bluuebunny/crossref_metadata_2025 huggingface dataset:
THE INFLUENCE OF X-RAYS ON ORGANELLE INDUCTION AND DIFFERENTIATION IN GRASSHOPPER SPERMATOGENESIS
Authors: Tahmisian T. N. , Devine R. L.
Published: May 2004

The effect of x-irradiation on grasshopper spermatogenesis was studied with the aid of light and electron microscopy. The insects were irradiated at the second instar prior to the presence of maturation stages and observed at the last instar and imago stages. Dosages of 100 to 600 roentgens were found to retard the differentiation of the nucleus and mitochondrial nebenkern in spermatids. Evidence is presented that irradiation causes a curtailment and disorganization in the differentiation of the nebenkern from mitochondria. The above doses also induced the formation of supernumerary centrioles, flagellar filaments and acrosomes; nuclear disorganization as well as pycnosis and fragmentation also occur. The nucleus appears to be drawn toward each radiation-induced supernumerary acrosome, with consequent multipolarity of the nucleus. Induction of a set of flagellar filaments is seen only where the centriolar structure is in contact with the nucleus. Details are given of an organelle, heretofore not described, that is composed of anastomosed and interwoven cytoplasmic strands.
MITOCHONDRIAL LOCALIZATION OF OXIDATIVE ENZYMES: STAINING RESULTS WITH TWO TETRAZOLIUM SALTS
Authors: Novikoff Alex B. , Shin Woo-Yung , Drucker Joan
Published: May 2004

A comparison is made of the staining results obtained with Nitro-BT and MTT-Co++ as acceptors when DPNH is the substrate in frozen sections of cold formol-calcium-fixed rat kidney (normal and following ligation of the blood vessels) and human liver containing lipofuscin granules. The kidney results are evaluated in terms of mitochondrial morphology seen after classical mitochondrial stains and in electron micrographs. It is concluded that, except for formazan deposition on lipid-aqueous interfaces, Nitro-BT staining indicates the intracellular localization of oxidative enzymes, at least at the level of light microscopy. In contrast, the use of MTT-Co++ is not reliable for such intracellular localizations. The deposition of the formazan of MTT-Co++ is determined in large part by physicochemical factors other than enzyme localization. Despite marked abnormalities of the mitochondria in cells of the ligated kidney, MTT-Co++ formazan is generally deposited in the same dotlike fashion as in cells of normal kidney.
A STUDY OF THE PENETRATION OF MAMMALIAN CELLS BY DEOXYRIBONUCLEIC ACIDS
Authors: Borenfreund Ellen , Bendich Aaron
Published: May 2004

Tritium-labeled deoxyribonucleic acid (DNA) from pneumococci and from human leukocytes was added to growing cultures of HeLa cells at 37°C. Autoradiography revealed an extensive localization of tritium in the nuclear regions. The label could not be removed by treatment with ribonuclease or dilute perchloric acid, but quantitative removal from the cells could be effected with deoxyribonuclease. Chemical and radioactivity determinations on nucleic acids isolated from the exposed HeLa cells revealed the presence of tritium in all 4 DNA bases. About 12 µg. of tritiated DNA was recovered from 6 x 106 HeLa cells which had been exposed for 24 hours to 240 µg. of the human DNA. From this, it is concluded that the amount of DNA, or its degradation products, taken up by the cells was equivalent to at least 10 per cent of the normal HeLa cell complement.
DEAMINATION OF DEOXYCYTIDINE AND 5-METHYLDEOXYCYTIDINE IN DEVELOPING ANTHERS OF LILIUM LONGIFLORUM (VAR. CROFT)
Authors: Hotta Yasuo , Stern Herbert
Published: May 2004

A study has been made of developmental variations in anthers of Lilium longiflorum with respect to the deamination of deoxycytidine and 5-methyldeoxycytidine. Although a periodicity of enzymatic activities surrounding the mitotic cycle has been found, the over-all evidence is against the significance of such periodicity to chromosome duplication. On the basis of evidence obtained a distinction has been drawn between enzyme systems functioning in a broad supporting role and those immediately tied to morphogenetic events.
ELECTRON MICROSCOPY OF OSTEOCLASTS IN HEALING FRACTURES OF RAT BONE
Authors: Gonzales Federico , Karnovsky Morris J.
Published: May 2004

Osmium-fixed, undecalcified, callus tissue from healing fractures of rat tibias was sectioned with a diamond knife for study with the electron microscope. Large multinucleated cells were found adjacent to bone. A characteristic labyrinthine infolded border was consistently seen in parts of the cells close to the bone surface. The innermost parts of this "ruffled border" gave rise to vacuoles. The bone surface was always disrupted under the "ruffled border" of the cells. Needle-like crystals were seen at the osseous fringe, within folds in the ruffled border as well as within vacuoles deeper in the cells. Collagen fibers denuded of crystals were never observed. Mitochondria, containing clusters of fine granules, were abundant. The part of the cell away from bone contained rough endoplasmic reticulum and the cell membrane was thrown into irregular microvilli. These observations are discussed in relation to current concepts of osteoclastic resorption of bone.
ELECTRON MICROSCOPY OF PLASMA-CELL TUMORS OF THE MOUSE
Authors: Parsons D. F. , Darden E. B. , Lindsley D. L. , Pratt Guthrie T.
Published: May 2004

