bluuebunny/crossref_metadata_2025 huggingface dataset:
A HISTOCHEMICAL METHOD FOR DISTINGUISHING BETWEEN SIDE-CHAIN AND TERMINAL (α-ACYLAMIDO) CARBOXYL GROUPS OF PROTEINS
Authors: Karnovsky Morris J. , Fasman Gerald D.
Published: May 2004

The specificity of the Barrnett-Seligman method for the histochemical demonstration of α-acylamido carboxyl groups (C terminal) of proteins is dependent on the conversion of such groups to ketones by the action of acetic anhydride and absolute pyridine. Studies on model compounds show that the side-chain carboxyl groups also react in the method and that most of the final color developed can be attributed to these carboxyls, rather than to the C terminal carboxyl groups. It is postulated that the side-chain carboxyls react by formation of mixed anhydrides in the presence of acetic anhydride and pyridine. This mixed anhydride then could link with a hydrazide to form a dihydrazide, which is capable of coupling with a diazo dye. Acetic anhydride treatment alone, without pyridine, also yields mixed anhydride. The mixed anhydride derived from the side-chain carboxyls can be destroyed by base, whereas the methyl ketone derived from the C terminal carboxyl is unaffected, and this treatment makes the method specific for C terminal carboxyl groups. Tissues treated in such a fashion demonstrate that all the color reaction obtained in the method is due to side-chain carboxyls, and that C terminal groups yield little or no staining as would be expected for "average" molecular weight proteins.
THE SEX CHROMATIN AND HETEROCHROMATIC BODIES IN HUMAN DIPLOID AND POLYPLOID NUCLEI
Authors: Klinger Harold P. , Schwarzacher Hans G.
Published: May 2004

The deoxyribonucleic acid (DNA) content of Feulgen-stained interphase nuclei from human amnion epithelium and liver parenchyma as well as the DNA content of their sex chromatin or heterochromatic bodies was determined histophotometrically. In female nuclei the ratio between the nuclear DNA content and that of their sex chromatin or heterochromatic bodies remains constant irrespective of the polyploid state of the nucleus. Thus, in polyploid nuclei the heterochromatic bodies double their DNA content with each duplication of the diploid nuclear DNA content. Therefore, it is assumed that the large or multiple heterochromatic bodies of female polyploid nuclei are conjugated or multiple sex chromatin bodies. Some male diploid nuclei show a distinct heterochromatic body whose DNA content is about one-half that of a sex chromatin. About one-half of the polyploid male nuclei have heterochromatic bodies whose DNA content is approximately one-half of those of female nuclei of the corresponding polyploid class. This would indicate that in male diploid nuclei the single X chromosome sometimes leaves a heterochromatic rest one-half the size of a sex chromatin and in polyploid male nuclei the two or more X's may leave larger heterochromatic bodies. However, many male nuclei, even when polyploid, do not have distinct heterochromatic bodies. Possibly the heterochromatic portions of the X's have failed to join and form a sex chromatin-like body; or the Y inhibits the X from leaving a heterochromatic rest. The proposal of other authors that the sex chromatin is derived from but one of the X chromosomes of the female nucleus, the X in the male leaving no heterochromatic rest, is also considered. The DNA content does not vary significantly between nuclei with and without sex chromatin or heterochromatic bodies. It remains clear that female nuclei with abnormally large or multiple sex chromatin bodies and male nuclei with large distinct heterochromatic bodies are always polyploid.
THE CONSISTENCY OF AMEBA CYTOPLASM AND ITS BEARING ON THE MECHANISM OF AMEBOID MOVEMENT
Authors: Allen Robert D.
Published: May 2004

Three species of common, free-living amebae, Amoeba proteus, Amoeba dubia, and Chaos chaos were directly observed and photographed while exposed to a range of centrifugal accelerations in two types of centrifuge microscopes. Cytoplasmic inclusions in all three species are displaced discontinuously (at a variable velocity) in apparently all parts of the cell, suggesting non-Newtonian behavior and/or heterogeneous consistency. The ectoplasm of all species shows the highest yield point of any region in the cell; the posterior ectoplasm is less rigid than that in the anterior part of the cell. The axial part of the endoplasm shows evidence of structure (a sharp viscosity transition if not a true yield point) by its: (a) resistance to the displacement of particles carried in that region of the cell, (b) hindrance to the passage through the cell of inclusions displaced from other regions, and its (c) support without visible back-slip of inclusion being resuspended in the axial endoplasm in a centripetal direction at accelerations as high as 170 g. At this acceleration, each crystal "weighs" the equivalent reduced weight of seven times its volume in gold at 1 g. The only regions of the normal, moving cell which show clear evidence of low apparent viscosity are the "shear zone" (see Fig. 8) and the "recruitment zone." Possible reasons for low apparent viscosity in these regions are discussed. A new scheme of ameba "structure" is presented on the basis of the combined results of velocity profile analysis and the present centrifugation study.
ETUDE AU MICROSCOPE ELECTRONIQUE DES TRANSFORMATIONS NUCLEAIRES DE E. COLI K12 S ET K12 S (λ26) APRES IRRADIATION AUX RAYONS ULTRAVIOLETS ET AUX RAYONS X
Authors: Ryter Antoinette
Published: May 2004

