bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell-to-cell communication and myogenesis.
Authors: Kalderon N , Epstein M L , Gilula N B
Published: May 2004

Cell-to-cell communication was characterized in prefusion chick embryo myoblast cultures, and it was determined that the prefusion myoblasts can interact via gap junctions, ionic coupling, and metabolic coupling. The biological relevance of this communication was supported by the detection of gap junctions between myoblasts in embryonic muscle. Communication was also examined in fusion-arrested cultures to determine its potential relationship to fusion competency. In cultures that were fusion arrested by treatment with either 1.8 mM ethyleneglycolbis-(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), 3.3 X 10(-6) M 5-bromodeoxyuridine (BUdR), or 1 microgram/ml cycloheximide (CHX), both gap junctions and ionic coupling were present. Therefore, it is possible to conclude that cell communication is not a sufficient property by itself, to generate fusion between myob-asts. The potential role of communication in myogenesis is discusssed with respect to these observations.
Sites of lipoprotein particles in normal rat hepatocytes.
Authors: Novikoff P M , Yam A
Published: May 2004

Very low density lipoprotein (VLDL) particles are packaged by the Golgi apparatus into vacuoles which move to the plasma membrane and empty the particles into the space of Disse, via exocytosis. Traditionally, all lipoprotein-containing cisternae and vacuoles are thought to be parts of this pathway. Observations reported here demonstrate that there is a second population of lipoprotein-containing cisternae and vacuoles. This population is part of GERL, an organelle we consider to be a specialized hydrolase-rich region of the endoplasmic reticulum (ER). To our knowledge, this is the first systematic study of GERL in normal rat hepatocytes.
Regulation of macronuclear DNA content in Paramecium tetraurelia.
Authors: Berger J D , Schmidt H J
Published: May 2004

The macronucleus of Paramecium divides amitotically, and daughter macronuclei with different DNA contents are frequently produced. If no regulatory mechanism were present, the variance of macronuclear DNA content would increase continuously. Analysis of variance within cell lines shows that macronuclear DNA content is regulated so that a constant variance is maintained from one cell generation to the next. Variation in macronuclear DNA content is removed from the cell population by the regulatory mechanism at the same rate at which it is introduced through inequality of macronuclear division. Half of the variation in macronuclear DNA content introduced into the population at a particular fission by inequality of division is compensated for during the subsequent period of DNA synthesis. Half of the remaining variation is removed during each subsequent cell cycle. The amount of variation removed in one cell cycle is proportional to the postfission variation. The cell's power to regulate DNA content is substantially greater than that required to compensate for the small differences that arise during division of wild-type cells. For example, a constant variance was still maintained when the mean difference between sister cells was increased to ten times its normal level in a mutant strain. The observations are consistent with a replication model that assumes that each cell synthesizes an approximately constant amount of DNA which is independent of the initial DNA content of the macronucleus. It is suggested that the amount of DNA synthesized may be largely determined by the mass of the cell.
Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure
Authors: Moore PL , Bank HL , Brissie NT , Spicer SS
Published: May 2004

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane.

During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.
A freeze-fracture and lanthanum tracer study of the complex junction between Sertoli cells of the canine testis.
Authors: Connell C J
Published: May 2004

What appear to be true septate junctions by all techniques currently available for the cytological identification of intercellular junctions are part of a complex junction that interconnects the Sertoli cells of the canine testis. In the seminiferous epithelium, septate junctions are located basal to belts of tight junctions. In thin sections, septate junctions appear as double, parallel, transverse connections or septa spanning an approximately 90-A intercellular space between adjacent Sertoli cells. In en face sections of lanthanum-aldehyde-perfused specimens, the septa themselves exclude lanthanum and appear as electron-lucent lines arranged in a series of double, parallel rows on a background of electron-dense lanthanum. In freeze-fracture replicas this vertebrate septate junction appears as double, parallel rows of individual or fused particles which conform to the distribution of the intercellular septa. Septate junctions can be clearly distinguished from tight junctions as tight junctions prevent the movement of lanthanum tracer toward the lumen, appear as single rows of individual or fused particles in interlacing patterns within freeze-fracture replicas, and are seen as areas of close membrane apposition in thin sections. Both the septate junction and the tight junction are associated with specializations of the Sertoli cell cytoplasm. This is the first demonstration in a vertebrate tissue of a true septate junction.
Particle arrays in earthworm postjunctional membranes.
Authors: Rosenbluth J
Published: May 2004

