bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of 5-bromodeoxyuridine on the ACTH-dependent mitochondrial biogenesis in cortical cells of fetal rat adrenals in tissue culture.
Authors: Kahri A I , Salmenperä M , Saure A
Published: May 2004

Cortical cells of fetal rat adrenals in tissue culture were treated with 5-bromodeoxyuridine (BrdU) during their proliferative phase and during ACTH stimulation when nuclear DNA synthesis has almost ceased. Pretreatment with 0.5 mug/ml/day of BrdU inhibited the ACTH-induced differentiation of cortical cells as well as the secretion of corticosterone and 18-OH-deoxycorticosterone (18-OHDOC). When nuclear DNA synthesis was suppressed and mitochondrial DNA synthesis was stimulated by ACTH BrdU addition (30 mug/ml/day) permitted normal untrastructural differentiation of cortical cells, except that the development of mitochondrial inner membranes was inhibited. Simultaneously mitochondrial inner membranes was inhibited. Simultaneously mitochondrial 11beta- and 18-hydroxylations were strongly inhibited while cytoplasmic 21-hydroxylation was not affected.
Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle.
Authors: Hodge L D , Mancini P , Davis F M , Heywood P
Published: May 2004

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS-polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000-75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.
Ultrastructural and biochemical evidence for a steroid-containing secretory organelle in the perfused cat adrenal gland.
Authors: Gemmell R T , Laychock S G , Rubin R P
Published: May 2004

A correlative study of the ultrastructural and biochemical effects of ACTH on fasciculata cells was carried out on the isolated cat adrenal gland perfused in situ with Locke's solution. The outstanding morphologic feature of cortical cells exposed to microunit ACTH concentrations for 40 min was the abundance of electron-dense granules (0.2-0.4 mum). These organelles were observed in small groups in close proximity to the Golgi region and to the cell membrane. Morphometric and biochemical analysis of control and ACTH-treated glands demonstrated that ACTH stimulation was associated with a fourfold increase in the number of these granules and a comparable increase in the corticosteroid content of the gland. By contrast, ACTH failed to augment cortical lysosomal enzyme activity. These findings, which link steroid release to the appearance of intracellular granules, extend further the parallels between the mechanism of release of newly synthesized steroid and the release of preformed hormones stored in secretory organelles. These results also lend support to the concept that a process related to exocytosis may be the underlying mechanism for extruding steroid from the cortical cell.
Identification of a sperm receptor on the surface of the eggs of the sea urchin Arbacia punctulata.
Authors: Schmell E , Earles B J , Breaux C , Lennarz W J
Published: May 2004

The possibility that the surface of the egg of the sea urchin Arbacia punctulata contains a species-specific receptor for sperm has been investigated. The extent of fertilization of eggs of A. punctulata, which is proportional to the number of sperm, is unaffected by the presence of either eggs or membranes prepared from eggs of Strongylocentrotus purpuratus. In marked contrast, membranes prepared from eggs of A. punctulata quantitatively inhibit fertilization of A. punctulata eggs by A. punctulata sperm. Several lines of evidence indicate that this inhibition is due to the presence of a membrane-associated glycoprotein that binds to the sperm, thus preventing them from interacting with receptor on the surface of the eggs. First, eggs treated with trypsin are incapable of being fertilized, although they can be activated with the Ca2+ ionophore A23187. Moreover, membranes prepared from eggs pretreated with trypsin do not inhibit fertilization of eggs. Second, receptor isolated in soluble form from surface membranes binds to sperm and thus prevents them from fertilizing eggs; the inhibition by soluble receptor is species-specific. Third, the soluble receptor binds to concanavalin A-Sepharose. Fourth, eggs are incapable of being fertilized if they are pretreated with concanavalin A. The specificity of inhibition, and the affect of trypsin and concanavalin A on intact eggs, suggest that the receptor is a species-specific macromolecule located on the surface of the eggs. The sensitivity of the receptor to trypsin, and its ability to bind to concanavalin A, indicate that it is a glycoprotein.
The electrophysiological mapping of compartments within a mammalian cell.
Authors: Giulian D , Diacumakos E G
Published: May 2004

The electrical properties of structures within an intact cell were examined by impalement with micropipette electrodes. Mean potential differences (PDs) measured from interphase HeLa cells showed that internal membrane-bounded compartments such as the nucleus, Golgi region, and the mitochondria were more negative than the cytoplasm with respect to an external grounding electrode. The nuclear PDs, unlike Golgi and cytoplasmic PDs, shifted during interphase and reached a peak value shortly before mitosis. The positioning of micropipettes was confirmed by electron microscope examination of marker solutions that were microinjected into specific intracellular regions. The combination of methods described here offers a new approach for the study of physiological events within intact, living cells.
Molecular events during membrane fusion. A study of exocytosis in rat peritoneal mast cells.
Authors: Lawson D , Raff M C , Gomperts B , Fewtrell C , Gilula N B
Published: May 2004