An electron microscope study was made of a series of transplanted MPC-1 plasma-cell tumors carried by BALB/c mice. Large numbers of particles similar in morphology to virus particles were present inside the endoplasmic reticulum of tumor plasma cells. Very few particles were seen outside the cells or in ultracentrifuged preparations of the plasma or ascites fluid. In very early tumors particles were occasionally seen free in the cytoplasm adjacent to finely granular material. In general, the distribution of these particles inside endoplasmic reticulum is similar in early and late tumors. A few transplanted X5563 tumors of C3H mice were also examined. Large numbers of particles were found in the region of the Golgi apparatus in late X5663 tumors. A newly described cytoplasmic structure of plasma cells, here called a "granular body," appears to be associated with the formation of the particles. Particles present in MPC-1 tumors are exclusively of a doughnut form, whereas some of those in the inclusions of the late X5563 tumors show a dense center. Normal plasma cells, produced by inoculation of a modified Freund adjuvant into BALB/c mice. have been compared morphologically with tumor plasma cells of both tumor lines.
ELECTRON MICROSCOPY OF PLASMA-CELL TUMORS OF THE MOUSE
Authors: Parsons D. F. , Bender M. A. , Darden E. B. , Pratt Guthrie T. , Lindsley D. L.
Published: May 2004

The X5563 tumor has been grown in tissue culture. Cells similar to those of the original tumor migrated from the explant and attached to the glass walls of the culture vessels. Electron microscopy showed that large numbers of particles, similar in morphology to virus particles, were associated with these cells after 7 days of culture. The two principal types of particles found in the tumor in vivo appear to be present in vitro. Many more of these particles, however, were larger and showed a more complex structure. Whereas the particles were mainly localized inside endoplasmic reticulum or the Golgi zone in the tumors in vivo, in the tissue culture the majority of the particles were associated with the plasma membrane and were found outside of the cells. The relation of the particles to the granular body is discussed as well as a possible relation to the mammary tumor agent.
NUCLEIC ACID AND PROTEIN METABOLISM DURING THE MITOTIC CYCLE IN VICIA FABA
Authors: Woodard John , Rasch Ellen , Swift Hewson
Published: May 2004

In order to investigate some of the cytochemical processes involved in interphase growth and culminating in cell division, a combined autoradiographic and microphotometric study of nucleic acids and proteins was undertaken on statistically seriated cells of Vicia faba root meristems. Adenine-8-C14 and uridine-H3 were used as ribonucleic acid (RNA) precursors, thymidine-H3 as a deoxyribonucleic acid (DNA) precursor, and phenylalanine-3-C14 as a protein precursor. Stains used in microphotometry were Feulgen (DNA), azure B (RNA), pH 2.0 fast green (total protein), and pH 8.1 fast green (histone). The autoradiographic data (representing rate of incorporation per organelle) and the microphotometric data (representing changes in amounts of the various components) indicate that the mitotic cycle may be divided into several metabolic phases, three predominantly anabolic (net increase), and a fourth phase predominantly catabolic (net decrease). The anabolic periods are: 1. Telophase to post-telophase during which there are high rates of accumulation of cytoplasmic and nucleolar RNA and nucleolar and chromosomal total protein. 2. Post-telophase to preprophase characterized by histone synthesis and a diphasic synthesis of DNA with the peak of synthesis at mid-interphase and a minor peak just preceding prophase. The minor peak is coincident with a relatively localized DNA synthesis in several chromosomal regions. This period is also characterized by minimal accumulations of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. 3. Preprophase to prophase in which there are again high rates of accumulation of cytoplasmic RNA, and nucleolar and chromosomal total protein and RNA. The catabolic phase is: 4. The mitotic division during which there are marked losses of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA.
LOCALIZATION OF MACROMOLECULES IN ESCHERICHIA COLI
Authors: Caro Lucien G. , Forro Frederick
Published: May 2004

The distribution of RNA in cells of E. coli 15 T-U- labeled with uridine-H3 was studied by methods involving the analysis of radioautographic grain counts over random thin cross-sections and serial sections of the cells. The results were correlated with electron microscope morphological data. Fractionation and enzyme digestion studies showed that a large proportion of the label was found in RNA uracil and cytosine, the rest being incorporated as DNA cytosine. In fully labeled cells the distribution of label was found to be uniform throughout the cell. The situation remained unchanged when labeled cells were subsequently treated with chloramphenicol. When short pulses of label were employed a localization of a large proportion of the radioactivity became apparent. The nuclear region was identified as the site of concentration. Similar results were obtained when cells were exposed to much longer pulses of uridine-H3 in the presence of chloramphenicol. If cells were subjected to a short pulse of cytidine-H3, then allowed to grow for a while in unlabeled medium, the label, originally concentrated to some extent in the nuclear region, was found dispersed throughout the cell. The simplest hypothesis which accounts for these results is that a large fraction of the cell RNA is synthesized in a region in or near the nucleus and subsequently transferred to the cytoplasm.
STUDIES ON THE OXIDATIVE METABOLISM OF SACCHAROMYCES CEREVISIAE
Authors: Vitols Eberhards , Linnane Anthony W.
Published: May 2004