Analysis of freeze-fractured earthworm body wall muscle reveals distinctive trough-shaped concavities in the protoplasmic leaflet of the muscle cell membrane which contain diagonally oriented rows of particles sometimes in highly ordered arrays. The troughs correspond to the concave postjunctional patches of sarcolemma seen previously in thin sections of myoneural junctions identified as cholinergic, and the intramembranous particles within the troughs correspond in concentration and arrangement to granular elements present in the outer dense lamina of the postjunctional membrane which were interpreted as acetylcholine receptors. The freeze-fracture data provide a more accurate picture of the arrangement of these putative receptors within the plane of the membrane, and indicate also that they extend into the membrane at least as far as its hydrophobic layer.
DNA polymerase activity in muscle cultures
Authors: Yeoh GC , Greenstein D , Holtzer H
Published: May 2004

Nuclei within myotubes do not synthesize DNA for replication. Accordingly, cultures of myotubes display low levels of DNA polymerase activity. The coincidental decline in DNA polymerase activity and increased formation of multinucleated myotubes during culture does not prove that the loss of capacity to synthesize DNA is a consequence of fusion. Tne experiments described demonstrate that myogenic cells prevented from fusing have low levels of DNA polymerase activity. This is consistent with the notion that, in myogenic cultures, there is a population of mononucleated cells, the myoblasts, which have withdrawn from the mitotic cycle before fusion.
Electrophorus acetylcholinesterase. Biochemical and electron microscope characterization of low ionic strength aggregates
Authors: Cartaud J , Bon S , Massoulié J
Published: May 2004

The "tailed" molecules of Electrophorus (electric eel) acetylcholinesterase aggregate under conditions of low ionic strength. These aggregates have been studied by sedimentation analysis and high-resolution electron microscopy. They consist of bundles of at least half a dozen molecules, the tails of which are packed side by side, to form the core of the structure. Although aggregation is normally fully reversible, aggregates were irreversibly stabilized by methylene blue-sensitized photo-oxidation. This process was shown to consist of a singlet oxygen oxidation reaction and probably involves methionine or histidine residues. It did not modify the structural or hydrodynamic characteristics of the aggregates.
Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid
Authors: Stoner CD , Sirak HD
Published: May 2004

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.
Actin in Xenopus oocytes
Authors: Clark TG , Merriam RW
Published: May 2004

It has been found that a high-speed supernatant fraction from Xenopus oocytes extracted in the cold will form a clear, solid gel upon warming. Gel formation occurs within 60 min at 18 degrees-40 degrees C, and is, at least initially, temperature reversible. Gelation is strictly dependent upon the addition of sucrose to the extraction medium. When isolated in the presence of ATP, the gel consists principally of a 43,000-dalton protein which co-migrates with Xenopus skeletal muscle actin on SDS-polyacrylamide gels, and a prominent high molecular weight component of approx. 250,000 daltons. At least two minor components of intermediate molecular weight are also found associated with the gel in variable quantities. Actin has been identified as the major consituent of the gel by ultrastructural and immunological techniques, and comprises roughly 47% of protein in the complex. With time, the gel spontaneously contracts to form a small dense aggregate. Contraction requires ATP. In the absence of exogenous ATP, a polypeptide which co-migrates with the heavy chain of Xenopus skeletal muscle myosin becomes a prominent component of the gel. This polypeptide is virtually absent from gels which have contracted in ATP-containing extracts. It has also been found that Ca++ is required for gelation in oocyte extracts. At both low and high concentrations of Ca++ (defined as a ratio of Ca++/EGTA in the extraction medium), gelation is inhibited.
Nuclear surface complex as observed with the high resolution scanning electron microscope. Visualization of the membrane surfaces of the neclear envelope and the nuclear cortex from Xenopus laevis oocytes
Authors: Schatten G , Thoman M
Published: May 2004

The nuclear envelope and associated structures from Xenopus laevis oocytes (stage VI) have been examined with the high resolution scanning electron microscope (SEM). The features of the inner and outer surfaces of the nuclear surface complex were revealed by manual isolation , whereas the membranes facing the perinuclear space (the space between the inner and outer nuclear membranes) were observed by fracturing the nuclear envelope in this plane and splaying the corresponding regions apart. Pore complexes were observed on all four membrane surfaces of this double-membraned structure. The densely packed pore complexes (55/micron2) are often clustered into triplets with shared walls (outer diameter = 90 nm; inner diameter = 25 nm; wall thickness = aproximately 30 nm), and project aproximately 20 nm above each membrane except where they are flush with the innermost surface. The pore complex appears to be an aggregate of four 30-nm subunits. The nuclear cortex, a fibrous layer (300 nm thickness) associated with the inner surface of the nuclear envelope, has been revealed by rapid fixation. This cortical layer is interrupted by funnel-shaped intranuclear channels (120-640 nm diam) which narrow towards the pore complexes. Chains of particles, arranged in spirals, are inserted into these intranuclear channels. The fibers associated with the innermost face of the nuclear envelope can be extraced with 0.6 MKI to reveal the pore complexes. A model of the nuclear surface complex, compiled from the visualization of all the membrane faces and the nuclear cortex, demonstrates relations between the intranuclear channels (3.2/micron2) and the numerous pore complexes, and the possibility of their role in nucleocytoplasmic interactions.
Analogous ultrastructure and surface properties during capping and phagocytosis in leukocytes
Authors: Berlin RD , Oliver JM
Published: May 2004