We have used thin section and freeze-fracture electron microscopy to study membrane changes occurring during exocytosis in rat peritoneal mast cells. By labeling degranulating mast cells with ferritin-conjugated lectins and anti-immunoglobulin antibodies, we demonstrate that these ligands do not bind to areas of plasma membrane or granule membrane which have fused with, or are interacting with, granule membrane. Moreover, intramembrane particles are also largely absent from both protoplasmic and external fracture faces of plasma and granule membranes in regions where these membranes appear to be interacting. Both the externally applied ligands and intramembrane particles are sometimes concentrated at the edges of fusion sites. The results indicate that membrane proteins are displaced laterally into adjacent membrane regions before the fusion process and that fusion occurs between protein-depleted lipid bilayers. The finding of protein-depleted blebs in regions of plasma and granule membrane interaction raises the interesting possibility that blebbing may be a process for exposing the granule contents to the extracellular space and for the elimination of excess lipid while conserving membrane proteins.
Conversion of lamellar body membranes into tubular myelin in alveoli of fetal rat lungs.
Authors: Williams M C
Published: May 2004

Fluid-filled lumina of fetal rat lungs contain lamellar bodies (LBs) as well as tubular myelin (TM), both of which are thought to be stores of phospholipid-rich pulmonary surfactant. The alveolar epithelium is believed to secrete LBs, but neither the origin nor the mechanism of TM formation is entirely certain. The main objective of this study was to determine the relationship between secreted LBs and TM and to define membrane phenomena which occur during TM formation. I examined lung tissues of 20-21 day-old fetuses (day 22 = term) using transmission and high voltage transmission electron microscopy and cytochemistry. My findings indicate that secreted LBs, identified by the presence of an acid-phosphatase reactive core, are the precursor of TM. Secreted LBs are highly organized structures which contain structurally specialized areas, one of which is a "mini-lattice" structure similar to TM. During TM formation, fuzzes or 8.0-nm diameter particles appear on transition membranes, although LB membranes appear to lack both structures. Similar particles are present on TM membranes and are generally associated with membrane intersections. My results provide evidence that TM is formed from LBs within the alveolar lumen by mechanisms which may be complex.
Thermotropic lateral translational motion of intramembrane particles in the inner mitochondrial membrane and its inhibition by artificial peripheral proteins.
Authors: Höchli M , Hackenbrock C R
Published: May 2004

Freeze fracturing and deep etching have been used to study thermotropic lateral translational motion of intramembrane particles and membrane surface anionic groups in the inner mitochondrial membrane. When the inner membrane is equilibrated at low temperature, the fracture faces of both halves of the membrane reveal a lateral separation between intramembrane particles and particle free, large smooth patches. Such separation is completely reversed through free lateral translational diffusion by reversing the temperature. The low temperature induced, particle-free, smooth membrane patches appear to represent regions of protein-excluding, ordered bilayer lipid which form during thermotropic liquid crystalline to gel state phase transitions. When polycationic ferritin is electrostatically bound to anionic groups exposed at the membrane surface at concentrations which inhibit the activities of cytochrome c oxidase and succinate permease, the bound ferritin migrates with intramembrane particles during the thermotropic lateral separation between the membrane particles and smooth patches. When bound polycationic ferritin is cross-bridged with native ferritin, an artificial peripheral protein lattice forms in association with the surface anionic groups and diminishes the thermotropic lateral translational motion of intramembrane particles in the membrane. These results reveal that the anionic groups of metabolically active integral proteins which are known to be exposed at the surface of the inner mitochondrial membrane migrate with intramembrane particles in the plane of the membrane under conditions which induce lipid-protein lateral separations. In addition, cross-bridging of the anionic groups through an artificial peripheral protein lattice appears to diminish such induced lipid protein lateral separations.
The role of the centriolar region in animal cell mitosis. A laser microbeam study.
Authors: Berns M W , Rattner J B , Brenner S , Meredith S
Published: May 2004

An argon ion laser microbeam (488 and 514 nm) was used to selectively irradiate one of the two centriolar regions of rat kangaroo Potorous tridactylis (PtK2) prophase cells in vitro. The cells were sensitized to the laser radiation by treatment with acridine orange (0.1-0.2 mug/ml). Ultrastructural examination of the irradiated centriolar regions demonstrated that the primary site of damage was the pericentriolar material. This result suggests that nucleic acid is present in the pericentriolar material. Behavioral and ultrastructural analysis demonstrated that cells with one damaged pericentriolar zone could undergo (a) nuclear membrane breakdown, (b) chromosome condensation, (c) metaphase plate formation, and (d) cytokinesis. However, the chromosomes neither separated nor exhibited any anaphase movements. Detailed ultrastructural analysis revealed the presence of kinetochore microtubules on both sides of chromosome mass and a lack of microtubules in the cytokinesis constriction. These results indicate that the pericentriolar material is important in spindle orgainization and essential for the formation of the interpolar microtubules.
Membrane changes during cartilage maturation. Increase in 5'-nucleotidase and decrease in adenosine inhibition of adenylate cyclase.
Authors: Rodan G A , Bourret L A , Cutler L S
Published: May 2004