A specially designed high-speed blendor and glass beads have been used to disintegrate yeast cells. The method enables large quantities of cells to be fragmented quickly at low temperature, and cell-free mitochondrial particles to be prepared in high yield. The particles are isolated in a sucrose-Tris-EDTA medium and extensively refractionated in the same medium. The success of the fractionation is dependent upon the presence of the Tris buffer, as the latter prevents the aggregation of the particulate material. Two morphologically and enzymatically different particle types have been obtained: a heavy fraction corresponding to mitochondria in size and internal organization, and a light fraction consisting of vesicular, single-membrane particles of a smaller size. The light particles oxidize DPNH and succinate, but do not oxidize pyruvate-malate, and lack the capacity for phosphorylation. The heavy particles oxidize pyruvate-malate as well as the citric acid cycle intermediates, although their α-ketoglutaric dehydrogenase activity is low. Oxidation by the heavy particles is coupled to phosphorylation, and P/O ratios of about 1.5 have been obtained. Lactic acid dehydrogenase is also present in the heavy fraction, and lactate is oxidized with a P/O ratio of about 0.7.
THE CELLULAR COMPLEMENT OF THE SKELETAL SYSTEM STUDIED AUTORADIOGRAPHICALLY WITH TRITIATED THYMIDINE (H3TDR) DURING GROWTH AND AGING
Authors: Tonna Edgar A.
Published: May 2004

An autoradiographic study has been made of mouse femora from birth to one year of age. Tritiated thymidine was used to study the proliferative rate of various skeletal tissues. The labeling index of the periosteum was found to be highest at birth (0.085). At 8 weeks of age the index fell sharply to 0.007 and remained low throughout the entire period of study. Chondro-osteogenic cells of the periosteum, especially those at the perichondrial zone, were most frequently labeled initially. The labeling index of the epiphyseal disk remained high (0.054 to 0.048) until about the 6th week of age. Following this age the index fell sharply to 0.018 at 8 weeks and 0.002 at 26 weeks of age. Autoradiographs showed that the cells of the articulating surface of the epiphysis and disk are derived at least in part from migrating chondro-osteogenic cells of the periosteum residing at the perichondrial region, and that these cells serve as the progenitor pool necessary for both circumferential growth and expansion of the epiphyseal disk.
ELECTRON MICROSCOPE REPLICAS FROM THE SURFACE OF A FRACTURE THROUGH FROZEN CELLS
Authors: Haggis G. H.
Published: May 2004

A technique is described, which is new in certain details, for fracturing frozen cells and taking replicas from the fracture surface. This technique has been developed as a cytological preparative technique complementary to those currently used in electron microscopy. The present paper describes results for human red cells, chosen for preliminary work to facilitate recognition of artifacts. The cells are suspended in 20 per cent glycerol, 0.9 per cent NaCl solution, allowed time to equilibrate, and rapidly frozen in a small chamber plunged into propane at -196°C. The chamber is kept at -196°C., and fractured in vacuo, in such a way that the fracture plane runs through the frozen suspension, and through the cells in its path. After fracture, the chamber is brought to -105°C., still in vacuo, to etch the fracture surface, and a platinum-carbon replica is deposited on the surface from a carbon arc. The replica is subsequently cleaned in concentrated alkaline solution and examined in the electron microscope. The outlines of the fractured cells can be recognised. There are indications that in areas of the replica which correspond to the interior of a cell, individual haemoglobin molecules can be seen, and in favourable cases the arrangement of some of the four molecular subunits.
Single bacteriorhodopsin molecules revealed on both surfaces of freeze-dried and heavy metal-decorated purple membranes.
Authors: Studer D , Moor H , Gross H
Published: May 2004

The flat sheets of the purple membrane from Halobacterium halobium contain only a single protein (bacteriorhodopsin) arranged in a hexagonal lattice. After freeze-drying at -80 degrees C (a method that is superior to air-drying), shadowing with tantalum/tungsten, and image processing, structural details on both surfaces are portrayed in the range of 2 nm. One surface is rough and lattice lines are clearly visible, whereas the other is smooth and the hexagonal order seems to be absent. The optical diffraction patterns, however, indicate a hexagonal lattice for both surfaces. In addition, these diffraction patterns are characteristic and easily distinguished. The orientation of the two surfaces was identified by silver decoration: partial condensation of silver on purple membranes enabled the smooth surface to be identified as the plasmatic and the rough surface as the exoplasmic surface. After image processing, the exoplasmic surface shows a triplet structure which exactly fits the projected structure determined by Unwin and Henderson (1975. Nature(Lond.). 257:28-32) at molecular resolution, whereas, on the plasmatic surface, four image details per unit cell are visible. Three of them match the arrangement of bacteriorhodopsin, whereas the fourth must be located over a lipidic array. Summarizing these results, it is possible to show the part of each single bacteriorhodopsin protein that is present in the surfaces of the purple membrane. By "shadowing" the membranes perpendicularly, we prove that these components of the surfaces are mainly portrayed by a decoration effect of the tantalum/tungsten condensate.
Encapsulation of "core" eIF3, regulatory components of eIF3 and mRNA into liposomes, and their subsequent uptake into myogenic cells in culture.
Authors: O'Loughlin J , Lehr L , Havaranis A , Heywood S M
Published: May 2004

