plasmalemma. The actin filaments disperse into the cytoplasmic processes during blebbing and now many appear to point toward the plasmalemma. In isolated epithelia that flatten in response to soluble collagens, laminin, and fibronectin, the actin filaments reform the basal cortical mat typical or epithelial in situ. Thus, extracellular macromolecules influence and organize not only the basal cell surface but also the actin-rich basal cell cortex of epithelial cells.
Central-pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants.
Authors: Adams G M , Huang B , Piperno G , Luck D J
Published: May 2004

Four mutants of Chlamydomonas reinhardtii representing independent gene loci have been shown to lack totally (pf-18, pf-19, and pf-15) or nearly totally (pf-20) the central microtubular pair complex in isolated axonemal preparations. Analysis of 35S-labeled axonemal proteins, using two methods of electrophoresis, reveals that all four mutants lack or are markedly deficient in 18 polypeptides, ranging in molecular weight from 360,000 to 20,000, that are regularly present in wild-type axonemes. Analyses of axonemal proteins labeled by cellular growth on 32P-labeled medium indicates that a subset of 8 of the 18 polypeptides are phosphorylated. Mutant and wild-type axonemes and flagella have been analyzed for their content of tubulin subunits using a high resolution two-dimensional electrophoresis system combined with agarose gel overlays containing either anti-alpha or anti-beta tubulin sera prepared from Chlamydomonas tubulins. The immunoprecipitates identify two major alpha tubulins, a major beta tubulin, and a minor component which is also precipitated by the anti-beta serum. None of these tubulins shows a specific defect in mutant axonemes, nor do the tubulin polypeptides show altered two-dimensional map positions in the mutant flagella. The 18 polypeptides provide a useful signature for identifying other mutants affecting the central-pair microtubular complex. Such mutants could be useful in defining the structural or functional role of these polypeptides in the central microtubules. Efforts to obtain additional central-pair mutants based on the motility phenotype of the four mutants analyzed here have yielded mutants which are allelic to three of the four mutants.
Crystalline actin sheets: their structure and polymorphism.
Authors: Aebi U , Fowler W E , Isenberg G , Pollard T D , Smith P R
Published: May 2004

Crystalline sheets of Acanthamoeba actin induced by the trivalent lanthanide gadolinium exist in three different polymorphic forms, which show different striation patterns and surface topographies. We have called these different forms "rectangular" and "square" sheets, and "cylinders" and have shown that each of the three forms is constructed from common "basic" lattices associated in different ways. We have used image processing of electron micrographs to obtain a model for the actin molecule in projection to a resolution of 1.5 nm. The overall dimensions observed in these images are 5.6 x 3.3 x 4.5 nm, and the molecule itself appears distinctly bilobed with the two lobes separated by a cleft. actin monomers in the sheets are arranged with P2 symmetry and are therefore packed in a manner different from that of the molecules in actin filaments. Because approximately 35% of the surface area of the actin molecule is exposed on the surface of these sheets, the sheets should be useful to study the stoichiometric binding of actin-binding proteins to the actin molecule.
Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins.
Authors: Kaslow H R , Groppi V E , Abood M E , Bourne H R
Published: May 2004

Cholera toxin catalyzes transfer of radiolabel from [32P]NAD+ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of Mr = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (Mr = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and [32P]NAD+ caused radiolabeling of purified microtubule and intermediate filament proteins.
Structural features of alveolar wall basement membrane in the adult rat lung.
Authors: Vaccaro C A , Brody J S
Published: May 2004

The ultrastructural characteristics of alveolar (ABM) and capillary (CBM) basement membranes in the adult rat lung have been defined using tannic acid fixation, ruthenium red staining, or incubation in guanidine HCl. ABM is dense and amorphous, has 3- to 5-nm filaments in the lamina rara externa (facing the alveolus) that run between the lamina densa and the basal cell surface of the epithelium, has an orderly array of ruthenium red-positive anionic sites that appear predominantly (79%) on the lamina rara externa, and has discontinuities beneath alveolar type II cells but not type I cells that allow penetration of type II cytoplasmic processes into the interstitium of the alveolar wall. The CBM is fibrillar and less compact than ABM, has no lamina rara filaments, and has one fifth the number of ruthenium red-positive anionic sites of ABM that appear predominantly (64%) overlying the lamina densa. Incubation of lung tissue with Flavobacterium heparinum enzyme or with chondroitinase has shown that ABM anionic sites represent heparan sulfate proteoglycans, whereas CBM anionic sites contain this and other sulfated proteoglycans. The CBM fuses in a local fashion with ABM, compartmentalizing the alveolar wall into a thick and thin side and establishing a thin, single, basement-membrane gas-exchange surface between alveolar air, and capillary blood. The potential implications of ABM and CBM ultrastructure for permeability, cell differentiation, and repair and morphogenesis of the lung are discussed.
Immunoelectron microscopic localization of the 210,000-mol wt microtubule-associated protein in cultured cells of primates.
Authors: De Brabander M , Bulinski J C , Geuens G , De Mey J , Borisy G G
Published: May 2004