To examine the potential participation of the plasma membrane in differentiation, we studied the enzymatic activities of 5'-nucleotidase and adenylate cyclase as a function of chondrocyte maturation. 16-day-old chick embryo tibiae epiphyses were dissected into proliferative, growing and hypertrophying zones. Partially purified membrane fractions prepared by differential centrifugation from the respective tissue segments were assayed for enzymatic activity. Cell suspensions from the same segments were examined cytochemically for the presence of 5'-nucleotidase. The findings show that the 5'-nucleotidase activity of the chick embryo epiphyseal cartilage has the following characteristics: (a) it has a Km of about 25 muM for 5'AMP, and is inhibited by a mixture of 2' and 3'AMP (apparent Ki about 10(-4) M) and by AOPCP; (b) it is predominantly localized at the cell surface but is also detected in the cytoplasm and in association with nuclear heterochromatin; and (c) it increases 10-fold (on a DNA basis) during the maturation of the epiphyseal cartilage cells. The adenylate cyclase activity has these characteristics: (a) it does not change during chondrocyte maturation (on a DNA basis); (b) its susceptibility to adenosine inhibition decreases at least 10-fold. The implication of these findings relative to a possible role of adenosine in cellular communication is discussed.
A comparison of the distribution of actin and tubulin in the mammalian mitotic spindle as seen by indirect immunofluorescence.
Authors: Cande W Z , Lazarides E , McIntosh J R
Published: May 2004

Rabbit antibodies against actin and tubulin were used in an indirect immunofluorescence study of the structure of the mitotic spindle of PtK1 cells after lysis under conditions that preserve anaphase chromosome movement. During early prophase there is no antiactin staining associated with the mitotic centers, but by late prophase, as the spindle is beginning to form, a small ball of actin antigenicity is found beside the nucleus; After nuclear envelope breakdown, the actiactin stains the region around each mitotic center, and becomes organized into fibers that run between the chromosomes and the poles. Colchicine blocks this organization, but does not disrupt the staining at the poles. At metaphase the antiactin reveals a halo of ill-defined radius around each spindle pole and fibers that run from the poles to the metaphase plate. Antitubulin shows astral rays, fibers running from chromosomes to poles, and some fibers that run across the metaphase plate. At anaphase, there is a shortening of the antiactin-stained fibers, leaving a zone which is essentially free of actin-staining fluorescence between the separating chromosomes. Antitubulin stains the region between chromosomes and poles, but also reveals substantial fibers running through the zone between separating chromosomes. Cells fixed during cytokinesis show actin in the region of the cleavage furrow, while antitubulin reveals the fibrous spindle remnant that runs between daughter cells. These results suggest that actin is a component of the mammalian mitotic spindle, that the distribution of actin differs from that of tubulin and that the distributions of these two fibrous proteins change in different ways during anaphase.
Myelinogenesis in optic nerve. A morphological, autoradiographic, and biochemical analysis.
Authors: Tennekoon G I , Cohen S R , Price D L , McKhann G M
Published: May 2004

Morphological, autoradiographic, and biochemical methods were used to study the time of appearance, distribution, and nature of sulfated constituents in the developing rat optic nerve. Electron microscope studies showed that myelination begins (6 days postnatal) shortly after the appearance of oligodendroglia (5 days postnatal). Over the ensuing 3 wk, myelination increased rapidly. During the 1st postnatal wk, mucopolysaccharides and glycoproteins were labeled with 35S and autoradiographs showed grains over arachnoidal cells, astroglia, and the glia limitans. These results indicated that astroglia synthesize sulfated mucopolysaccharides of the glia limitans. After the onset of myelination, however, the major portion of [35S]sulfate was incorporated into sulfatide. Autoradiographs showed a shift of radioactive grains from astroglia and arachnoidal cells to myelin, indicating that actively myelinating oligodendroglia incorporate [35S]sulfate into myelin sulfatide; there was a concomitant increase in the activity of cerebroside sulfotransferase. In addition, the increasing amounts of proteolipid protein and myelin basic protein corresponded with the morphological appearance of myelin. These results point to a strict correlation between the structural and biochemical changes occurring during myelination. This system provides a useful model for studies designed to evaluate the effects of various perturbations on the process of myelination.
Enzymic characteristics of fat globule membranes from bovine colostrum and bovine milk.
Authors: Powell J T , Järlfors U , Brew K
Published: May 2004

Fat globule membranes have been isolated from bovine colostrum and bovine milk by the dispersion of the fat in sucrose solutions at 4 degrees C and fractionation by centrifugation through discontinuous sucrose gradients. The morphology and enzymic characteristics of the separated fractions were examined. Fractions comprising a large proportion of the total extracted membrane were thus obtained having high levels of the Golgi marker enzymes UDP-galactose N-acetylglucosamine beta-4-galactosyltransferase and thiamine pyrophosphatase. A membrane-derived form of the galactosyltransferase has been solubilized from fat and purified to homogeneity. This enzyme is larger in molecular weight than previously studied soluble galactosyltransferases, but resembles in size the galactosyltransferase of lactating mammary Golgi membranes. In contrast, when fat globule membranes were prepared by traditional procedures, which involved washing the fat at higher temperatures, before extraction, galactosyltransferase was not present in the membranes, having been released into supernatant fractions, When the enzyme released by this procedure was partially purified and examined by gel filtration, it was found to be of a degraded form resembling in size the soluble galactosyltransferase of milk. The release is therefore attributed to the action of proteolytic enzymes. Our observations contrast with previous biochemical studies which suggested that Golgi membranes do not contribute to the milk fat globule membrane. They are, however, consistent with electron microscope studies of the fat secretion process, which indicate that secretory vesicle membranes, derived from the Golgi apparatus, may provide a large proportion of the fat globule membrane.
Induction of pinocytosis in rat hepatocytes by partial hepatectomy.
Authors: Mori M , Novikoff A B
Published: May 2004