Ultrastructural analyses have revealed striking similarities between Concanavalin A capping and phagocytosis in leukocytes. Both processes involve extensive membrane movement to form a protuberance or pseudopods; a dense network of microfilaments is recruited into both the protuberance and the pseudopods; microtubules are disassembled either generally (capping) or in the local region of the pseudopods (phagocytosis); and cells generally depleted of microtubules by colchicine show polarized phagocytosis via the microfilament-rich protuberance rather than uniform peripheral ingestion of particles via individual pseudopods. Cap formation can thus be viewed as occurring as an exaggeration of the same ultrastructural events that mediate phagocytosis. Similar changes in cell surface topography also accompany capping and phagocytosis. Thus, in nonfixed cells, Concanavalin A-receptor complexes aggregate into the region of the protuberance in colchicine-treated leukocytes (conventional capping) or into the region of pseudopod formation in phagocytizing leukocytes. In the latter case, the movement of lectin-receptor complexes occurs from membrane overlying peripheral microtubules into filament-rich pseudopods that exclude microtubules. These data provide evidence against a role for microtubules as "anchors" for lectin receptors. Rather, they indicate a preferential movement of cell surface Concanavalin A-receptor complexes towards areas of extensive (the protuberance) or localized (pseudopods) microfilament concentration. In conventional capping, Concanavalin A must be added to the colchicine-treated cells before fixation in order to demonstrate movement of receptors from a diffuse distribution into the protuberance. However, Convanavalin A receptors are enriched in the membrane associated with phagocytic particles as compared to the remaining membrane. This particle-induced redistribution of receptors is particularly prominent in colchicine-treated cells that phagocytize and are then fixed and Concanavalin A labeled; both lectin receptors and beads are concentrated over the protuberance. Thus, the final analogy between conventionally capped and phagocytic cells is that in both cases the properties of the plasma membrane in regions of microfilament concentration are modified by Concanavalin A itself (capping) or by the phagocytized particle, to limit locally the diffusion of Concanavalin A receptors.
Biochemical and stereological analysis of rat liver mitochondria in different thyroid states
Authors: Jakovcic S , Swift HH , Gross NJ , Rabinowitz M
Published: May 2004

The concentrations of the inner mitochondrial membrane markers cardiolipin and cytochrome alpha have been measured in liver homogenates and in purified mitochondria after thyroxine administration to thyroidectomized and normal rats. The biochemical results have been correlated with stereological electron micrographic analyses of hepatocytes in liver sections, and of isolated mitochondrial pellets. There were progressive and parallel increases in homogenate and mitochondrial cardiolipin concentration, and in mitochondrial cytochrome alpha concentration, after administration of 20 microgram of thyroxine on alternate days to thyroidectomized rats, and of 300 microgram on alternate days to normal rats. Electron microscope measurements showed marked differences in the shape of the mitochondria and in the number of cristae in different thyroid states. Hypothyroid mitochondria were shorter and wider than controls, and hyperthyroid mitochondria longer but of similar width. Mitochondrial volume per unit cell volume was virtually unchanged in hypo- and hyperthyroid animals. The most striking changes were a decrease in the area of the inner membrane plus cristae in thyroidectomized rats, and a substantial increase in membrane area after thyroxine administration. The biochemical and electron micrographic results indicate that, in rat liver, thyroid hormone administration leads to a selective increase in the relative amount of mitochondrial inner membranes, with little or no change in the mitochondrial volume per unit cell volume, or in total mitochondrial protein per unit total cell protein.
Membrane potentials and resistances of giant mitochondria. Metabolic dependence and the effects of valinomycin.
Authors: Maloff B L , Scordilis S P , Reynolds C , Tedeschi H
Published: May 2004

The membrane potentials and resistances of giant mitochondria from mice fed cuprizone have been studied. They were found to correspond approx. 10-20 mV, positive inside, and 2 M omega, respectively. These properties were found to be independent of the metabolic state. The microelectrodes were in the inner mitochondrial space since (a) the potentials in the presence of valinomycin depended on the K+ concentration of the medium and magnitude of the K+ diffusion potentials was consistent with the presence of a high internal concentration of K+, (b) almost identical results were obtained with mitochondria from which the external membrane had been removed and the cristae were evaginated, and (c) punch-through experiments, in which the microelectrodes were advanced until they emerged through the other side of the mitochondria, showed an identical membrane potential both in the presence and in the absence of valinomycin. The potentials were stable under a variety of conditions and showed no sign of decay of membrane leakiness. Detailed evidence that the impaled mitochondria are metabolically viable will be presented in a separate publication.
Derivatized silica spheres as immunospecific markers for high resolution labeling in electron microscopy.
Authors: Peters K R , Rutter G , Gschwender H H , Haller W
Published: May 2004