Nuclear transformations induced in E. coli K12S and K12S(λ26) by ultraviolet radiations and x-rays have been studied with ultrathin sections in the electron microscope. The nucleoplasm keeps its normal aspect during "fragmentation" and during "condensation" of the nucleus into the "vesicular" form. Serial sections show that the "fragmented" nucleus consists in reality of only one very tortuous vacuole. No difference either in the shape or in the fine structure of the nucleus could be observed between the lysogenic strain and the non-lysogenic strain. A high concentration of NaCl has a "condensation" effect on the fragmented nuclei and decreases the induction rate.
STAINED PECTIN AS SEEN IN THE ELECTRON MICROSCOPE
Authors: Albersheim Peter , Mühlethaler K. , Frey-Wyssling A.
Published: May 2004

This paper describes electron microscopic studies on the distribution of pectin within young plant cells. Dark-grown onion roots, from 1 to 3 mm. in length, were used. In order to make the pectic substances selectively dense to electrons, they were first reacted with basic hydroxylamine. This treatment produces pectic hydroxamic acids, which in turn were treated with ferric ion to form insoluble complexes. The tissue was imbedded, sectioned, and then observed by electron microscopy. Dense deposits of iron were found in the region of the middle lamella and in a second area near the surface of the primary wall. Transverse walls of varying maturity were noted. The pectin of the more frequent, immature cross-walls, leads directly into the inner reacting layer of the axillary (longitudinal) wall. The pectin of the more mature transverse walls becomes, on the other hand, intimately associated with the middle lamella pectin of the axillary wall. It is shown that the pectin of the middle lamella represents the hot water-soluble portion of the pectic substance, while the internal layer of the axillary wall and the transverse wall pectin represent the so called residual fraction. Hot versene extraction removes essentially all electron-dense material.
AN ELECTRON MICROSCOPIC STUDY OF PINOCYTOSIS IN AMEBA
Authors: Brandt Philip W. , Pappas George D.
Published: May 2004

The attachment to the surface of the ameba (Chaos chaos L. (Pelomyxa carolinensis, Wilson)) of two proteins, ribonuclease and ferritin, and two colloidal suspensions, thorium dioxide and gold, was studied in the electron microscope. The initial step in the pinocytosis of ferritin and thorium dioxide particles by amebas is shown to be the attachment of these substances to the "hairlike" extensions of the plasmalemma. Ribonuclease caused alterations in the structure of the plasmalemma, but on account of its relative lack of density, it could not be definitely localized. Colloidal gold did not appear to be active with respect to pinocytosis in amebas. Since molecules in solution and particles in suspension are taken up by the same mechanism, the first step of which is their attachment to the cell surface, it is suggested that a single mechanism underlies phagocytosis, pinocytosis, ropheocytosis, cytopempsis, and potocytosis.
PERMANGANATE FIXATION OF THE GOLGI COMPLEX AND OTHER CYTOPLASMIC STRUCTURES OF MAMMALIAN TESTES
Authors: Mollenhauer Hilton H. , Zebrun William
Published: May 2004

Observations on the fine structure of KMnO4-fixed testes of small mammals (guinea pig, rat, and mouse) reveal certain morphological differences between the spermatogenic and Sertoli cells which have not been demonstrated in the same tissue fixed with OsO4. Aggregates of minute circular profiles, much smaller than the spherical Golgi vesicles, are described in close association with the Golgi complex of developing spermatids. Groups of dense flattened vesicles, individually surrounded by a membrane of different dimensions than that which bounds most of the other cell organelles, appear dispersed within the cytoplasm of some spermatogenic cells. Flattened vesicles of greater density than those belonging to the Golgi complex are reported confined to the inner Golgi zone of developing guinea pig spermatids between the Golgi cisternae and the head cap. The profiles of endoplasmic reticulum within spermatocytes appear shorter, wider, and more tortuous than those of Sertoli cells. Minute cytoplasmic particles approximately 300 A in diameter and of high electron opacity appear randomly disposed in some Sertoli cells. Groups of irregular-shaped ovoid bodies within the developing spermatids are described as resembling portions of cytoplasm from closely adjacent spermatids. Interpretation is presented regarding the fine structure of KMnO4-fixed testes in view of what has already been reported for mammalian testes fixed in OsO4.
IRRADIATION ULTRA-VIOLETTE DES ORGANITES CELLULAIRES AVEC OBSERVATION CONTINUE EN CONTRASTE DE PHASE
Authors: Bessis Marcel , Nomarski Georges
Published: May 2004

The apparatus described is designed to permit the observation of the smallest cellular components with the phase contrast microscope (which requires an objective of a high numerical aperture) while a part of the cell is irradiated by a very small spot of ultraviolet radiation. Theoretical considerations show a good distribution of the energy for a diameter of the irradiation spot as small as 0.2 microns. Indications are given about the limitations of the brightness caused by diffraction at the spot diaphragm and the energy absorption by volume-unit of the irradiated object. The practical applications of the apparatus are discussed.
Sexual conjugation in yeast. Cell surface changes in response to the action of mating hormones.
Authors: Tkacz J S , MacKay V L
Published: May 2004