Rat hepatocytes, normally not highly pinocytic cells, becomes so after partial hepatectomy when about two-thirds of the liver is removed. Droplets, up to 20 mum in diameter, develop, initially by addition to smaller pinocytic structures and later by fusion with lysosomes. The droplets contain a material with an electron microscope periodicity characteristic of fibrin; they are periodic acid Schiff-positive as is plasma. It is therefore reasonable to consider plasma glycoproteins to be major components of the droplets. The droplets are at all times membrane delimited, an observation possible only after perfusion fixation. The droplets are positive for three lysosomal hydrolases identified cytochemically: acid phosphatase, N-acetyl-beta-glucosaminidase, and beta-glucuronidase. From light and electron microscopy it is evident that these activities are acquired by fusion with lysosomes, mostly autophagic vacuoles and residual bodies both of which become very numerous after partial hepatectomy. Pinocytic structures are seen relatively infrequently in the hepatocytes of normal rats but a great many are present after partial hepatectomy. They are most easily observed if horseradish peroxidase (HRP) is intravenously injected before sacrifice and sections are incubated for HRP cytochemistry. The low dose of HRP employed (10 mg/100 g body weight) does not induce pinocytosis in controls, either untreated rats or rats subjected to laparotomy, including palpation of the liver. However, in partially hepatectomized rats even a much smaller dose of intravenous HRP (3.3 mg/100 g) visualizes the pinocytic structures in hepatocytes (coated vesicles, channels, cuplike bodies, and droplets). Kupffer cells pinocytose much HRP in both control and partially hepatectomized rats.
Freeze-fracture of monolayer cultures.
Authors: Pauli B , Weinstein R S , Soble L W , Alroy J
Published: May 2004

This paper describes a simple method for the freeze-fracturing of cells in monolayers or multi-layer tissue cultures. The method produces high quality replicas and is applicable to the study of virtually any tissue culture or organ culture system. It uses standard materials and equipment for both tissue culture and freeze-fracturing.
Different sensitivity of DNA in situ in interphase and metaphase chromatin to heat denaturation.
Authors: Darzynkiewicz Z , Traganos F , Sharpless T , Melamed M R
Published: May 2004

Heat denaturation of DNA in situ, in unbroken cells, was studied in relation to the cell cycle. DNA in metaphase cells denatured at lower temperatures (8 degrees-10 degrees C lower) than DNA in interphase cells. Among interphase cells, small differences between G1, S, and G2 cells were observed at temperatures above 90 degrees C. The difference between metaphase and interphase cells increased after short pretreatment with formaldehyde, decreased when cells were heated in the presence of 1 mM MgCl2, and was abolished by cell pretreatment with 0.5 N HCl. The results suggest that acid-soluble constituents of chromatin confer local stability to DNA and that the degree of stabilization is lower in metaphase chromosomes than in interphase nuclei. These in situ results remain in contrast to the published data showing no difference in DNA denaturation in chromatin isolated from interphase and metaphase cells. It is likely that factors exist which influence the stability of DNA in situ are associated with the super-structural organization of chromatin in intact nuclei and which are lost during chromatin isolation and solubilization. Since DNA denaturation is assayed after cell cooling, there is also a possibility that the extent of denatured DNA may be influenced by some factors that control strand separation and DNA reassociation. The different stainability of interphase vs. metaphase cells, based on the difference in stability of DNA, offers a method for determining mitotic indices by flow cytofluorometry, and a possible new parameter for sorting cells in metaphase.
Characterization of mitochondrial and cytoplasmic ribosomes from Paramecium aurelia.
Authors: Tait A , Knowles J K
Published: May 2004

The ribosomes extracted from the mitochondria of the ciliate, Paramecium aurelia, have been shown to sediment at 80S in sucrose gradients. The cytoplasmic ribosomes also sediment at 80S but can be distinguished from their mitochondrial counterparts by a number of criteria. Lowering of the Mg++ concentration, addition of EDTA, or high KCl concentrations results in the dissociation of the cytoplasmic ribosomes into 60S and 40S subunits, whereas the mitochondrial ribosomes dissociate into a single sedimentation class at 55S. Furthermore, the relative sensitivity of the two types of ribosome to dissociating conditions can be distinguished. Electron microscopy of negatively stained 80S particles from both sources has also shown that the two types can be differentiated. The cytoplasmic particles show dimensions of 270 X 220 A whereas the mitochondrial particles are larger (330 X 240 A). In addition, there are several distinctive morphological features. The incorporation of [14C]leucine into nascent polypeptides associated with both mitochondrial and cytoplasmic ribosomes has been shown: the incorporation into cytoplasmic 80S particles is resistant to erythromycin and chloramphenicol but sensitive to cycloheximide, whereas incorporation into the mitochondrial particles is sensitive to erythromycin and chloramphenicol but resistant to cycloheximide.
An ultrastructural study of cross-fertilization (Arbacia female x Mytilus male).
Authors: Longo F J
Published: May 2004

