bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthesis of membrane glycoproteins in rat hepatoma tissue culture cells
Authors: Baumann Heinz
Published: May 2005

The early steps in the biosynthesis of glycoproteins associated with the plasma membranes of rat hepatoma tissue culture cells has been analyzed. By measuring the effect of tunicamycin on the incorporation of [3H] mannose and [3H] fucose into cell glycoproteins, it was determined that an interval of about 1 h was required to transfer the glycoprotein from the site of mannosylation to the site of fucosylation. This result was corroborated by an analysis of the time required for the appearance of either mannose or fucose‐labeled glycoproteins at the cell surface. The separation of membrane glycoproteins by a two‐dimensional gel system allowed the visualization of the modifications leading to both size and charge heterogeneity of these proteins. By following the changes in electrophoretic mobility introduced into membrane glycoproteins during a chase period after a pulse labeling, the time course of these molecular alterations could be estimated. Several glycoproteins have apparently higher rates of synthesis than the bulk of membrane‐associated glycoproteins. Most of these glycoproteins were released within 2 h after biosynthesis from the intracellular membrane fraction and appear after 3 h in the medium. In addition to the glycoproteins that contain both mannose and fucose and that show a high degree of charge heterogeneity, there are other membrane‐bound species that are not noticeably modified by the in corporation of fucose or sialic acids. These glycoproteins could represent constituents limited to the internal membrane system of the HTC cell.
Direct linkage of thrombin to its cell surface receptors in different cell types
Authors: Simmer Robert L. , Baker Joffre B. , Cunningham Dennis D.
Published: May 2005

When 125I‐thrombin was incubated with foreskin fibroblasts, cervical carcinoma cells or fibrosarcoma cells of human origin, or with secondary chick embryo cells or Chinese hamster lung cells, it became directly linked to its cell surface receptors. The thrombin‐receptor complex (TH‐R) was derived exclusively from a pool of 125I‐thrombin that had become specifically bound to the cell surface. The linkage was probably covalent, since the complex was resistant to boiling in sodium dodecyl sulfate and 2‐mercaptoethanol. Raising the pH to 12 disrupted TH‐R, but did not affect a similar complex between epidermal growth factor and its receptor, suggesting that the linkage of these mitogens to their receptors was different. Mild trypsin treatment removed the ability of cells to form TH‐R; however, after a 24‐h incubation in serum‐free medium, trypsin‐treated cells recovered the capacity to form TH‐R, suggesting that TH‐R resulted from interaction of 125I‐thrombin with a cellular rather than a serum component. The mitogenic response of cells to thrombin was inversely related to the fraction of specifically bound 125I‐thrombin represented by TH‐R. The role of TH‐R in mitogenesis may be clarified in future studies by obtaining clones of Chinese hamster lung cells that vary in their capacities to form TH‐R and to respond to the mitogenic action of thrombin.
Role of the microfibrillar system in knob action of transformed cells
Authors: Meek William D. , Puck Theodore T.
Published: May 2005

Transformed cells often display knobs (or blebs) distributed over their surface throughout most of interphase. Scanning electron microscopy (SEM) and time‐lapse cinematography on CHO‐K1 cells reveal roughly spherical knobs of 0.5–4 μm in diameter distributed densely around the cell periphery but sparsely over the central, nuclear hillock and oscillating in and out of the membrane with a period of 15–60 sec. Cyclic AMP derivatives cause the phenomenon of reverse transformation, in which the cell is converted to a fibroblastic morphology with disappearance of the knobs. A model was proposed attributing knob formation to the disorganization of the jointly operating microtubular and microfilamentous structure of the normal fibroblast. Evidence for this model includes the following: (1) Either colcemid or cytochalasin B (CB) prevents the knob disappearance normally produced by cAMP, and can elicit similar knobs from smooth‐surfaced cells; (2) knob removal by cAMP is specific, with little effect on microvilli and lamellipodia; (3) immunofluorescence with antiactin sera reveals condensed, amorphous masses directly beneath the membrane of CB‐treated cells instead of smooth, parallel fibrous patterns of reversetransformed cells or normal fibroblasts; (4) transmission electron microscopy (TEM) of sections show dense, elongated microfilament bundles and microtubules parallel to the long axis of the reverse‐transformed CHO cell, but sparse, random microtubules throughout the transformed cell and an apparent disordered network of 6‐nm microfilaments beneath the knobs; (5) cell membranes at the end of telophase, when the spindle disappears and cleavage is complete, display typical knob activity as expected by this picture.
Selective protein transport: Characterization and solubilization of the phosvitin receptor from chicken oocytes
Authors: Woods John W. , Roth Thomas F.
Published: May 2005

Phosvitin (PV), a subunit of a female‐specific protein, vitellogenin, binds to oocyte membranes with a KD of 10−6 M. Binding reaches equilibrium within 30 min after incubation at 25°C. Bound 125I‐PV dissociates from the membrane with a t1/2 of 13 h when incubated in buffer. However, when 125I‐PV‐labeled membranes are incubated in buffer containing 10−5 M unlabeled PV, 50% of the initially bound 125I‐PV dissociates from the membrane within 10 min. These results support the conclusion that PV binds to a membrane‐associated receptor.