In the yeast Saccharomyces cerevisiae, sexual conjugation between haploid cells of opposite mating type results in the formation of a diploid zygote. When treated with fluorescently labeled concanavalin A, a zygote stains nonuniformly, with the greatest fluorescence occurring at the conjugation bridge between the two haploid parents. In the mating mixture, unconjugated haploid cells often elongate to pear-shaped forms ("shmoos") which likewise exhibit asymmetric staining with the most intense fluorescence at the growing end. Shmoo formation can be induced in cells of one mating type by the addition of a hormone secreted by cells of the opposite mating type; such shmoos also stain asymmetrically. In nearly all cases, the nonmating mutants that were examined stained uniformly after incubation with the appropriate hormone. Asymmetric staining is not observed with vegetative cells, even those that are budded. These results suggest that, before and during conjugation, localized cell surface changes occur in cells of both mating types; the surface alterations facilitate fusion and are apparently mediated by the hormones in a manner that is mating-type specific.
Nucleation of polar actin filament assembly by a positively charged surface.
Authors: Brown S S , Spudich J A
Published: May 2004

Polylysine-coated polystyrene beads can nucleate polar assembly of monomeric actin into filamentous form. This nucleation has been demonstrated by a combination of biochemical and structural experiments. The polylysine-coated beads accelerate the rate of actin assembly as detected by two different biochemical assays. Subsequent examination of the beads by electron microscopy reveals numerous actin filaments of similar length radiating from the beads. ATP promotes this bead-induced acceleration of assembly. Decoration of the filaments with the myosin fragment S1 shows that these filaments all have the same polarity, with the arrowhead pattern pointing toward the bead. The relevance of the system to in vitro mechanisms and its usefulness in other studies are discussed.
Ligatin from embryonic chick neural retina.
Authors: Jakoi E R , Marchase R B
Published: May 2004

Ligatin, a filamentous protein previously found in suckling rat ileum, has been purified from plasma membranes of embryonic chick neural retina. The isolated plasma membranes are covered in part by 4.5-nm filaments that can be released from the membranes by treatment with Ca++. Subsequent dialysis against EGTA followed by sieve chromatography results in purification of the 10,000-dalton ligatin monomer. When labeled either with radioisotopes or with fluorescamine, the monomer is shown to electrophorese as a single discrete band in polyacrylamide gels. However, during standard fixing and staining procedures it diffuses from the gels and thus is not visualized. Ligatin's amino acid composition is distinguished by its high content of polar residues, especially Glx and Asx, and by the presence of phosphorylated serine. Upon re-addition of Ca++, purified ligatin monomers polymerize to form filaments 3 nm in Diam, identical to those formed by purified ileal ligatin. However, in both retina and ileum, the filaments observed on plasma membranes are greater than 3 nm in Diam. In ileum, this enlargement results from ligatin's function as a baseplate for the attachment of another protein, a beta-N-acetylhexosaminidase, to the cell surface. In retina, a corresponding difference in diameter between filaments seen in vivo and those formed from repolymerized ligatin alone and the co-solubilization of other proteins with ligatin suggest that ligatin may also function there as a baseplate for other cell surface proteins. The proteins associated with ligatin in retina differ morphologically from beta-N-acetylhexosaminidase and do not possess this enzymatic activity.
Bridging structures spanning the junctioning gap at the triad of skeletal muscle.
Authors: Somlyo A V
Published: May 2004

The membrane systems of skeletal muscle were examined after tannic acid fixation. A new structure consisting of bridges spanning the junctional gap is described, and a model is proposed in which the cytoplasmic but not the luminal membrane leaflets of the transverse tubule and of the junctional sarcoplasmic reticulum (SR) are continuous. The globular particles (presumably the Ca-binding proteins) within the terminal cisternae were arranged in longitudinal rows and appeared adherent to the junctional membrane. The junctional gap was present in negatively stained, frozen thin sections of fixed muscles. Negatively staining material occured within the junctional gap. The cytoplasmic leaflets of the longitudinal, intermediate, and terminal cisterna regions of the SR exhibited a thick coat of densely staining material compatible with the presence of the Ca-ATPase. Similar bridges were also observed at the surface membrane-SR close coupling sites of vascular smooth muscle.
Large species differences in the pattern of snPI RNA which can distinguish ape from human.
Authors: Benecke B J , Penman S
Published: May 2004

The snPI RNA species are a recently described set of molecules whose sizes range from 5S to 10S. They can be labeled in vitro in isolated nuclei and are apparently formed by an RNA polymerase I type of activity. However, in contrast to ribosomal precursor RNA, the usual polymerase I product, they are not found in the nucleolus but rather are located in the nucleoplasm. The snPI RNAs have been found in all mammalian cell types studied. The spectrum seen in gel electrophoresis is unique to each animal species studied but is essentially the same in different cell types within a species. The differences in snPI patterns are quite large between even closely related species and are clearly distinguishable in gorilla and human cells.
Nucleosome packing in interphase chromatin.
Authors: Rattner J B , Hamkalo B A
Published: May 2004