Insemination of sea urchin (Arbacia) ova with mussel (Mytilus) sperm has been accomplished by treating eggs with trypsin and suspending the gametes in seawater made alkaline with NaOH. Not all inseminated eggs undergo a cortical granule reaction. Some eggs either elevate what remains of their vitelline layer or demonstrate no cortical modification whatsoever. After its incorporation into the egg, the nucleus of Mytilus sperm undergoes changes which eventually give rise to the formation of a male pronucleus. Concomitant with these transformations, a sperm aster may develop in association with the centrioles brought into the egg with the spermatozoon. Both the male pronucleus and the sperm aster may then migrate centrad to the female pronucleus. Evidence is presented which suggests that fusion of the male pronuclei from Mytilus sperm with female pronuclei from Arbacia eggs may occur, although this was not directly observed. These results demonstrate that Mytilus sperm nuclei are able to react to conditions within Arbacia eggs and differentiate into male pronuclei.
Large-sized polysomes in Chironomus tentans salivary glands and their relation to Balbiani ring 75S RNA.
Authors: Daneholt B , Anderson K , Fagerlind M
Published: May 2004

Polysomes from the salivary glands of Chironomus tentans were investigated to determine whether Balbiani ring 75S RNA is incorporated into polysomal structures, and thus probably acts as messenger RNA. A new extraction technique for obtaining ribonucleoproteins was applied that gives a high yield of polysomes with only moderate degradation of the cytoplasmic, high molecular weight RNA. The polysomes sedimented in a broad region (200-2,000S) with a peak value of about 700S, which suggested that they were partly of very large sizes. This was confirmed by visualization of the polysomes in the electron microscope: 400S polysomes contained mainly 11-16 ribosomes, and 1,500S polysomes about 60 ribosomes per polysome. However, polysomes containing 100 or more ribosomes were also observed. It was further established that most of the cytoplasmic 75S RNA was located in polysomes, preferentially in the most rapidly sedimenting ones. From the available information on Balbiani ring RNA in cytoplasm and the present demonstration of 75S RNA molecules in polysomes, it was concluded that at least some Balbiani ring RNA, generated as 75S RNA within the Balbiani rings, eventually enters polysomes without being measurably changed in size. The present information on the potential amino acid coding sequences in 75S RNA is discussed in relation to the large size of the polysomes observed.
Properties of an antiserum against native dynein 1 from sea urchin sperm flagella.
Authors: Ogawa K , Asai D J , Brokaw C J
Published: May 2004

Effects of an antiserum against native dynein 1 from sperm flagella of the sea urchin Strongylocentrotus purpuratus were compared with effects of an antiserum previously obtained against an ATPase-active tryptic fragment (fragment 1A) of dynein 1 from sperm flagella of the sea urchin, Anthocidaris crassispina. Both antisera precipitate dynein 1 and do not precipitate dynein 2. Only the fragment 1A antiserum precipitates fragment 1A and produces a measurable inhibition of dynein 1 ATPase activity. Both antisera inhibit the movement and the movement-coupled ATP dephosphorylation of reactivated spermatozoa. The inhibition of movement by the antiserum against dynein 1 is much less than by the antiserum against fragment 1A, suggesting that a specific interference with the active ATPase site may be required for effective inhibition of movement. Both antisera reduce the bend angle as well as the beat frequency of reactivated S. purpuratus spermatozoa, suggesting that the bend angle may depend on the activity of the dynein arms which generate active sliding.
Concentration of elements in mitotic chromatin as measured by x-ray microanalysis.
Authors: Cameron I L , Sparks R L , Horn K L , Smith N R
Published: May 2004

Unfixed frozen-dried and uncoated tissue sections of the mouse duodenum were placed on carbon planchets and analyzed in a scanning electron microscope fitted with energy dispersive X-ray equipment. Computer analysis of the X-ray spectra allowed elemental microanalysis of the nucleus, cytoplasm, and mitotic chromatin regions in the cryptal and villus enterocytes. The peak to continuum ratio of S, Cl, K, and Ca were higher in mitotic chromatin than any of the other sites measured. The redistribution of Ca at mitosis is postulated to help explain both chromosome condensation and assembly of the mitotic spindle apparatus.
Continued initiation of DNA synthesis in arginine-deprived Chinese hamster ovary cells.
Authors: Weissfeld A S , Rouse H
Published: May 2004