Solubilization studies show that Triton X‐100 solubilizes up to 45% of the total membrane‐bound 125I‐PV. Gel‐exculsion chromatography of the solubilized material yields a 500,000 dalton 125I‐PV‐containing complex separated from free 125I‐PV. The 500,000 dalton complex completely dissociates to yield free 125I‐PV when incubated with excess unlabeled PV. However, when incubated with (1) no addition, (2) IgG, or (3) serum albumin, the extent of dissociation is significantly reduced and is consistent with that which would be predicted on the basis of the observed dissociation rate in the absence of unlabeled PV.

These results suggest that bound 125I‐PV can only be displaced by unlabeled PV. These results also indicate that the 500,000 dalton species is a solubilized PV‐receptor complex and that it is possible to solubilize the PV‐receptor in an active form.
Uptake and binding of riboflavin by membrane vesicles of bacillus subtilis
Authors: Cecchini Gary , Kearney Edna B.
Published: May 2005

Riboflavin uptake and membrane‐associated riboflavin‐binding activity have been investigated in Bacillus subtilis. The uptake and binding activity of the vitamin were found to be repressed coordinately by riboflavin present in the growth medium. The uptake of riboflavin has been shown to have properties of a carrier‐mediated process, and membrane vesicles have been shown to demonstrate riboflavin counterflow and exchange. The membrane‐associated binding activity for riboflavin has been solubilized with detergents, and a procedure for the partial purification of this component is described. The partially purified riboflavin‐binding component has properties expected for a carrier involved in riboflavin uptake, as it shows saturation kinetics and is inhibited by riboflavin analogues. Evidence is also presented showing that reduced riboflavin binds to a greater extent than oxidized riboflavin, and the possible role of the reduced riboflavin in riboflavin uptake is discussed.
A proton nuclear magnetic resonance investigation of histine‐binding protein J of salmonella typhimurium: A model for transport of L‐histidine across cytoplasmic membrane
Authors: Ho Chien , Giza Yueh‐hua , Takahashi Seizo , Ugen Kenneth E. , Cottam Patricia F. , Dowd Susan R.
Published: May 2005

Genetic evidence suggests that the high‐affinity L‐histidine transport in Salmonella typhimurium requires the participation of a periplasmic binding protein (histidine‐binding protein J) and two other proteins (P and Q proteins). The histidine‐binding protein J binds L‐histidine as the first step in the high‐affinity active transport of this amino acid across the cytoplasmic membrane. High‐resolution proton nuclear magnetic resonance spectroscopy at 600 MHz is used to investigate the conformations of this protein in the absence and presence of substrate. Previous nuclear magnetic resonance results reported by this laboratory have shown that there are extensive spectral changes in this protein upon the addition of L‐histidine. When resonances from individual amino acid residues of a protein can be resolved in the proton nuclear magnetic resonance spectrum, a great deal of detailed information about substrate‐induced structural changes can be obtained. In order to gain a deeper insight into the nature of these structural changes, deuterated phenylalanine or tyrosine has been incorporated into the bacteria. Proton nuclear magnetic resonance spectra of selectively deuterated histidine‐binding protein J were obtained and compared to the normal protein. Several of the proton resonances have been assigned to the various aromatic amino acid residues of this protein. A model for the high‐affinity transport of L‐histidine across the cytoplasmic membrane of S typhimurium is proposed. This model, which is a version of the pore model, assumes that both P and Q proteins are membrane‐bound and that the interface between these two proteins forms the channel for the passage of substrate. The histidine‐binding protein J serves as the “key” for the opening of the channel for the passage of L‐histidine. In the absence of substrate, this channel or gate is closed owing to a lack of appropriate interactions among these three proteins. The channel can be opened upon receiving a specific signal from the “key”; namely, the substrate‐induced conformational changes in the histidine‐binding protein J molecule. This model is consistent with available experimental evidence for the high‐affinity transport of L‐histidine across the cytoplasmic membrane of S typhimurium.
5′‐p‐Fluorosulfonylbenzoyladenosine as an ATP site affinity probe for Na+,K+‐ATPase
Authors: Cooper James B. , Winter Charles G.
Published: May 2005