Higher-order chromatin fibers (200--300 A in diameter) are reproducibly released from nuclei after lysis in the absence of formalin and/or detergent. Electron microscope analysis of these fibers shows that they are composed of a continuous array of closely apposed nucleosomes which display several distinct packing patterns. Analysis of the organization of nucleosomes within these arrays and their distribution along long stretches of chromatin suggest that the basic 100-A chromatin fiber is not packed into discrete superbeads and is not folded into a uniform solenoid within the native 250-A fiber. Furthermore, because similar higher-order fibers have been visualized in metaphase chromosomes, the existence of this fiber class appears to be independent of the degree of in vivo chromatin condensation.
Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. II. Internalization of proteins.
Authors: Willinger M , Gonatas N , Frankel F R
Published: May 2004

The distribution of surface proteins during phagocytosis by rabbit peritoneal polymorphonuclear leukocytes was studied to determine whether the proteins of the phagocytic vesicles of these differentiated cells were representative of the entire set of plasma membrane proteins. Phagocytosis of bovine serum albumin-diisodecylphthalate emulsion by lactoperoxidase-iodinated rabbit neutrophils was linear over 15-20 min at a rate of 96 microgram oil/min/mg cell protein. This rate was similar to that of unlabeled cells. Incorporation of cell-associated free iodine by endogenous myeloperoxidase during phagocytosis was inhibited by 1 mM cyanide, which had no effect on the rate of particle uptake. The surface of intact neutrophils contained at least 13 iodinated proteins distinguishable by polyacrylamide gel electrophoresis followed by autoradiography. Isolated phagosomes were deficient in six of these proteins. The plasma membrane fraction of these cells was missing five of these same proteins which, however, were enriched in a dense surface fraction (Willinger, M., and F. R. Frankel. J. Cell Biol. 82: 32-44). When experimental conditions were reversed, and the PMNs were labeled after phagocytosis, these five proteins remained on the cell surface, while at least three of the major proteins found on resting cells were depleted. Incubating the cells with colchicine, which has been shown to affect the distribution of some plasma membrane constituents during phagocytosis, had no effect on the distribution of surface proteins in our system. These results indicate that a nonrandom interiorization of lactoperoxidase-labeled surface proteins of polymorphonuclear leukocytes occurs during phagocytosis.
N-ethylmaleimide-modified heavy meromyosin. A probe for actomyosin interactions.
Authors: Meeusen R L , Cande W Z
Published: May 2004

Treatment of rabbit skeletal muscle heavy meromyosin (HMM) with the sulfhydryl reagent N-ethylmaleimide (NEM) produces a species of HMM which remains tightly bound to actin in the presence of MgATP. NEM-HMM forms characteristic "arrowhead" complexes with actin which persist despite rinses with MgATP. NEM-HMM inhibits the actin activation of native HMM-ATPase activity, the superprecipitation of actomyosin, the contraction of glycerinated muscle myofibrils, and the contraction of cytoplasmic strands of the soil amoeba Chaos carolinensis. However, NEM-HMM does not interfere with in vitro microtubule polymerization or beating of demembranated cilia.
Clusters of intramembrane particles associated with binding sites for alpha-bungarotoxin in cultured chick myotubes.
Authors: Cohen S A , Pumplin D W
Published: May 2004

Developing chick myotubes in tissue culture were freeze-fractured to yield complementary replicas of large areas of membrane. Regions of muscle fibers with high concentrations of acetylcholine receptors were identified by binding of fluorescent-labeled alpha-bungarotoxin. Membranes in such regions contained clusters of large (100 A Diam) angular particles, similar in appearance to particles found in postsynaptic membranes of cholinergic synapses. Particles appeared in apposing areas of cytoplasmic and external leaflets but were more prevalent in the cytoplasmic leaflet. The areas of high particle concentration were coextensive with the fluorescence due to bound toxin. Treatment of cultures with tetrodotoxin increased the size of fluorescent spots and areas of high concentration of particles relative to those found in control cultures. In muscle cultures grown in the presence of spinal cord explants, some neurites contacted and innervated nearby myotubes. Intense fluorescence due to binding or alpha-bungarotoxin was present at portions of such neurite-myotube contacts. At these same portions, a high concentration of large angular particles was present in the sarcolemma adjacent to the neurite. In addition, an ordered arrangement of large particles was seen in the cytoplasmic leaflet of the neuronal plasmalemma directly apposing the muscle. The possible significance of these arrangements is discussed. Clusters on myotubes tended to be larger (contain more particles) when they occurred in groups, defined as three or more clusters with an intercluster distance of less than 0.5 micrometers. Clusters were also larger in myotubes treated with tetrodotoxin and in myotubes adjacent to some neurites in nerve-muscle cocultures. Several depressions containing particles similar to those in the clusters were found in the sarcolemma. The implications of these depressions are discussed in light of current theories of incorporation of proteins into cell membranes.
Quantitative reflection contrast microscopy of living cells.
Authors: Bereiter-Hahn J , Fox C H , Thorell B
Published: May 2004