Eukaryotic initiation factor 3 (eIF3), encapsulated in liposomes, is taken up by chick muscle cells in culture. The exogenously supplied factor (isolated from 14-d embryonic muscle) rapidly associated with 40S ribosomal subunits and particles sedimenting at 80-120S (the known sedimentation value of myosin heavy chain [MHC] mRNPs). In addition, exogenously supplied eIF3 has a specific stimulatory effect on myofibrillar protein synthesis. This stimulation is most apparent at the onset of cell fusion and after the accumulation of MHC-mRNPs. As previously reported (8), total eIF3 can be fractionated on an MHC-mRNA affinity column into a "core" eIF3 and a high affinity component (HAF) which dictates the discriminatory activity of core eIF3. Liposome-encapsulated core eIF3 delivered to cells is found predominantly in 40S ribosomal subunits and gives only a slight stimulation of total protein synthesis. When 3H-MHC-mRNA, preincubated with HAF, is introduced into myoblasts via liposomes, the mRNA is found in heavy polysomes. On the other hand, when the messenger alone or with core eIF3 is taken up by the cells, it is found only on small polysomes. Similar experiments, using viral RNA with the HAF, show no increase in the size class of polysomes. These results mimic the differences observed between myoblast and myotube utilization of MHC-mRNA previously observed (17). These results demonstrate the mRNA discriminatory activity of specific proteins associated with muscle eIF3 and suggest that these proteins play a role in mRNA activation and translation during muscle differentiation.
Mechanism of retraction of the trailing edge during fibroblast movement.
Authors: Chen W T
Published: May 2004

Retraction of the taut, trailing portion of a moving chick heart fibroblast in vitro is an abrupt dynamic process. Upon retraction, the fibroblast tail always ruptures, leaving a small amount of itself attached to the substratum by focal contacts. Time-lapse cinemicrography shows that retraction produces a sudden, massive movement of both surface and cytoplasmic material toward a cluster of focal contacts near the main body of the cell. The appearance of folds on the upper cell surface at this time and the absence of endocytotic vesicles are consistent with this forward movement. Retraction of the trailing edge, either occurring naturally or produced artificially with a microneedle, consists of an initial fast component followed and overlapped by a slow component. Upon artificial detachment in the presence of iodoacetate, dinitrophenol, and sodium fluoride, and at 4 degrees C, the slow component is strongly inhibited and the fast one only slightly inhibited. Moreover of the bundles of microfilaments oriented parallel to the long axis of the tail seen in TEM. Most of the birefringence is lost during the fast phase and the rest during the slow phase of retraction. Concurrently, the bundles of microfilaments disappear during the fast phase of retraction and are replaced by a microfilament meshwork. All of these results are consistent with the hypothesis that the initial fast component of retraction is a passive elastic recoil, associated with the oriented bundles of microfilaments, and that the slow component of retraction is an active contraction, associated with a meshwork of microfilaments.
In vitro biosynthesis, core glycosylation and membrane integration of opsin
Authors: Goldman BM , Blobel G
Published: May 2004

A membrane-integrated , core-glycosylated form of bovine opsin was synthesized in vitro when bovine retina mRNA was translated in a wheat germ cell-free system supplemented with dog pancreas microsomal vesicles; glycosylation and integration of opsin into membranes were coupled to translation. Proteolysis with themolysin was used to probe the orientation of opsin within the dog pancreas microsomal membrane, and to compare it with that of opsin in rod cell disk membranes isolated from bovine retina. Intact microsomal or disk vesicles were required for production of discrete, membrane-associated thermolysin fragments of opsin; no discrete opsin fragments were detected when membranes were incubated with thermolysin in the presence of the nonionic detergent, Triton X-100. The major opsin fragments produced by themosylin treatment of intact microsomal vesicles resembled those from disk vesicles in their size, oligosaccharide content, and order of appearance. In each case, the first cleavage of opsin took place at the COOH-terminus, generating a glycosylated fragment, O', which was only slightly smaller than intact opsin. Both the microsomal and disk membrane forms of O' were next cleaved internally; glycosylated fragments of similar sizes in both cases were detected which were derived from the NH(2)-terminal portion of O'. Several smaller NH(2)-terminal fragments of opsin were detected only in thermolysin-treated microsomal membranes, and not in disk membranes. The data suggest that the topology of opsin integrated into dog pancreas microsomal vesicles is similar to that in rod cell disk vesicles, although not identical. In each case, the glycosylated NH(2)-terminal region of opsin is located within the lumen of the vesicle, while discrete COOH-terminal and internal segments of opsin apparently emerge at the outer, cytoplasmic face of the membrane. Thus, opsin in the heterologous microsomal membrane, like its counterpart in the native disk membrane, may cross the bilayer at least three times. The internal domain of the polypeptide that emerges at the outer membrane surface is apparently more highly exposed in the case of opsin in microsomal membranes, evidenced by the additional internal thermolysin cleavage sites detected.
Isolation of the intercellular glycoproteins of desmosomes.
Authors: Gorbsky G , Steinberg M S
Published: May 2004