When exponentially growing CHO cells were deprived of arginine (Arg), cell multiplication ceased after 12 h, but initiation of DNA synthesis continued: after 48 h of starvation with continuous [3H]thymidine exposure, 85% of the population had incorporated label, as detected autoradiographically. Consideration of the distribution of exponential cells in the various cell cycle phases leads to a calculation that most cells in G1 at the time that Arg was removed, as well as those in S, engaged in some DNA synthesis during starvation. In contrast, isoleucine (Ile)-starved cells did not initiate DNA synthesis, as has been reported by others. Experiments with cells synchronized by mitotic selection confirmed this difference in Arg- and Ile- deprived behavior, but also showed that cells which underwent the mitosis leads to G1 transition during Arg starvation remained arrested in G1 (G0?). The results suggest that Arg-deprived cells continue to maintain some proliferative function(s) while Ile-deprived cells do not.
Biochemical characterization of RNA and protein synthesis in erythrocyte development.
Authors: Grasso J A , Chromey N C , Moxey C F
Published: May 2004

Newts (Triturus cristatus) made anemic with acetylphenylhydrazine (APH) fail to regenerate erythrocytes (RBC's) immediately and exhibit a latent period of 1.5-2 wk during which animals lack RBC's and are aplastic. With the establishment of erythroid regeneration at 10-14 days, relatively homogeneous populations of successive erythropoietic stages occur in the blood. This feature makes possible biochemical analyses of events in early, intermediate, and late developmental stages, respectively, each of which can be obtained in vivo with minimal contamination by other stages. Previous studies have described a primitive cell population referred to as "erythroid precursor cells" (EPC's) which precedes the appearance of definitive erythroid elements. The present studies show that EPC's and early erythroid cells are engaged mainly in ribosomal production, including synthesis of rRNA and ribosomal proteins. Moreover, EPC's and early erythroid cells also synthesize tRNA and a presumed Hb-mRNA which has been identified by its sedimentation rate at 9-12 s and its content of polyadenylic acid. In intermediate stages, there occurs a fourfold decrease in the level of RNA synthesis and, while rRNA continues to be formed, there is a disproportionate accumulation of the two major cytoplasmic rRNA species in favor of the large ribosomal subunit RNA. In late developmental stages, the level of RNA synthesis is markedly diminished with little or no evidence of formation of defined RNA classes. Correlated radioautographic and biochemical studies with radioactive delta-aminolevulinic acid and leucine indicate that EPC's and other early erythroid elements synthesize not only hemoglobin but also ferritin and ribosomal proteins. It is concluded that: (a) erythroid RNA synthesis is most pronounced in the early developmental stages, being manifested predominantly by rRNA production but including tRNA and Hb-mRNA; (b) intermediate developmental stages show both "ribosomal wastage" and decreased growth rate, marking a pivotal point between the transcriptional activities of early stages and translational activities of late stages; (c) EPC's represent a cell population already committed to RBC formation and are excluded from a role as the pluripotential stem cell.
Redox constituents in milk fat globule membranes and rough endoplasmic reticulum from lactating mammary gland.
Authors: Jarasch E D , Bruder G , Keenan T W , Franke W W
Published: May 2004

Milk fat globule membranes (MFGM) and rough endoplasmic reticulum (RER) membranes were isolated from milk and lactating mammary gland from the cow and were characterized by biochemical and electron microscope methods in terms of gross composition (proteins, phospholipids, neutral lipids, cholesterol, RNA, and DNA) and purity. Both fractions contained significant amounts of a b-type cytochrome with several properties similar to those of cytochrome b5 from liver, as well as a rotenone-insensitive NADH- and NADPH-cytochrome c reductase. The b-type cytochrome content in the apical plasma membrane-derived MFGM was of the same order of magnitude as it was in RER membranes. It was characterized by a high resistance to extraction by low- and high-salt concentrations and nonionic detergents. MFGM contained much more flavin and much higher activities of xanthine oxidase than the RER membranes. The same redox components were found in MFGM and mammary RER from women, rats, mice, and goats, but in absolute contents great differences between the species were noted. The cytochromes described here differed from liver cytochrome b5 in some spectral properties. The alpha-band of the reduced hepatic cytochrome b5 is asymmetric with a maximum at 555 nm that is split into two distinct peaks at low temperatures. The alpha-band of the b-type cytochromes from MFGM and mammary RER appears as one symmetrical peak at about 560 nm that is not split at low temperatures. When treated with cyanide, MFGM and mammary microsomes showed difference spectra of a reduced b-type cytochrome. Under the same conditions, liver microsomes gave a completely different spectrum. These findings demonstrate the presence of a b-type cytochrome and associated redox enzymes in MFGM, i.e., a derivative of the apical cell surface membrane that is regularly used for envelopment of the milk fat globule during secretion.
Muscle actin filaments bind pituitary secretory granules in vitro.
Authors: Ostlund R E , Leung J T , Kipnis D M
Published: May 2004