For high resolution labeling of influenza virus cell surface antigens on HeLa cells, an immunospecific marker is used with silica sphere cores of 13--14 nm average diameter. These markers are formed using commercially available silica sphere sols. Two other size ranges are available, 7--8 nm and 22--25 nm. The steps for chemical derivatization are described in detail. Amino and aldehyde functions are covalently introduced onto the sphere surface. Sols of these derivatized silica spheres (DSS) are physicochemically stable and therefore usable for years. Coupling of IgG to DSS followed by permeation chromatography on controlled pore glass results in size-defined immunospecific silica sphere markers (DSS-markers). Saturation labeling of cell surface antigens on HeLa cells on cover slips is obtained with the final sphere concentration of 10(14) DSS-marker/cm3 within 20 min. With usual protective conditions, the marker stability and labeling ability are preserved for months. The visibility and the fine structure of the DSS-marker on cell surfaces are shown by using transmission electron microscopy (TEM) with stereo replicas and ultrathin sections.
Tyrosyltubulin ligase and colchicine binding activity in synchronized Chinese hamster cells.
Authors: Forrest G L , Klevecz R R
Published: May 2004

Tyrosyltubulin ligase (TTL) was found to be present in CHO and V79 Chinese hamster cells grown in tissue culture. The enzyme is soluble and requires potassium, magnesium, and ATP for maximum activity and requires tubulin as a substrate. TTL was analyzed through the cell cycle of V79 and CHO Chinese hamster cells. The enzyme showed two peaks of activity in V79 cells at 4 h and 7 h after mitotic selection, corresponding to the early S and mid to late S phases of the cell cycle. In CHO cells the enzyme displayed a major peak of activity at mid S and a minor peak or plateau during early S. Tubulin, as measured by (3H)colchicine binding, was shown to increase through S phase and reach a maximum late in the cycle during G2 approx. 3 h after maximum TTL activity.
Intracellular transport of proteins in active and resting secretory cells of the venom gland of Vipera palaestinae.
Authors: Oron U , Bdolah A
Published: May 2004

The intracellular transport of venom proteins has been studied in active and resting venom glands of the snake Vipera palaestinae by electron microscope radioautography after an intra-arterial injection of [3H]leucine. In the active gland, most of the label is initially (10 min) found over the RER. By 30 min, the relative grain density of the Golgi complex reaches its maximum, with concomitant increase in the labeling of the condensing vacuoles. Later on, a steep increase in radioactivity of the secretory granules is observed. At 3 h, these granules, which comprise about 2% of the cell volume, contain 22% of the total grains. At the following hour, their labeling declines and at the same time the radioactivity of the secreted venom is increased. It is concluded that, in the active cell, venom proteins are transported via the Golgi apparatus into membrane-bounded granules which are the immediate source of the secreted venom. An alternative pathway, which involves the RER cisternae as a storage compartment, seems unlikely, since incorporated label does not accumulate in this compartment after prolonged postpulse intervals. The route of intracellular transport of proteins in the resting glands is similar to that of the active ones, but the rate of synthesis and transport is much slower. The present results and earlier data, thus, show that the increase in the rate of secretion after initiation of a new venom regeneration cycle is the result of accelerated rates of both synthesis and transport.
Dynamics of toxin and lectin receptors on a lymphoma cell line and its toxin-resistant variant using ferritin-conjugated, 125I-labeled ligand.
Authors: Nicolson G L , Smith J R , Hyman R
Published: May 2004

The dynamics of the toxin Ricinus communis agglutinin II (RCAII or ricin) on cells of a murine lymphoma line (BW5147) and a toxin-resistant variant line (BW5147RicR.3) that is 200 times more resistant than the parent to direct RCAII cytotoxicity were examined using ferritin-conjugated, affinity purified, 125I-labeled RCAII (ferritin-125I-RCAII). Ferritin-125I-RCAII was indistinguishable from native RCAII in quantitative binding and cytotoxicity experiments. When RCAII-sensitive BW5147 and -resistant BW5147RicR.3 cells were labeled with ferritin-125I-RCAII at various toxin concentrations (1--10 microgram/ml), no differences in toxin binding were observed. These same cells were examined by electron microscopy. At low ferritin-125I-RCAII concentrations (1-3 microgram/ml RCAII) where only the parental BW5147 cells were significantly more sensitive to RCAII, toxin receptors were internalized by ferritin-125I-RCAII-induced endocytosis. In parallel experiments, ferritin-125I-RCAII that bound to the resistant BW5147RicR.3 cells remained relatively dispersed or clustered, and there was little evidence of transport into cells via endocytosis. At higher ferritin-125I-RCAII concentrations (greater than 7 microgram/ml RCAII) where both parental and resistant variant cells are sensitive to the cytotoxic effects of RCAII, more ferritin-conjugated toxin was bound, and subsequent endocytosis occurred to a similar degree in both cell types. Endocytosis of ferritin-conjugated concanavalin A was indistinguishable on RCAII-sensitive parental and resistant variant cells at all concentrations tested. The results suggest that a specific defect on the selected BW5147RicR.3 cells prevents RCAII entry into these cells a low toxin concentrations, rendering them more resistant to the cytotoxic effects of RCAII.
Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes.
Authors: Elias P M , Goerke J , Friend D S , Brown B E
Published: May 2004