To characterize the desmosome components that mediate intercellular adhesion and cytoskeletal-plasma membrane attachment, we prepared whole desmosomes and isolated desmosomal intercellular regions (desmosomal "cores") from the living cell layers of bovine muzzle epidermis. The tissue was disrupted in a nonionic detergent at low pH, sonicated, and the insoluble residue fractionated by differential centrifugation and metrizamide gradient centrifugation. Transmission electron microscopic analyses reveal that a fraction obtained after differential centrifugation is greatly enriched in whole desmosomes that possess intracellular plaques. Metrizamide gradient centrifugation removes most of the plaque material, leaving the intercellular components and the adjoining plasma membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with methods that reveal carbohydrate-containing moieties on gels demonstrate that certain proteins present in whole desmosomes are glycosylated. These glycoproteins are specifically and greatly enriched in the desmosome cores of which they are the principal protein constituents, and thus may function as the intercellular adhesive of the desmosome.
Detection and characterization of monoclonal antibodies to platelet membrane proteins.
Authors: Newman P J , Kahn R A , Hines A
Published: May 2004

We have devised a solid-phase radioimmunoassay for the detection and characterization of monoclonal antibodies directed against platelet surface antigens. Platelet membrane proteins, solubilized with 0.1% Triton X-100, were covalently coupled to cyanogen bromide (CNBr)-activated filter paper disks that were than used as the support in antibody binding assays. SDS PAGE of solubilized membrane proteins taken immediately before and after incubation with activated disks indicated that representative amounts of each membrane protein were bound to the disks. Either monoclonal or heterologous anti-platelet antibody could be detected on disks that had been prepared using as little as 50 micrograms of membrane protein per 100 disks. For the detection of antibody, disks were incubated with test sera for 2 h, washed, and incubated with 125I-labeled anti-immunoglobulin G, and the amount of bound radioactivity was determined. The sensitivity of the disk assay in detecting monoclonal antibodies was far greater than that of a corresponding radioimmunoassay that used whole platelets as the solid phase. By linking other proteins such as fibrinogen or anti-mouse subclass specific antisera to CNBr-activated disks, the method was adapted for antibody characterization. The sensitivity and ease with which the assay can be performed make this technique most suitable for screening and characterizing monoclonal antibodies.
Distribution of kinetochore (centromere) antigen in mammalian cell nuclei.
Authors: Moroi Y , Hartman A L , Nakane P K , Tan E M
Published: May 2004

Antigens associated with mammalian centromeres were localized at the high and electron microscopic levels using the peroxidase-labeled antibody method. The antibody used was of a type naturally occurring in the sera of patients with scleroderma. At the light microscopic level, it reacts specifically with the centromere regions of chromosomes in a variety of mammalian species and strains in discrete foci in interphase nuclei. We find that the number of foci approximates the number of chromosomes present in the various cell types. At the ultrastructural level, the antigenic foci are confirmed to lie in the kinetochore regions of each chromosome. In interphase nuclei, the antigenic foci were usually associated either with the inner surfaces of the nuclear envelope or with the nucleoli. These observations indicate that the centromere regions of the chromosomes in interphase are not randomly distributed within the nucleus but are usually fixed either to the inner surface of the nuclear envelope or to nucleoli.
Phagocytosis of gelatin-latex particles by a murine macrophage line is dependent on fibronectin and heparin.
Authors: van de Water L , Schroeder S , Crenshaw E B , Hynes R O
Published: May 2004

It has been suggested that fibronectin plays a role in clearing particles from the circulation by promoting binding to phagocytes of the reticuloendothelial system. By use of a well-defined system to investigate the possible opsonic role of fibronectin, we have studied the uptake of gelatin-coated latex particles by a murine macrophage cell line (P388D1). Fibronectin promotes binding of gelatin-coated beads to these cells in both suspension and monolayer cultures. In both cases there is a requirement for heparin as a cofactor. Other glycosaminoglycans (chondroitin sulfates A and C, dermatan sulfate, and keratan sulfate) were inactive, whereas heparan sulfate was somewhat active. Proof that beads were actually endocytosed was obtained by electron microscopy, which showed beads internalized in membrane-bounded vesicles, and by immunofluorescence analyses, using antibodies to fibronectin to stain external beads. Two rapid assays for the opsonic activity of fibronectin were developed based on differential centrifugation of cell-associated beads and on the immunofluorescence procedure. Binding and endocytosis were time- and temperature-dependent and varied with the amount of gelatin on the beads and with the concentrations of fibronectin and heparin added, and could be inhibited by F(ab')2 antifibronectin. These studies provide a sound basis for a detailed analysis of the interaction of fibronectin with the cell surface and of its involvement in endocytosis.
Beginning of exocytosis captured by rapid-freezing of Limulus amebocytes.
Authors: Ornberg R L , Reese T S
Published: May 2004