Mammalian cells in culture (BHK-21, PtK2, Friend, human flia, and glioma cells) have been observed by reflection contrast microscopy. Images of cells photographed at two different wavelengths (546 and 436 nm) or at two different angles of incidence allowed discrimination between reflected light and light that was both reflected and modulated by interference. Interference is involved when a change in reflected intensity (relative to glass/medium background reflected intensity) occurs on changing either the illumination wavelength or the reflection incidence angle. In cases where interference occurs, refractive indices can be determined at points where the optical path difference is known, by solving the given interference equation. Where cells are at least 50 nm distant from the glass substrate, intensities are also influenced by that distance as well as by the light's angle of incidence and wavelength. The reflected intensity at the glass/medium interface is used as a standard in calculating the refractive index of the cortical cytoplasm. Refractive indices were found to be higher (1.38--1.40) at points of focal contact, where stress fibers terminate, than in areas of close contact (1.354--1.368). In areas of the cortical cytoplasm, between focal contacts, not adherent to the glass substrate, refractive indices between 1.353 and 1.368 were found. This was thought to result from a microfilamentous network within the cortical cytoplasm. Intimate attachment of cells to their substrate is assumed to be characterized by a lack of an intermediate layer of culture medium.
Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses.
Authors: Frank E , Fischbach G D
Published: May 2004

The development of clusters of acetylcholine (ACh) receptors at newly formed synapses between embryonic chick spinal cord and muscle cells grown in vitro has been studied by iontophoretic mapping with ACh. A semi-automated technique using on-line computer analysis of ACh responses and a photographic system to record the position of each ACh application permit the rapid construction of extensive and detailed maps of ACh sensitivity. Clusters of receptors, evident as peaks of ACh sensitivity, are present on many uninnervated myotubes. The distribution of ACh sensitivity closely parallels the distribution of 125I-alpha-bungarotoxin binding sites on the same muscle cell. In all cases where individual myotubes were adequately mapped before and after synapse formation, ingrowing axons induced new clusters of receptors rather than seeking out preexisting clusters. Synapses can form at active growth cones within 3 h of nerve-muscle contact. New receptor clusters can appear beneath neurites within a few hours. Many of the uninnervated clusters on innervated myotubes disappear with time. In contrast, receptor clusters on uninnervated myotubes remain in the same location for many hours. Synaptic clusters and clusters on uninervated myotubes are stable even though individual receptors are metabolized rapidly. The morphology of several identified sites of transmitter release was examined. At the scanning EM level, synapses appeared as small, rough-surfaced varicosities with filopodia that radiated outwards over the muscle surface. One synapse was studied by transmission EM. Acetylcholinesterase and a basement lamina were present within the synaptic cleft.
Involvement of membrane calcium in the response of rabbit neutrophils to chemotactic factors as evidenced by the fluorescence of chlorotetracycline.
Authors: Naccache P H , Showell H J , Becker E L , Sha'afi R I
Published: May 2004

We have utilized the fluorescent chelate probe chlorotetracycline to investigate the possible involvement of membrane calcium in the response of rabbit peritoneal neutrophils to chemotactic factors. Two chemotactic factors, the small molecular weight fragment of the fifth component of complement C5a and the synthetic peptide formyl-methionyl-leucyl-phenylalanine (F-Met-Leu-Phe), were tested and found to decrease the fluorescence of cell-associated chlorotetracycline in a manner strongly suggesting stimulus-induced displacement of membrane calcium. The time-course, concentration dependence, and receptor specificity of the calcium redistribution induced by the stimuli are consistent with its early role in the initiation of the various neutrophil functions. F-Met-Leu-Phe and C5a appear to interact with the same pool of membrane calcium and to release it to the cytoplasmic side of the plasma membrane. Intracellular calcium then binds back to the membrane(s) from where it can be displaced by additional stimulation. The release of membrane calcium, experimentally defined here, appears to play a central role in the initiation of the various neutrophil functions.
Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy.
Authors: Summers K , Kirschner M W
Published: May 2004

We describe here the continuous observations of the polymerization of individual microtubules in vitro by darkfield microscopy. In homogeneous preparations we verify that polymerization can occur onto both ends of microtubules. The assembly of microtubules is polar, with one end growing at three times the rate of the other. The differential rate of elongation can be used to determine the polarity of growth off cellular nucleating centers. We show that the microtubules grow off the proximal end of ciliary axonemes at a growth rate equal to that of the slow growing end of free microtubules, while growth off the distal end proceeds at the same rate as the fast growing end. Applying this technique to microtubule growth from metaphase chromosomes isolated from HeLa and CHO cells, we demonstrate that chromosomes initiate polymerization with the fast growing end facing away from the chromosome nucleation site. The opposite ends of free microtubules show different sensitivities to microtubule depolymerizing agents such as low temperature, Ca++ or colchicine as measured directly by darkfield microscopy. The differing rates of assembly and disassembly of each end of a microtubule suggest that a difference in polarity of growth off nucleating sites could serve as one basis for regulating the polymerization of different groups of microtubules in the same cell.
Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin.
Authors: Thoma F , Koller T , Klug A
Published: May 2004