To advance our understanding of the organization of cholesterol within cell membranes, we used digitonin in freeze-fracture investigations of model lipid vesicles and tissues. Cholesterol suspensions or multilamellar liposomes composed of phosphatidylcholine with and without cholesterol were exposed to digitonin. Freeze-fracture replicas of those multilamellar liposomes containing cholesterol displayed either 50--60-nm wide intramembrane corrugations or extramembrane tubular complexes. Comparable intramembrane hemitubular scallops and extra-cellular free tubular complexes were observed in thin sections. Exposure of sperm, erythrocytes (whole and ghosts), and intact tissues (skin, liver, adrenal gland, epididymis) to digitonin produced the same types of intra- and extramembrane complexes or furrows as were formed in liposomes. The plasma membrane of guinea pig serum tail had two unfurrowed regions: the annulus and the zipper. Incubating erythrocyte membranes with digitonin resulted in rapid displacement of cholesterol, accompanied by intramembrane particle clustering and membrane faceting, a feature which we did not see in the intact epithelia studied. In freeze-fractured epithelia, we found that plasma membranes, lysosomes, and some vesicular organelles commonly furrowed, but that mitochondrial membranes and nuclear envelopes were generally spared, correlating well with their known cholesterol content. Finally, plasma membrane corrugations approached but did not impinge on either gap or tight junctions, or on coated vesicles. We conclude that freeze-fracture of membranes exposed to digitonin: (a) reveals distinctive cholesterol-digitonin structural complexes; (b) distinguishes cholesterol-rich and -poor organelle membranes; and (c) demonstrates membrane domains rich or poor in cholesterol.
Unusual responses of rat hepatic and renal peroxisomes to RMI 14, 514, a new hypolipidemic agent.
Authors: Svoboda D J
Published: May 2004

RMI 14, 514 ([5-tetradecycloxy]-2-furancarboxylic acid) represents a new class of hypolipidemic agents which cause unusual ultrastructural changes in liver of male rats and in selected peroxisomal enzymes in liver and kidney of both sexes. Among the principal ultrastructural changes in peroxisomes of male rat liver were (a) cavitation and compartmentalization of the matrix, often giving the appearance of a peroxisome-within-a-peroxisome, and (b) narrow, dense extensions of canaliculi or cisterns from the periphery of the peroxisome, forming partial circlets or surrounding irregular areas of cytoplasm. The unusual enzyme responses were (a) elevation of catalase activity in liver and kidney in female rats, (b) increased activity of three hydrogen peroxide-producing oxidases (urate oxidase, L-alpha-hydroxy acid oxidase, and D-amino acid oxidase) in the liver of both sexes, and (c) elevation of activity of the last two oxidases in male kidney. The peculiar ultrastructural changes in liver peroxisomes combined with the responses of selected peroxisomal enzymes represent unusual modulations or adaptations of these organelles to a hypolipidemic agent, the effects of which have not been reported extensively.
Sulfation of fucoidin in focus embryos: III. Required for localization in the rhizoid wall
Authors: Hogsett WE , Quatrano RS
Published: May 2004

Zygotes of the brown alga Fucus distichus L. Powell accumulate a sulfated polysaccharide (fucoidin) in the cell wall at the site of rhizoid formation. Previous work indicated that zygotes grown in seawater minus sulfate do not sulfate the preformed fucan (an unsulfated fucoidin) but form rhizoids. Under these conditions, we determined whether sulfation of the fucan is required for its localization in the rhizoid wall. This was accomplished by developing a specific stain for both the fucan and fucoidin. Using a precipitin assay, we demonstrated in vitro that the lectin ricin (RCA(I)) specifically complexes with both the sulfated and desulfated polysaccharide. No precipitate is observed when either is incubated in 0.1 M D-galactose or when RCA(I) is mixed with laminarin or alginic acid, the other major polysaccharides in Fucus. RCA(I) conjugated with fluorescein isothiocyanate (FITC) is also shown to bind specifically to fucoidin using a filter paper (DE81) assay. When added to zygotes, RCA(I)-FITC binds only to the site of fucoidin localization, i.e., the rhizoid cell wall. However, RCA(I)-FITC is not observed in the rhizoid wall of zygotes grown in the absence of sulfate. This observation is not due to inability of RCA(I)-FITC to bind to the fucan in vivo. Chemically desulfated cell walls that contained fucoidin in the rhizoid wall bind RCA(I)-FITC only in the rhizoid region. Also, the concentration of fucose-containing polymers and polysaccharides that form precipitates with RCA(I) is the same in embryos grown in the presence or absence of sulfate. If sulfate is added back to cultures of zygotes grown without sulfate, fucoidin is detected at the rhizoid tip by RCA(I)-FITC several hours later. These results support the conclusion that the enzymatic sulfation of the fucan is a modification of the polysaccharide required for its localization and/or assembly into a specific region of the cell wall.
Fibroblasts and macrophages of mice with the Chediak-Higashi-like syndrome have microtubules and actin cables.
Authors: Frankel F R , Tucker R W , Bruce J , Stenberg R
Published: May 2004