Structural changes underlying exocytosis evoked by the application of endotoxin to Limulus amebocytes were studied at the level of detail afforded by freeze-fracture and freeze-substitution techniques combined with the time resolution of direct rapid-freezing. The results with amebocytes prepared in this manner differed from those with other secretory cells prepared by conventional means. Exocytosis begins within seconds of endotoxin treatment when the plasmalemma invaginates to form pedestallike appositions with peripheral secretory granules. The juxtaposed membranes at these pedestal appositions form several punctate pentalaminar contacts, but examination of freeze-fractured pedestals failed to reveal any corresponding changes in the intramembrane particle distribution. Small secretory granule openings or pores, which are very infrequent, appear within the first 5 s after endotoxin treatment. These pores rapidly widen and this widening is immediately followed by the sequential dissolution of the granule contents, which then move into the surrounding extracellular space. Cytoplasmic filaments connecting the plasmalemma with the granule membrane are suitably deployed to be responsible for the plasmalemma invaginations. How pores begin is not certain, but the appearance of clear spaces between the granule core and the granule membrane at this point in exocytosis supports the possibility of a role of osmotic forces.
Bead rings at the endoplasmic reticulum-golgi complex boundary: morphological changes accompanying inihibition of intracellular transport of secretory proteins in arthropod fat body tissue
Authors: Brodie DA
Published: May 2004

Golgi complex beads are 10-nm particles arranged in rings on the smooth surface of rough endoplasmic reticulum (ER) makind the forming face of the Golgi complex (GC). In arthropod cells they stain specifically with bismuth. Their morphology has been studied after treatment with reagents known to interfere with GC function. Inhibitors of oxidative phosphorylation (antimycin A, cyanide, and anoxia), but not an inhibitor of glycolysis (iodoacetate), both cause the bead rings to collapse and the GC saccules to round up, and inhibit transition vesicle (TV) formation. Cycloheximide blocks protein synthesis on ribosomes but does not stop TV formation or disrupt bead rings, even after prolonged treatment (6 h) to allow emptying of the rough ER cisternae. Thus the collapse of bead rings is not attributable to inhibition of protein synthesis, and the ring structure of beads does not require continued protein synthesis and secretion for its maintenance. Valinomycin has effects on the GC similar to those of antimycin A, but A23187, monensin, and lasalocid do not affect bead ring structure or TV formation. These results are consistent with valinomycin's secondarily uncoupling mitochondria, which collapses bead rings and prevents TV formation. Thus inhibitors of oxidative phosphorylation do not influence the beads through cation movement. Because mononsin and lasalocid block secretion at the level of the condensing vacuoles, bead rings are not influenced by blocks in secretion distal to them or by the backup of secretory material. These experiments are consistent with inhibitors of oxidative phosphorylation collapsing bead rings by decreasing intracellular ATP. The concomitant block to TV formation and the collapse of bead rings suggests that integrity of the bead rings is essential for the transport of secretory material from the rough ER to the GC.
Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules.
Authors: Fletcher D J , Quigley J P , Bauer G E , Noe B D
Published: May 2004

The conversion of proglucagon and proinsulin by secretory granules isolated from both prelabeled and unlabeled anglerfish islets was investigated. Either granules isolated from tissue labeled with [3H]tryptophan and [14C]isoleucine or [35S]cysteine, or lysed granules from unlabeled tissue to which exogenously labeled prohormones had been added were incubated under various conditions. Acetic acid extracts of these granule preparations were analyzed for prohormone and hormone content by gel filtration. Both prelabeled and lysed, unlabeled secretory granules converted radiolabeled precursor peptides (Mr 8,000-15,000) to labeled insulin and glucagon. The accuracy of the cleavage process was established by demonstrating comigration of products obtained from in vitro cleavage with insulin and glucagon extracted from intact islets using electrophoresis and high-pressure liquid chromatography (HPLC). The pH optimum for granule-mediated conversion was found to be in the range of pH 4.5-5.5. Conversion of both proglucagon and proinsulin by secretory granules was significantly inhibited in the presence of antipain, leupeptin, p-chloromercuribenzoate (PCMB) or dithiodipyridine (DDP) but not chloroquine, diisopropyl fluorophosphate, EDTA, p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, or N-p-tosyl-L-lysine chloromethyl ketone HCl. The inhibitory action of PCMB and DDP was reversed in the presence of dithiothreitol. Both membranous and soluble components of the secretory granules possessed significant converting activity. HPLC and electrophoretic analysis of cleavage products demonstrated that the converting activities of the membranous and soluble components were indistinguishable. The amount of inhibition of proinsulin and proglucagon conversion caused by 600 micrograms/ml porcine proinsulin was significantly lower than that caused by the same concentration of unlabeled anglerfish precursor peptides. These results indicate that the proinsulin and proglucagon converting enzyme(s) in the anglerfish pancreatic islet is a unique intracellular thiol proteinase(s) that may be granule membrane-associated and may require the presence of prohormone sequences in addition to the dibasic residues at cleavage sites for substrate recognition and/or binding.
In vitro transcription of cloned 5S RNA genes of the newt Notophthalmus.
Authors: Kay B K , Schmidt O , Gall J G
Published: May 2004