Hog anterior pituitary secretory granules sediment at 3,000 g. When rat or rabbit skeletal muscle actin filaments are present with the granules, the sedimentation decreases markedly. Depolymerized actin or viscous solutions of Ficoll and collagen have no effect on granule sedimentation. With this assay, actin filaments bind secretory granules (consisting of the proteinaceous core plus limiting membrane), secretory granule membranes, mitochondria, artificial lecithin liposomes, and styrene-butadiene microspheres, but have little or no interaction with membrane-free secretory granule cores and albumin microspheres. A secretory granule-actin complex sedimentable between 3,000 g and 25,000 g can be isolated. Metal ions, nucleotides, salts, dithiothreitol, or pretreatment of the granules with trypsin do not destroy the binding, which appears to be a lipophilic interaction.
Localization of Na+-pump sites in frog skin.
Authors: Mills J W , Ernst S A , DiBona D R
Published: May 2004

The localization of Na+-pump sites (Na+-K+-ATPase) in the frog skin epithelium was determined by a freeze-dry radioautographic method for identifying [3H]ouabain-binding sites. Ventral pelvic skins of Rana catesbeiana were mounted in Ussing chambers and exposed to 10(-6) M [3H]ouabain for 120 min, washed in ouabain-free Ringer's solution for 60 min, and then processed for radioautography. Ouabain-binding sites were localized on the inward facing (serosal) membranes of all the living cells. Quantitative analysis of grain distribution showed that the overwhelming majority of Na+-pump sites were localized deep to the outer living cell layer, i.e., in the stratum spinosum and stratum germinativum. Binding of ouabain was correlated with inhibition of Na+ transport. Specificity of ouabain binding to Na+-K+-ATPase was verified by demonstrating its sensitivity to the concentration of ligands (K+, ATP) that affect binding of ouabain to the enzyme. Additional studies supported the conclusion that the distribution of bound ouabain reflects the distribution of those pumps involved in the active transepithelial transport of Na+. After a 30-min exposure to [3H]ouabain, Na+ transport declined to a level that was significantly less than that in untreated paired controls, and analysis of grain distribution showed that over 90% of the ouabain-binding sites were localized to the inner cell layers. Furthermore, in skins where Na+ transport had been completely inhibited by exposure to 10(-5) M ouabain, the grain distribution was identical to that in skins exposed to 10(-6) M. The results support a model which depicts all the living cell layers functioning as a syncytium with regard to the active transepithelial transport of Na+.
Intramolecular heterogeneity of mitochondrial DNA of Drosophila melanogaster.
Authors: Goldring E S , Peacock W J
Published: May 2004

DNA from purified mitochondria of Drosophila melanogaster can be isolated as supercoiled molecules which when nicked have a contour length of 5.9 micron. Partial denaturation mapping shows regional heterogeneity of base composition with one early denaturing region, with a calculated GC content close to zero, extending over 20% of the genome. DNA isolated from unfertilized eggs shows nuclear and mitochondrial DNA in equal proportions; we found no evidence of other cytoplasmic species.
Chitinous fibrils in the lorica of the flagellate chrysophyte Poteriochromonas stipitata (syn. Ochromonas malhamensis).
Authors: Herth W , Kuppel A , Schnepf E
Published: May 2004

Ordered microfibrils are formed on the membrane of the cytoplasmic tail of the alga Poteriochromonas after attachment to a substrate. The ultrastructure of native and extracted stalk fibrils was studied with electron microscope methods. In addition, the structural polysaccharide was characterized by hydrolyses, separation of the monomers by thin-layer chromatography, gas-liquid chromatography and amino acid analysis, and by X-ray diffraction. The alkali-resistant fibrils yielded mostly glucosamine upon extensive hydrolysis, and showed X-ray diffraction patterns similar to those of fugal chitin. It is concluded that the resistant core of the fibrils is chitinous.
Lipogenesis and the synthesis and secretion of very low density lipoprotein by avian liver cells in nonproliferating monolayer culture. Hormonal effects.
Authors: Tarlow D M , Watkins P A , Reed R E , Miller R S , Zwergel E E , Lane M D
Published: May 2004

The nonproliferating chicken liver cell culture system described yields cell monolayers with morphological and lipogenic properties characteristic of the physiological-nutritional state of donor animals. Synthesis and secretion of fatty acid, cholesterol, and very low density lipoprotein (VLDL) occur at in vivo rates and respond to hormones and agents which affect these processes in vivo. Cells derived from fed chickens maintain high rates of synthesis of fatty acid and cholesterol for several days if insulin is present in the medium. High rates of fatty acid synthesis are correlated with the appearance of membrane-enclosed triglyceride-rich vesicles in the cytoplasm; deletion of insulin causes a decrease (T1/2 = 22 h) in fatty acid synthetic activity. Addition of glucagon or cyclic AMP (cAMP) causes an immediate cessation of fatty acid synthesis and blocks the appearance of the triglyceride-rich vesicles. Fatty acid synthesis in liver cells prepared from fasted chickens is less than 5% that of cells from fed animals. After 2-3 days in culture with serum-free medium containing insulin +/- triiodothyronine, fatty acid synthesis is restored to normal; glucagon or dibutyryl cAMP blocks this recovery. Liver cells derived from estradiol-treated chickens synthesize and secrete VLDL for at least 48 h in culture. Electron micrographs of these cells reveal more extensive development of the rough endoplasmic reticulum and Golgi complex compared to cells from untreated chickens. Whereas [3H]leucine incorporation into total protein is unaffected by estrogen treatment, [3H]leucine incorporation into cellular and secreted immunoprecipitable VLDL is markedly increased indicating specific activation of VLDL apopeptide synthesis; 8-10% of the labeled protein synthesized and secreted is VLDL. Dodecyl sulfate-acrylamide gel electrophoresis of immunoprecipitated 3H-VLDL reveals three major apopepetides of 300,000, 11,000, and 8,000 daltons corresponding to those of purified chicken VLDL.
Biosynthesis and axonal transport of rat neurohypophysial proteins and peptides.
Authors: Gainer H , Sarne Y , Brownstein M J
Published: May 2004