Cells of the beige mouse contain abnormally large lysosomes and show enhanced capping of concanavalin A. It has been suggested that these phenomena may be secondary to a defect in microtubule polymerization. We have examined the cytoskeleton of beige mouse cells by indirect immunofluorescence and find the number and distribution of microtubules and actin cables to be indistinguishable from those of normal control cells.
Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis.
Authors: Drochmans P , Freudenstein C , Wanson J C , Laurent L , Keenan T W , Stadler J , Leloup R , Franke W W
Published: May 2004

Complexes of plasma membrane segments with desmosomes and attached tonofilaments were separated from the stratum spinosum cells of calf muzzle by means of moderately alkaline buffers of low ionic strength and mechanical homogenization. These structures were further fractionated by the use of various treatments including sonication, sucrose gradient centrifugation, and extraction with buffers containing high concentrations of salt, urea, citric acid, or detergents. Subfractions enriched in desmosome-tonofilament-complexes and tonofilament fragments were studied in detail. The desmosome structures such as the midline, the trilaminar membrane profile, and the desmosomal plaque appeared well preserved and were notably resistant to the various treatments employed. Fractions containing desmosome-tonofilament complexes were invariably dominated by the nonmembranous proteins of the tonofilaments which appeared as five major polypeptide bands (apparent molecular weights: 48,000; 51,000; 58,000; 60,000; 68,000) present in molar ratios of approx. 2:1:1:2:2. Four of these polypeptide bands showed electrophoretic mobilities similar to those of prekeratin polypeptides from bovine hoof. However, the largest polypeptide (68,000 mol wt) migrated significantly less in polyacrylamide gels than the largest component of the hoof prekeratin (approximately 63,000 mol wt). In addition, a series of minor bands, including carbohydrate-containing proteins, were identified and concluded to represent constituents of the desmosomal membrane. The analysis of protein-bound carbohydrates (total 270 microgram/mg phospholipid in desmosome-enriched subfractions) showed the presence of relatively high amounts of glucosamine, mannose, galactose, and sialic acids. These data as well as the lipid composition (e.g., high ratio of cholesterol to phospholipids, relatively high contents of sphingomyelin and gangliosides, and fatty acid pattern) indicate that the desmosomal membrane is complex in protein and lipid composition and has a typical plasma membrane character. The similarity of the desmosome-associated tonofilaments to prekeratin filaments and other forms of intermediate-sized filaments is discussed.
Decoration of specific sites on freeze-fractured membranes.
Authors: Gross H , Kuebler O , Bas E , Moor H
Published: May 2004

Fracturing under ultrahigh vacuum (UHV, P less than or equal to 10(-9) Torr) produces membrane fracture faces devoid of contamination. Such clean surfaces are a prerequisite for studies of interactions between condensing gases and distinct regions of a surface. For the study of water condensation, a device has been developed which enables production of pure water vapor and controlled variation of its partial pressure in an UHV freeze-fracture apparatus. Experiments with yeast plasmalemma fracture faces, produced at -196 degrees C and exposed to pure water vapor before replication, resulted in a "specific decoration" with ice crystals of those pits in the extraplasmic face where the corresponding particles of the plasmic face had been removed. Because water condenses as discrete ice crystals which resemble intramembrane particles, ice crystals might easily be misinterpreted as actual membrane structures. At low specimen temperature (T less than or equal to 110 degrees C) the structural features of membrane fracture faces produced under high vacuum (P approximately 10(-6) Torr) should, therefore, be interpreted with caution.
Analysis of the distribution of spindle microtubules in the diatom Fragilaria.
Authors: Tippit D H , Schulz D , Pickett-Heaps J D
Published: May 2004