Recombinant plasmids that carried genes coding for 5S ribosomal RNA of the newt, Notophthalmus viridescens, were transcribed in vitro with extracts of Xenopus laevis oocyte nuclei. Plasmids containing multiple repeats of the 5S gene and spacer directed accurate transcription of 5S RNA (120 bases). Individual repeat units were recloned by inserting Sau 3A restriction fragments into the Bam HI site of plasmid pBR322. Because each repeat was cut by the enzyme within the coding region, the inserts had incomplete coding regions at their ends and spacer sequences in the middle. The DNA of these subclones directed synthesis of a 5S-size RNA that contained both plasmid and 5S RNA sequences. Transcription initiated in the vector, proceeded through the gene segment coding for nucleotides 41-120, and terminated at the end of the gene. The initiation of in vitro transcription required neither the original 5' flanking sequences of the spacer nor the first third of the gene. We conclude that intragenic DNA sequences control the initiation of transcription. Other subclones that include pseudogenes gave rise to some transcripts 156 nucleotides long. These long transcripts represented continuation of transcription through the 36-base-pair pseudogene that is located immediately downstream from the 5S gene. However, most transcripts of these subclones terminated at the end of the normal gene before the beginning of the pseudogene. It is probable that a run of four or more Ts serves as part of the termination signal.
Immunofluorescence analysis of the time-course of extinction, reexpression, and activation of albumin production in rat hepatoma-mouse fibroblast heterokaryons and hybrids.
Authors: Mével-Ninio M , Weiss M C
Published: May 2004

We have used a combination of a sensitive immunocytochemical stain for intracellular albumin, and Hoechst 33258 dye for identification of parental nuclei to investigate the time-course of extinction, reexpression, and activation of albumin production in fusion products of 1s (hyperdiploid) or 2s (hypertetradiploid) rat hepatoma cells with mouse fibroblasts (L cells or embryonic cells). In all combinations, the initial event is extinction of albumin production. Extinction occurs immediately after fusion when the mouse fibroblast is a normal embryonic (senescent?) cell. In the case of an L cell, rat albumin is synthesized and secreted during the first 12 h after fusion; no production of mouse albumin occurs. Thereafter, albumin production ceases. 8-12 d after fusion, young hybrid colonies are found to resume the synthesis of rat albumin (reexpression), and several days later the production of mouse albumin begins (activation). The patterns of reexpression and activation indicate (a) that chromosome loss is not necessary for either event to occur and (b) that the cells active in the synthesis of mouse albumin are a subpopulation of those cells already engaged in the production of rat albumin. We conclude that (a) extinction is mediated by diffusible factor(s) from the L-cell parent that act in the hepatoma nucleus to prevent the formation of new albumin messenger RNA; (b) reexpression and activation are gene dosage-dependent but extinction is not; and (c) previously active genes are more rapidly expressed than previously silent ones.
Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth
Authors: Castellot JJ , Addonizio ML , Rosenberg R , Karnovsky MJ
Published: May 2004

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.
Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells.
Authors: Mayrand S , Setyono B , Greenberg J R , Pederson T
Published: May 2004

The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A)+ hnRNA in intact HeLa cells, as revealed by a novel in situ RNA-protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the greater than 42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A)+ hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in Cs2SO4 density gradients. These results establish that poly(A)+ hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo. This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modulating hnRNA structure and processing in the cell.
In vitro uptake and processing of prezein and other maize preproteins by maize membranes.
Authors: Burr F A , Burr B
Published: May 2004

A cell-free, mRNA-dependent system has been developed for the translation and processing of zein preproteins. A rough endoplasmic reticulum (RER)-enriched fraction, isolated by sucrose density gradients, can be treated with micrococcal nuclease to destroy endogenous messages. When these membranes are added to a wheat germ protein-synthesizing system together with zein mRNA, synthesis and processing of the polypeptides to the mature products takes place. The RER fraction from the endosperm has a different protein composition than that prepared from either the shoot or nucellar tissue and processes prezein more efficiently. The cleavage of the preproteins appears to be a cotranslational step as the completed preprotein chains cannot be processed, although they can be taken up to a limited extent. This small uptake, or absorption, or unprocessed zein seems to be an artifact and may be related to the unusual solubility properties of zein. Finally a sodium dodecyl sulfate (SDS)-urea polyacrylamide gel system has been developed which is particularly suited for the separation of low molecular weight proteins (less than 10,000 daltons). Using this method, we examined the products of in vitro zein processing and detected no presequence polypeptides. This suggests that the zein cleavage proteinase is probably an exopeptidase.
Golgi apparatus, GERL, and secretory granule formation within neurons of the hypothalamo-neurohypophysial system of control and hyperosmotically stressed mice.
Authors: Broadwell R D , Oliver C
Published: May 2004