Results from ultrastructural immunocytochemistry on glutaraldehyde-fixed cells confirmed and extended findings previously obtained with immunofluorescence. A microtubule-associated protein (MAP) of 210,000 molecular weight was shown to be specifically associated with all cytoplasmic and mitotic microtubules along their entire length in primate cells. Specific labeling with the anti-MAP antibody could not be detected on any other subcellular structures, notably the centrosomes, kinetochores, microfilaments, and intermediate filaments. Treatment with the microtubule-disrupting drug, nocodazole, induced diffusion of the MAP throughout the cytoplasm. During repolymerization of microtubules following disassembly by nocodazole, the association of the MAP with the microtubules was intermediate and complete. When cells were treated with vinblastine, the tubulin paracrystals formed were heavily stained by the antibody. Neither sodium azide nor taxol affected the association of the MAP with microtubules.
Polymorphonuclear leukocyte migration through human amnion membrane
Authors: Russo RG , Liotta LA , Thorgeirsson U
Published: May 2004

A new in vitro model has been developed for studying migration of human polymorphonuclear leukocytes (PMN) through living native cellular and matrix barriers. Human amnion membrane consists of a single layer of epithelium bound to a continuous basement membrane interfacing an avascular collagenous stroma. Living amnion was placed in plastic chambers with separate compartments on each side of the membrane. PMN were introduced on the epithelial side of the amnion, and a Millipore filter (Millipore Corp., Bedford, Mass.) was placed against the stromal side. In response to N-formylmethionyl-leucyl- phenylanlanine (FMLP) chemoattractant, PMN penetrated the full thickness of the amnion and were collected and counted on the filter. The rate of PMN traversal of the amnion was dependent on the concentration of FMLP (optimal at 10(-8)M) as well as the slope of the FMLP gradient across the amnion. The route of PMN migration was studied by transmission electron microscopy. PMN first attached to the epithelial surface, then infiltrated between intercellular junctions. PMN migrated around or through tight junction and hemidesmosome attachments. The PMN then penetrated the basement membrane and migrated through the dense collagenous stroma.

The present amnion migration system has characteristics of the in vivo inflammatory state not described in any previous method for monitoring PMN migration in vitro. Prior methods have not used native epithelium, whole basement membrane, or collagenous stroma. PMN penetration of these barriers occurs in the normal inflammatory response and probably involves biochemical mechanisms not required for simple migration through the pores of an artificial filter. The amnion system can be useful for future biochemical and morphological studies of PMN penetration of these barriers and possible repair processes that may follow.
Taxol binds to polymerized tubulin in vitro
Authors: Parness J , Horwitz SB
Published: May 2004

Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5'-guanylyl-α,β-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.
Differentiation of troponin in cardiac and skeletal muscles in chicken embryos as studied by immunofluorescence microscopy.
Authors: Toyota N , Shimada Y
Published: May 2004

The differentiation of troponin (TN) in cardiac and skeletal muscles of chicken embryos was studied by indirect immunofluorescence microscopy. Serial sections of embryos were stained with antibodies specific to TN components (TN-T, -I, and -C) from adult chicken cardiac and skeletal muscles. Cardiac muscle began to be stained with antibodies raised against cardiac TN components in embryos after stage 10 (Hamburger and Hamilton numbering, 1951, J. Morphol. 88:49-92). It reacted also with antiskeletal TN-I from stage 10 to hatching. Skeletal muscle was stained with antibodies raised against skeletal TN components after stage 14. It also reacted with anticardiac TN-T and C from stage C from stage 14 to hatching. It is concluded that, during embryonic development, cardiac muscle synthesizes TN-T and C that possess cardiac-type antigenicity and TN-I that has antigenic determinants similar to those present in cardiac as well as in skeletal muscles. Embryonic skeletal muscle synthesizes TN-I that possesses antigenicity for skeletal muscle and TN-T and C which share the antigenicities for both cardiac and skeletal muscles. Thus, in the development of cardiac and skeletal muscles, a process occurs in which the fiber changes its genomic programming: it ceases synthesis of the TN components that are immunologically indistinguishable from one another and synthesizes only tissue-type specific proteins after hatching.
Effects of cytochalasin B on membrane-associated microfilaments in a cell-free system.
Authors: Phillips M J , Oda M , Yousef I M , Funatsu K
Published: May 2004