The spindle of the colonial diatom Fragilaria contains two distinct sets of spindle microtubules (MTs): (a) MTs comprising the central spindle, which is composed of two half-spindles interdigitated to form a region of "overlap"; (b) MTs which radiate laterally from the poles. The central spindles from 28 cells are reconstructed by tracking each MT of the central spindle through consecutive serial sections. Because the colonies of Fragilaria are flat ribbons of contiguous cells (clones), it is possible, by using single ribbons of cells, to compare reconstructed spindles at different mitotic stages with minimal intercellular variability. From these reconstructions we have determined: (a) the changes in distribution of MTs along the spindle during mitosis; (b) the change in the total number of MTs during mitosis; (c) the length of each MT (measured by the number of sections each traverses) at different mitotic stages; (d) the frequency of different classes of MTs (i.e., free, continuous, etc.); (e) the spatial arrangement of MTs from opposite poles in the overlap; (f) the approximate number of MTs, separate from the central spindle, which radiate from each spindle pole. From longitudinal sections of the central spindle, the lengths of the whole spindle, half-spindle, and overlap were measured from 80 cells at different mitotic stages. Numerous sources of error may create inaccuracies in these measurements; these problems are discussed. The central spindle at prophase consists predominantly of continuous MTs (pole to pole). Between late prophase and prometaphase, spindle length increases, and the spindle is transformed into two half-spindles (mainly polar MTs) interdigitated to form the overlap. At late anaphase-telophase, the overlap decreases concurrent with spindle elongation. Our interpretation is that the MTs of the central spindle slide past one another at both late prophase and late anaphase. These changes in MT distribution have the effect of elongating the spindle and are not involved in the poleward movement of the chromosomes. Some aspects of tracking spindle MTs, the interaction of MTs in the overlap, formation of the prophase spindle, and our interpretation of rearrangements of MTs, are discussed.
Effects of adenylyl imidodiphosphate, a nonhydrolyzable adenosine triphosphate analog, on reactivated and rigor wave sea urchin sperm.
Authors: Penningroth S M , Witman G B
Published: May 2004

A nonhydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP), has been used to study the role of ATP binding in flagellar motility. Sea urchin sperm of Lytechinus pictus were demembranated, reactivated, and locked in "rigor waves" by a modification of the method of Gibbons and Gibbons (11). Rigor wave sperm relaxed within 2 min after addition of 4 micrometer ATP, and reactivated upon addition of 10-12 micrometer ATP. The beat frequency of the reactivated sperm varied with ATP concentration according to Michaelis-Menten kinetics ("Km" = 0.24 mM; "Vmax" = 44 Hz) and was competitively inhibited by AMP-PNP (Ki" approximately to 8.1 mM). Rigor wave sperm were completely relaxed (straightened) within 2 min by AMP-PNP at concentrations of 2-4 mM. The possibilities that relaxation in AMP-PNP was a result of ATP contamination, AMP-PNP hydrolysis, or lowering of the free Mg++ concentration were conclusively ruled out. The results suggest that dynein cross-bridge release is dependent upon ATP binding but not hydrolysis.
ELECTRON MICROSCOPE AND X-RAY DIFFRACTION STUDIES OF THE EFFECTS OF DEHYDRATION ON THE STRUCTURE OF NERVE MYELIN
Authors: Finean J. B.
Published: May 2004

The dehydration of frog sciatic nerve has been studied by allowing specimens to become partially or fully dried before fixation and preparation for electron microscopy. Low magnification electron micrographs of OsO4-fixed preparations showed marked tissue shrinkage which could be correlated quantitatively with the loss of water during the preliminary drying. KMnO4-fixation appeared to cause a rehydration of the dried tissue. Higher magnification electron micrographs of the OsO4-fixed preparations showed a sequence of modifications of the myelin layers which could be correlated with changes in the small-angle x-ray diffraction data which were recorded during drying. An intermediate stage of drying was characterised by a partial collapse of layers and a disappearance of the intraperiod dense line in some regions of the myelin sheath. Continuity between collapsed and non-collapsed layers was maintained throughout the sheath. The fully dried preparation showed two main modifications of the myelin layers. In many regions the layers (principal layers) resembled those of normal preparations, but showed an intensification and frequently a doubling of the intraperiod dense line. In addition, there was a very extensive system of fine (40 A periodicity) dense layers, some of which could be demonstrated to be continuous with the principal layers. In such cases it was observed that two of the fine layers were related to each principal layer. The correlation between diffraction data and electron microscope data is discussed, and some speculations are made concerning the molecular significance of the observations.
ELECTRON MICROSCOPIC STUDIES OF MITOSIS IN AMEBAE
Authors: Roth L. E. , Obetz S. W. , Daniels E. W.
Published: May 2004