We describe the results of a systematic study, using electron microscopy, of the effects of ionic strength on the morphology of chromatin and of H1-depleted chromatin. With increasing ionic strength, chromatin folds up progressively from a filament of nucleosomes at approximately 1 mM monovalent salt through some intermediate higher-order helical structures (Thoma, F., and T. Koller, 1977, Cell 12:101-107) with a fairly constant pitch but increasing numbers of nucleosomes per turn, until finally at 60 mM (or else in approximately 0.3 mM Mg++) a thick fiber of 250 A diameter is formed, corresponding to a structurally well-organized but not perfectly regular superhelix or solenoid of pitch approximately 110 A as described by Finch and Klug (1976, Proc. Natl. Acad. Sci. U.S.A. 73:1897-1901). The numbers of nucleosomes per turn of the helical structures agree well with those which can be calculated from the light-scattering data of Campbell et al. (1978, Nucleic Acids Res. 5:1571-1580). H1-depleted chromatin also condenses with increasing ionic strength but not so densely as chromatin and not into a definite structure with a well-defined fiber direction. At very low ionic strengths, nucleosomes are present in chromatin but not in H1-depleted chromatin which has the form of an unravelled filament. At somewhat higher ionic strengths (greater than 5 mM triethanolamine chloride), nucleosomes are visible in both types of specimen but the fine details are different. In chromatin containing H1, the DNA enters and leaves the nucleosome on the same side but in chromatin depleted of H1 the entrance and exit points are much more random and more or less on opposite sides of the nucleosome. We conclude that H1 stabilizes the nucleosome and is located in the region of the exit and entry points of the DNA. This result is correlated with biochemical and x-ray crystallographic results on the internal structure of the nucleosome core to give a picture of a nucleosome in which H1 is bound to the unique region on a complete two-turn, 166 base pair particle (Fig. 15). In the formation of higher-order structures, these regions on neighboring nucleosomes come closer together so that an H1 polymer may be formed in the center of the superhelical structures.
Dynamics of nuclear actin bundle induction by dimethyl sulfoxide and factors affecting its development.
Authors: Fukui Y , Katsumaru H
Published: May 2004

We have reported in a previous paper that dimethyl sulfoxide (DMSO) induces the formation of huge bundles of actin filaments in the nuclei of Dictyostelium mucoroides. The present study was performed to provide electron microscope data on the induction of nuclear actin bundles, illustrating both their formation and their reversion, as well as on the effects of various factors on the induction. The large nuclear bundles of actin appeared after 20--30 min of treatment with 10% DMSO. A DMSO concentration of 5 or 10% was optimal for the induction of the bundles. The nuclear actin bundle reverted to the original morphology within 5 min after removing DMSO. Induction of nuclear actin bundles was inhibited by Mg++ and low temperatures, but not by Na+, K+, Ca++, ATP, 3'5'-cyclic adenosine monophosphate (cAMP), phosphate buffer, or cytochalasin B. Neither NaN3 nor cycloheximide totally inhibited the induction of the bundles.
Immunocytochemical localization of calmodulin and a heat-labile calmodulin-binding protein (CaM-BP80) in basal ganglia of mouse brain.
Authors: Wood J G , Wallace R W , Whitaker J N , Cheung W Y
Published: May 2004

Antisera to calmodulin, a Ca2%-dependent modulator protein, and a heat-labile calmodulin-binding protein have been used to localize these proteins in mouse caudate-putamen. The two proteins appear to be located at identical sites in this brain area. At the light microscopic level, calmodulin and calmodulin-binding protein are found within the cytoplasm and processes of large cells. At the electron microscopic level the proteins are associated with neuronal elements only, primarily at postsynaptic sites within neuronal somata and dendrites. Within the dendrites the immunocytochemical label is associated predominantly with the postsynaptic density and dendritic microtubules. These results are in accord with recent biochemical and immunihistochemical studies of calmodulin in brain and in dividing cells. Thus, calmodulin and the heat-labile calmodulin-binding protein may play a role in the nervous system at the site of neurotransmitter action and at the level of microtubular function.
Rows of dimeric-particles within the axolemma and juxtaposed particles within glia, incorporated into a new model for the paranodal glial-axonal junction at the node of Ranvier.
Authors: Wiley C A , Ellisman M H
Published: May 2004

Using freeze-fracture techniques, we have analyzed the glial-axonal junction (GAJ) between Schwann cells and axons in the peripheral nervous system, and between oligodendrocytes and axons in the central nervous system of the rat. We have identified a new set of dimeric-particles arranged in circumferential rows within the protoplasmic fracture faces (P-faces) of the paranodal axolemma in the region of glial-axonal juxtaposition. These particles, 260 A in length, composed of two 115-A subunits, are observed in both aldehyde-fixed and nonfixed preparations. The rows of dimeric-particles within the axonal P-face are associated with complementary rows of pits within the external fracture face (E-face) of the paranodal axolemma. These axonal particles are positioned between rows of 160-A particles that occur in both fracture faces of the glial loops in the same region. We observed, in addition to these previously described 160-A particles, a new set of 75-A glial particles within the glial P-faces of the GAJ. These 75-A particles form rows that are centered between the rows of 160-A particles and are therefore superimposed over the rows of dimeric-particles within the paranodal axolemma. Our new findings are interpreted with respect to methods of specimen preparation as well as to a potential role for the paranodal organ in saltatory conduction. We conclude that this particle-rich junction between axon and glia could potentially provide an intricate mechanism for ion exchange between these two cell types.
Demonstration of vasoproliferative activity from mammalian retina.
Authors: Glaser B M , D'Amore P A , Michels R G , Patz A , Fenselau A
Published: May 2004