The mode of action of cytochalasin B was examined in vitro using bile canaliculus-enriched plasma membrane fractions isolated from rat liver. The pericanalicular microfilaments, which are mainly actin filaments and which are normally attached to the canalicular membranes, were dissociated from the membranes by cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin-treated specimens and a number of polypeptides, of which a polypeptide corresponding in molecular weight to actin was a notable member. These results suggest that actin filaments become detached from the canaliculus membranes by cytochalasin B.
Filamentous structures in adherent Mycoplasma pneumoniae cells treated with nonionic detergents.
Authors: Göbel U , Speth V , Bredt W
Published: May 2004

Mycoplasma pneumoniae cells adhering to glass or Parlodion-coated grids were extracted with Triton X-100. The extracted cells showed a cytoskeleton consisting of a rodlike tip structure and a filamentous network in the cytoplasm. The tip structure was up to 300 nm long and approximately 40 nm wide ending at the distal end in a bleb-like structure, and seemed to consist of filaments arranged in parallel, 4.8 +/- 0.5 nm wide. In the cytoplasm the filaments formed an irregular lattice. Similar filaments were found in platinum replicated critical-point dried extracted cells. An actinlike nature of the filaments is suggested by some of their properties, but the degree of homology with respect to eucaryotic actin is still unknown. The filaments were sensitive to protease treatment but stable in high molar KCl solutions. They were apparently destroyed by incubation in high molar KI solution, leaving only some parts of the tip structure. Formaldehyde-fixed M. pneumoniae cells treated with Triton X-100 bound rhodamine-labeled phalloidin specifically. Furthermore, they could be stained with antiactin antibodies. Binding of myosin subfragment 1 to the filaments was not observed.
Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein.
Authors: Walter P , Ibrahimi I , Blobel G
Published: May 2004

An 11S protein composed of six polypeptide chains was previously purified from a salt extract of dog pancreas microsomal membranes and shown to be required for translocation of nascent secretory protein across the microsomal membrane (Wistar and Blobel 1980 Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). This 11S protein, termed signal recognition protein (SRP), has been shown here (a) to inhibit translation in the wheat germ cell-free system selectively of mRNA for secretory protein (bovine preprolactin) but not of mRNA for cytoplasmic protein (alpha and beta chain of rabbit globin); (b) to bind with relatively low affinity (apparent KD less than 5 x 10(-5)) to monomeric wheat germ ribosomes; and (c) to bind selectively and with 6,000-fold higher affinity (apparent KD less than 8 x 10(-9)) to wheat germ ribosomes engaged in the synthesis of secretory protein but not to those engaged in the synthesis of cytoplasmic protein. Low- and high-affinity binding as well as the selective translation-inhibitory effect were abolished after modification of SRP by N-ethyl maleimide. High-affinity binding and the selective translation-inhibitory effect of SRP were largely abolished when the leucine (Leu) analogue beta-hydroxy leucine was incorporated into the nascent secretory polypeptide.
Externalization of an endogenous chicken muscle lectin with in vivo development.
Authors: Barondes S H , Haywood-Reid P L
Published: May 2004

Chicken-lactose-lectin-I (CLL-I), an endogenous lectin that is developmentally regulated in embryonic muscle, was localized by immunohistochemical techniques in tissue samples taken at various stages of in vivo development and in primary muscle cultures. Lectin, which was diffusely distributed in myoblasts, became localized in myotubes in a distribution similar to that of the sarcoplasmic reticulum and T tubules. Later in development, lectin was predominantly extracellular. This sequence suggests that externalization may have occurred by migration in the T tubules, which are continuous with the extracellular space, although alternative explanations are possible. Only traces of lectin were found in the adult. These studies did not reveal the function of CLL-I in muscle development. However, we infer that it acts by organizing complementary glycoconjugates in the intracellular tubular network, on the muscle surface, and/or in extracellular materials.
Rapid and reversible reduction of junctional permeability in cells infected with a temperature-sensitive mutant of avian sarcoma virus.
Authors: Atkinson M M , Menko A S , Johnson R G , Sheppard J R , Sheridan J D
Published: May 2004

The transformed or normal phenotype of cultured normal rat kidney cells infected with a temperature-sensitive mutant of avian sarcoma virus is conditional on the temperature at which the cells are grown. Using dye injection techniques, we show that junction-mediated dye transfer is also temperature-sensitive. The extent and rate of transfer between infected cells grown at the transformation-permissive temperature (35 degrees C) is significantly reduced when compared to infected cells grown at the nonpermissive temperature (40.5 degrees C) or uninfected cells grown at either temperature. Infected cells subjected to reciprocal temperature shifts express rapid and reversible alterations of dye transfer capacities, with responses evident by 15 min and completed by 60 min for temperature shifts in either direction. These results suggest that altered junctional capacities may be fundamental to the expression of the ASV-induced, transformed phenotype.