The vasopressin-producing neurons of the hypothalamo-neurohypophysial system are a particularly good model with which to consider the relationship between the Golgi apparatus nd GERL and their roles in secretory granule production because these neurons increase their synthesis and secretion of vasopressin in response to hyperosmotic stress. Enzyme cytochemical techniques for acid phosphatase (AcPase) and thiamine pyrophosphatase (TPPase) activities were used to distinguish GERL from the Golgi apparatus in cell bodies of the supraoptic nucleus from normal mice, mice hyperosmotically stressed by drinking 2% salt water, and mice allowed to recover for 5-10 d from hyperosmotic stress. In nonincubated preparations of control supraoptic perikarya, immature secretory granules at the trans face of the Golgi apparatus were frequently attached to a narrow, smooth membrane cisterna identified as GERL. Secretory granules were occasionally seen attached to Golgi saccules. TPPase activity was present in one or two of the trans Golgi saccules; AcPase activity appeared in GERL and attached immature secretory granules, rarely in the trans Golgi saccules, and in secondary lysosomes. As a result of hyperosmotic stress, the Golgi apparatus hypertrophied, and secretory granules formed from all Golgi saccules and GERL. Little or no AcPase activity could be demonstrated in GERL, whereas all Golgi saccules and GERL-like cisternae were TPPase positive. During recovery, AcPase activity in GERL returned to normal; however, the elevated TPPase activity and secretory granule formation seen in GERL-like cisternae and all Golgi saccules during hyperosmotic stress persisted. These results suggest that under normal conditions GERL is the predominant site for the secretory granule formation, but during hyperosmotic stress, the Golgi saccules assume increased importance in this function. The observed cytochemical modulations in Golgi saccules and GERL suggest that GERL is structurally and functionally related to the Golgi saccules.
Demonstration of contractility of circumferential actin bundles and its morphogenetic significance in pigmented epithelium in vitro and in vivo.
Authors: Owaribe K , Kodama R , Eguchi G
Published: May 2004

Each pigmented epithelial cell bears circumferential actin bundles at its apical level when the pigmented epithelium is established in eyes in situ or in culture in vitro. Well-differentiated pigmented epithelia in culture were treated with a 50% glycerol solution containing 0.1 M KCl, 5 mM EDTA, and 10 mM sodium phosphate buffer, pH 7.2, for 24 h or more at 4 degrees C. When the glycerinated epithelium was transferred to the ATP solution, each cell constituting the epithelium began to contract. The epithelium was cleaved into many cell groups as a result of contraction of each cell. The periphery of each cell group was lifted to form a cup or vesicle and eventually detached from the substratum. However, those cells that had not adhered tightly and not formed a monolayer epithelium with typical polygonal cellular pattern contracted independently as observed in the glycerinated fibroblasts. Contraction of the glycerinated pigmented epithelial cells was inhibited by N-ethylmaleimide but not by cytochalasin B. ITP and UTP also effected the contraction of the glycerinated cells, but GTP and ADP did not. Ca2+ was not required. This contractile model of pigmented epithelium provides a useful experimental system for analyzing the function of actin in cellular morphogenesis.
Tandemly repeated C-C-C-C-A-A hexanucleotide of Tetrahymena rDNA is present elsewhere in the genome and may be related to the alteration of the somatic genome.
Authors: Yao M C , Blackburn E , Gall J
Published: May 2004

The ribosomal RNA genes of the Tetrahymena macronucleus exist as extrachromosomal, linear molecules. The termini of these molecules have been shown to contain the tandemly repeated hexanucleotide (C-C-C-C-A-A)n. In this study the same or related sequences were found in other locations of the genome. Using the depurination method, we showed that macronuclear DNA contained this sequence even after rDNA had been removed. The sequence was found mainly in the repetitive fraction of the DNA. The presence of this sequence in both the macronucleus and the micronucleus was also shown by Southern hybridization using C-C-C-C-A-A repeat as a probe. Comparison between the hybridization patterns of macronuclei and micronuclei reveals interesting differences. Whereas the two nuclei share the same genetic origin, the majority of the restriction enzyme digestion sites flanking the C-C-C-C-A-A repeat appear to be different. Such a difference was found to be specific for this sequence, because it was not detected when other sequences were used for hybridization. These results suggest that some kind of alteration has occurred in the genome during the formation of the macronucleus, and that the C-C-C-C-A-A repeat may be related to this process.