Individual organisms of Amoeba proteus have been fixed in buffered osmium tetroxide in either 0.9 per cent NaCl or 0.01 per cent CaCl2, sectioned, and studied in the electron microscope in interphase and in several stages of mitosis. The helices typical of interphase nuclei do not coexist with condensed chromatin and thus either represent a DNA configuration unique to interphase or are not DNA at all. The membranes of the complex nuclear envelope are present in all stages observed but are discontinuous in metaphase. The inner, thick, honeycomb layer of the nuclear envelope disappears during prophase, reappearing after telophase when nuclear reconstruction is in progress. Nucleoli decrease in size and number during prophase and re-form during telophase in association with the chromatin network. In the early reconstruction nucleus, the nucleolar material forms into thin, sheet-like configurations which are closely associated with small amounts of chromatin and are closely applied to the inner, partially formed layer of the nuclear envelope. It is proposed that nucleolar material is implicated in the formation of the inner layer of the envelope and that there is a configuration of nucleolar material peculiar to this time. The plasmalemma is partially denuded of its fringe-like material during division.
ON A RETICULAR DERIVATIVE FROM GOLGI BODIES IN THE MERISTEM OF Anthoceros
Authors: Manton Irene
Published: May 2004

Micrographs of dictyosomes in face view and in profile, together with serial sections representing both these planes, are reproduced from three sample cells at different developmental stages in the meristem of Anthoceros. The stages are: a vegetative cell at anaphase of a mitotic division, a vegetative cell in an early stage of postmitotic extension growth, and a young spore mother cell in the act of rounding up before the onset of meiosis. The observations suggest that proliferation of tubules from the edges of the dictyosomal cisternae into the cytoplasm is occurring with varying intensity and with slightly different morphological expression in all three cells. In all, the tubules are joined into a reticulum which exhibits local swellings at varying distances from the unfenestrated part of the subtending cisterna. A comparison is suggested between the observed reticulum and "smooth" endoplasmic reticulum of animals but it is not claimed that all the cytoplasmic tubules detectable in Anthoceros need have arisen in this way. Morphological differences discernible between tubules near their point of attachment to dictyosomes and others apparently involved in the formation of the new nuclear membrane at the end of a cell division could mean that more than one category of tube may exist in these cells. A plea is registered for restraint in the formulation of far reaching theories until more facts are available on unequivocal evidence.
HISTOCHEMICAL DEMONSTRATION OF DEHYDROGENASE ACTIVITY IN THE CELLS OF NORMAL HUMAN BLOOD AND BONE MARROW
Authors: Ackerman G. Adolph
Published: May 2004

Endogenous and succinic dehydrogenase activity was demonstrated in the living cells of normal human blood and bone marrow using a buffered nitro BT-succinate incubating solution. With this technique dehydrogenase activity was localized primarily in the granular leukocytes and the sites of enzymatic activity appeared to be non-mitochondrial. The addition of a non-ionic surface active agent to the incubating solution resulted in marked differences in the cellular and intracellular localization of dehydrogenase activity. With this method it was possible to demonstrate dehydrogenase activity in the mitochondria of most of the formed elements of the blood and bone marrow, including developing granulocytes and erythroid cells, agranulocytes, and blood platelets. Mature erythrocytes also exhibited a minimal dehydrogenase reaction with this procedure. This investigation indicated that in order adequately to demonstrate and evaluate dehydrogenase activity in the cells of the blood and bone marrow it was necessary to have increased cellular and mitochondrial permeability, as well as partially viable cells with an intact dehydrogenase system.
CHANGES IN NUCLEAR HISTONES DURING FERTILIZATION, AND EARLY EMBRYONIC DEVELOPMENT IN THE PULMONATE SNAIL, Helix aspersa
Authors: Bloch David P. , Hew Howard Y. C.
Published: May 2004

Calf thymus histories comprising two fractions, one rich in lysine, the other having roughly equal amounts of lysine and arginine, Loligo testes histones rich in arginine, and salmine, are compared with respect to their amino acid compositions, and their staining properties when the proteins are fixed on filter paper. The three types of basic proteins; somatic, arginine-rich spermatid histones, and protamine can be distinguished on the following basis. Somatic and testicular histones stain with fast green or bromphenol blue under the same conditions used for specific staining of histones in tissue preparations. The former histones lose most or all of their stainability after deamination or acetylation. Staining of the arginine-rich testicular histones remains relatively unaffected by this treatment. Protamines do not stain with fast green after treatment with hot trichloracetic acid, but are stained by bromphenol blue or eosin after treatment with picric acid. These methods provide a means for the characterization of nuclear basic proteins in situ. Their application to the early developmental stages of Helix aspersa show the following: After fertilization the protamine of the sperm is lost, and is replaced by faintly basic histones which differ from adult histones in their inability to bind fast green, and from protamines, by both their inability to bind eosin, and their weakly positive reaction with bromphenol blue. These "cleavage" histones are found in the male and female pronuclei, the early polar body chromosomes, and the nuclei of the cleaving egg and morula stages. During gastrulation, the histone complement reverts to a type as yet indistinguishable from that of adult somatic cells.