Vasoproliferative activity has been demonstrated in extracts of retinas from human, bovine, and feline sources. These retinal extracts are capable of stimulating (a) proliferation and thymidine uptake of bovine vascular endothelial cells in culture and (b) neovascularization on the chick chorioallantoic membrane. Extracts of skeletal muscle, cardiac muscle, and liver lack similar stimulatory activity. The activity is nondialyzable, stable at 56 degrees C, and inactivated at 100 degrees C. Retinal extracts stimulate the proliferation of corneal fibroblasts but have no effect on the proliferation of vascular smooth muscle cells. Indirect evidence suggests the liberation of a vasoproliferative factor from retina in several ocular disorders. The data in this report represent the first direct demonstration of vasoproliferative activity from mammalian retina.
Glutamine synthetase localization in cortisol-induced chick embryo retinas.
Authors: Norenberg M D , Dutt K , Reif-Lehrer L
Published: May 2004

We report here for the first time, in chick retina, Muller cell localization of glutamine synthetase (GS) activity by an immunohistochemical technique, in agreement with previous reports of glial localization of this enzyme in rat brain and retina. Age-dependent changes in the endogenous enzyme activity as well as cortisol-induced changes in GS activity, both in ovo and in vitro, measured biochemically, reflect the changes observed by staining.
Involement of an acrosinlike proteinase in the sulfhydryl-induced degradation of rabbit sperm nuclear protamine
Authors: Zirkin BR , Chang TSK , Heaps J
Published: May 2004

Previous studies demonstrated that proteolytic activity is associated with isolated rabbit sperm nuclei and is responsible for the degradation of nuclear protamine that occurs during thiol-induced in vitro decondensation of the nuclei (Zirkin and Chang, 1977; Chang and Zirkin, 1978). In this study, we present the results of experiments designed to characterize this proteolytic activity. Basic protein isolated from rabbit sperm nuclei incubated with 5 mM dithiothreitol (DTT) and 1 percent Triton X-100 for increasing periods of time exhibited progressively faster migrating bands on acid-urea polyacrylamide gels, reflection the progressive degradation of protamine. Ultimately, a specific and characteristic peptide banding pattern resulted. When sperm nuclei were treated with the esterase inhibitor nitrophenyl-p-guanidino benzoate (NPGB) to inhibit the nuclear-associated proteolytic activity and then incubated with one of several exogenous proteinases in addition to DTT and Triton X-100, characteristic peptide banding patterns were seen for each exogenous proteinase employed. For trypsin, chymotrypsin, pronase, and papain, the peptide banding patterns differed from one another and from the pattern characteristic of protamine degradation by the nuclear-associated proteinase. By contrast, when rabbit acrosin served as the exogenous proteinase, the peptide banding pattern seen was identical to the pattern characteristic of the nuclear-associated proteinase. These results demonstrate directly that the proteinase associated with rabbit sperm nuclei and involved in sperm nuclear decondensation in vitro is acrosinlike.
Regulation of the Chlamydomonas cell cycle by light and dark.
Authors: Spudich J L , Sager R
Published: May 2004

By growing cells in alternating periods of light and darkness, we have found that the synchronization of phototrophically grown Chlamydomonas populations is regulated at two specific points in the cell cycle: the primary arrest (A) point, located in early G1, and the transition (T) point, located in mid-G1. At the A point, cell cycle progression becomes light dependent. At the T point, completion of the cycle becomes independent of light. Cells transferred from light to dark at cell cycle position between the two regulatory points enter a reversible resting state in which they remain viable and metabolically active, but do not progress through their cycles. The photosystem II inhibitor dichlorophenyldimethylurea (DCMU) mimics the A point block induced by darkness. This finding indicates that the A point block is mediated by a signal that operates through photosynthetic electron transport. Cells short of the T point will arrest in darkness although they contain considerable carbohydrate reserves. After the T point, a sharp increase occurs in starch degradation and in the endogenous respiration rate, indicating that some internal block to the availability of stored energy reserves has now been released, permitting cell cycle progression.
Ribosome binding sites visualized on freeze-fractured membranes of the rough endoplasmic reticulum.
Authors: Giddings T H , Staehelin L A
Published: May 2004

Freeze-fracture micrographs of cells of the green alga Micrasterias denticulata stabilized by ultrarapid freezing reveal imprints of polysomes on the rough endoplasmic reticulum membranes. The imprints appear as broad, spiral ridges on the P faces and as corresponding wide grooves on the E faces of the membranes. Distinct 110-A particles with a spacing of 270 +/- 45 A are associated with the P-face ridges. Where imprints of individual ribosomes can be discerned, it is seen that there is a 1:1 relationship between the ribosomes and the 110-A particles, and that the 110-A particles are located in a peripheral position with respect to the polysome spirals. We propose that the 110-A particles could be structural equivalents of ribosome-binding sites, consisting of a molecule each of ribophorins I and II and a nascent polypeptide chain. These observations suggest that the spiral form of polysomes could result from the forces generated by the extrusion of the growing polypeptide chains to one side of the polysome.
Interferon effects on microfilament organization cellular fibronectin distribution, and cell motility in human fibroblasts
Authors: Pfeffer LM , Wang E , Tamm I
Published: May 2004