35S-cysteine injected adjacent to the supraoptic nucleus (SON) of the rat is rapidly incorporated into proteins. These 35S-cysteine-labeled proteins in the SON (1-24 h after injection) were separated by polyacrylamide gel electrophoresis, and the distribution of radioactive proteins on the gels was analyzed. 1 h after injection, about 73% of the radioactivity appeared in two peaks (both about 20,000 mol wt). With time, these peaks (putative precursors of neurophysin) decreased, as a 12,000 mol wt peak (containing two distinct neurophysins) increased in radioactivity. Both the 20,000- and 12,000-mol wt proteins are transported into the axonal (median eminence) and nerve terminal (posterior pituitary) regions of the rat hypothalamo-neurohypophysial system. Conversion of the larger precursor protein to the smaller neurophysin appears to occur, in large part, intra-axonally during axonal transport. Six distinct 35S-cysteine-labeled peptides (less than 2500 mol wt), in addition to arginine vasopressin and oxytocin, are also synthesized in the SON and transported to the posterior pituitary where they are released together with labeled neurophysin by potassium depolarization in the presence of extracellular calcium. These data provide support for the hypothesis that the neurohypophysial peptides (vasopressin and oxytocin) and neurophysins are derived from the post-translational clevage of protein precursors synthesized in the SON, and that the conversion process can occur in the neurosecretory granule during axonal transport.
Basal lamina of embryonic salivary epithelia. Production by the epithelium and role in maintaining lobular morphology.
Authors: Banerjee S D , Cohn R H , Bernfield M R
Published: May 2004

The role of the basal lamina in maintaining the normal morphology of mouse embryo submandibular epithelia was assessed by examining its production as well as the cellular and organ culture changes associated with its removal and replacement. The lamina was removed from epithelia isolated free of mesenchyme by brief treatment with testicular hyaluronidase in the absence of calcium. The treatment causes rounding-up of the cells, loss of cellular cohesion, appearance of microvilli, and changes in the organization of cytoskeletal structures. The lamina is not removed and the cellular alterations do not occur in the absence of hyaluronidase in calcium-free medium or when both enzyme and calcium are present, possibly because digestion of chondroitin sulfate, a component of the lamina, is inhibited by calcium. Within 2 h after treatment, in the absence of mesenchyme or biological substrata, the epithelia deposits a new lamina, which is identical by several criteria to the preexisting lamina, and reverses the cellular alterations. Epithelia treated with hyaluronidase lose lobular morphology during culture with mesenchyme. Delaying culture with mesenchyme, to allow restoration of the lamina and of normal cellular architecture, prevents the loss of lobular morphology. The results indicate that the basal lamina imposes morphologic stability on the epithelium, while the mesenchyme apparently affects processes involved in changes in morphology, possibly by selective degradation of the basal lamina.
Basal lamina of embryonic salivary epithelia. Nature of glycosaminoglycan and organization of extracellular materials.
Authors: Cohn R H , Banerjee S D , Bernfield M R
Published: May 2004

The ultrastructural organization and the composition of newly synthesized glycosaminoglycan (GAG) in the epithelial basal lamina of mouse embryo submandibular glands were assessed. The labeled GAG accumulating in the lamina is distinct from that in its tissue of origin, the epithelium, or from that in the surrounding mesenchyme. In the lamina, hyaluronic acid accounts for approximately 50% of the labeled GAG, chondroitin-4-sulfate is twice the chondroitin-6-sulfate, and there is a low proportion of chondroitin. This composition is constant regardless of whether the lamina is labeled by whole glands or, in the absence of mesenchyme, by isolated epithelia retaining a lamina and by isolated epithelia generating a lamina de novo. The results andicate that the labeled GAG are bona fide components of the lamina, and suggest that laminar GAG is deposited in units of constant composition. Ultrastructural observations following ruthenium red staining or tannic acid fixation extablish that the lamina is a highly ordered specialization of the basal cell surface. Discrete structures in macroperiodic arrays apparently attached to the plasmalemma are visualized. This organization is seen in intact glands and in the laminae produced by epithelia in the absence of mesenchyme or biological substrate. The data are interpreted as indicating that the basal lamina contains supramolecular complexes of hyaluronic acid and proteoglycan which are organized into an extracellular scaffolding which imposes structural form on the epithelium.