We have shown previously (Pfeffer et al., 1979, Exp. Cell Res. 121:111-120) that treatment of human fibroblasts, planted at a density of 2x10(3) cells/cm(2), with purified human fibroblasts interferon (640 U/ml) for 3 d at 37 degrees C decreases the overall rate of cell proliferation to 35-40 percent of the control value. In the present experiments we have characterized the phenotype of interferon-inhibited fibroblasts. The mean volume of trypsinized, interferon-treated cells was increased 31 percent abover that of control cells. The interferon-treated population was much more heterogeneous than the control population with respect to volume, and there was a considerable overlap in the volume distributions of the two populations. The cell surface area was, on the average, increased 65 percent after interferon treatment. More than 80 percent of the treated cells had enlarged nuclei, many of which were lobed, and the fraction of binucleated cells was increased fivefold. After interferon treatment, over 40 percent of the cells showed large actin-containing fibers in the form of multiple parallel arrays. Fewer than 5 percent of the control cells contained such large actin fibers. The number of actin fibers of all sizes was tripled in the treated fibroblasts on a per cell basis and, calculated per unit surface area of the cells, the number was increased 82 percent. In contrast, 10-nm filaments and microtubules did not appear to be increased in number per unit surface area of the cells. The increases per cell in the abundance of these structures were directly related to increased cell size. After interferon treatment, fibronection was distributed in arrays of long filaments covering most portions of the cell surface. Interferon treatment markedly decreased the rate of cell locomotion as well as membrane ruffling and saltatory movements of intracellular granules.
Calcium in epithelial cell contraction.
Authors: Lee H C , Auersperg N
Published: May 2004

Epithelial morphogenesis in many organs involves asymmetric microfilament-mediated cellular contractions. Similar contractions, in terms of ultrastructure and cytochalasin B sensitivity, can be induced in the carcinoma cell line C-4II in culture. This line was used to compare total intracellular calcium levels ([Ca]i) in contracted monolayer fragments and in control cultures, and to determine whether epithelial cell contraction depends on influx of extracellular Ca. [Ca]i, defined as Ca not displaceable by lanthanum, was measured by atomic absorption spectrophotometry. Degrees of contraction were determined from shape changes of monolayer fragments. Detachment from the growth surface initiated cellular contractions and caused an immediate increase in [Ca]i, from 1.0 to 4.0-5.0 micrograms Ca/mg protein in early confluent cultures, and from 0.3 to 1.0-2.0 micrograms Ca/mg protein in crowded cultures. This increase was followed by a gradual decline in [Ca]i, though Ca levels remained higher than in controls and contraction progressed for 30 min. Contraction was inhibited completely by cold (7 degrees C) and by Ca-free medium, and in a dose-dependent manner by papaverine (2.5 x 10(-6) M-2.5 x 10(-4) M), lanthanum (1.0 x 10(-6) M-1.0 x 10(-4) M); and D-600 (1.0-2.0 x 10(-4) M). The Ca ionophore A23187 had no effect at 5.0 x 10(-6) M and was inhibitory at higher concentrations. The results provided direct evidence for increased [Ca]i in contracting epithelial cells, and suggest that Ca influx is required for such contraction to take place.
Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors.
Authors: MacLean-Fletcher S D , Pollard T D
Published: May 2004

We have studied the kinetics of the gelation process that occurs upon warming cold extracts of Acanthamoeba using a low-shear falling ball assay. We find that the reaction has at least two steps, requires 0.5 mM ATP and 1.5 mM MgCl2, and is inhibited by micromolar Ca++. The optimum pH is 7.0 and temperature, 25 degrees-30 degrees C. The rate of the reaction is increased by cold preincubation with both MgCl2 and ATP. Nonhydrolyzable analogues of ATP will not substitute for ATP either in this "potentiation reaction" or in the gelation process. Either of two purified or any one of four partially purified Acanthamoeba proteins will cross-link purified actin to form a gel, but none can account for the dependence of the reaction in the crude extract on Mg-ATP or its regulation by Ca++. This suggests that the extract contains, in addition to actin-cross-linking proteins, factors dependent on Mg-ATP and Ca++ that regulate the gelation process.
Codistribution of collagen types IV and AB2 in basement membranes and mesangium of the kidney. an immunoferritin study of ultrathin frozen sections.
Authors: Roll F J , Madri J A , Albert J , Furthmayr H
Published: May 2004

Affinity-purified rabbit antibodies specific for collagen types I, III, AB2 and for a partially characterized type IV collagen derived from a murine tumor were used to study the distribution of collagens in the normal mouse kidney. Immunofluorescence staining of conventional frozen sections demonstrated that types I and III were present in bundles around large vessels and in fibers surrounding glomeruli and tubules, whereas types IV and AB2 were distributed in a linear fashion along basement membranes of tubules, glomeruli, and Bowman's capsule and in the mesangial stalk. The distribution of types IV nd AB2 was examined at the ultrastructural level by staining of 600- to 800-A thick frozen sections with a three-stage procedure employing specific collagen antibodies, biotinyl sheep antirabbit IgG, and avidin-ferritin conjugates. Labeling by this procedure demonstrated codistribution of types AB2 and the putative type IV in all three basement membranes. In addition, mesangial matrix was shown to contain both of these collagen types. These results support recent biochemical evidence of collagen heterogeneity in basement membranes, and also support the concept of a structural relationship between mesangial matrix and glomerular basement membranes.