We have investigated the suitability of 5′‐p‐fluorosulfonylbenzoyladenosine (FSBA) as an ATP site affinity probe for the canine kidney Na+,K+‐ATPase. The purified enzyme is slowly inactivated by this compound in suitable buffers, losing about half of its activity over a two‐hour period. The rate of inactivation is more rapid in 0.1 M KCl than in 0.1 M NaCl. Low concentrations of ATP protect the enzyme against inactivation, with half‐maximal effects at 4 μM ATP in 0.1 M NaCl and 350 μM ATP in 0.1 M KCl. ADP also protects against FSBA inhibition, but AMP is ineffective when present at 100 μM levels. This pattern is consistent with the previously described nucleotide specificity of the Na+,K+‐ATPase. Addition of protective amounts of ATP after inactivation has occurred does not restore enzyme activity, indicating that inhibition is irreversible.

Measurement of the concentration‐dependence of FSBA inactivation suggests an apparent Kd for binding of this compound well above 1 mM, the solubility limit of the analog. This finding is reinforced by the failure of 1 mM FSBA to compete effectively with ATP for the high‐affinity ATP site of the enzyme. Nevertheless, attachment of the analog to this site is indicated by its ability to prevent [3H]‐ADP binding in proportion to the number of sites it has inactivated. Studies with [3H]‐FSBA show that about 1 mole of the analog attaches specifically to the α subunit per mole of enzyme inactivated. A similar amount of nonspecific labeling also occurs with negligible effect on enzyme activity. These findings suggest that FSBA may be useful in probing the topography of the high‐affinity ATP binding site of the Na+,K+‐ATPase and related enzymes.
Release of adenosine by C1300 neuroblastoma cells in tissue culture
Authors: Green Richard D.
Published: May 2005

Previous work in our laboratory led us to postulate that N2a cells release adenosine into growth medium, where it acts at the extracellular adenosine receptors to modulate the sensitivity of the cells to the cyclic AMP‐elevating effect of adenosine [Green, RD, J Pharmacol Exp Ther 201:610, 1977]. We have now devised a high‐performance liquid chromatographic (HPLC) procedure capable of quantitating the concentrations of adenosine in cells and tissue culture media. Growth media of N2a cells and a variant of N2a cells deficient in hypoxanthine‐guanine phosphoribosyltransferase (HGPRT−) contain 10–20 nM adenosine, while that of a variant deficient in adenosine kinase (AK−) is elevated severalfold. It appears that the concentration of adenosine in growth media is determined by both the rate at which it is released by cells into the medium and the rate at which it is metabolized by adenosine deaminase present in the serum in the growth medium. Both N2a and AK− cells release considerable amounts of adenosine into serum‐free medium (SFM) over a short period. Adenosine release is greater from AK− cells and is accelerated by erythro‐9‐(2‐hydroxy‐3‐nonyl)‐adenine (EHNA), a potent adenosine deaminase inhibitor. This accelerated release is retarded by dipyridamole and homocysteine. Surprisingly, dipyridamole and 4‐(3‐butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone (Ro 20 1724), a potent phosphodiesterase inhibitor, stimulate basal adenosine release from N2a but not from AK− cells. It remains to be determined if this is due to an effect of these compounds on adenosine kinase. These results give further support for the hypothesis that adenosine in growth medium modulates the sensitivity of the cells to the cyclic AMP‐elevating affect of adenosine, and furthermore they suggest that adenosine in growth media may tonically stimulate adenylate cyclase and affect processes controlled by the cyclic AMP:cyclic AMP‐dependent protein kinase system.
Control of animal cell proliferation
Authors: Holley Robert W.
Published: May 2005

Present understanding of the control of animal cell proliferation is summarized briefly. Major gaps in present knowledge are listed. Models of growth control are discussed.
Cell‐cell contact and growth regulation of pinocytosis in 3T3 cells
Authors: Davies Peter F.
Published: May 2005

In sub‐confluent cultures of Balb/c‐3T3 cells, pinocytosis rates were increased after exposure to specific growth factors (serum; platelet‐derived growth factor, PDGF; epidermal growth factor, EGF). Conversely, as cells became growth‐inhibited with increasing culture density, there was a corresponding decline in pinocytosis rate per cell. In order to test whether density‐inhibition of pinocytosis was influenced either by the growth cycle or by cell contact independently of growth, cells were induced into a quiescent state at a range of subconfluent and confluent densities. Under such conditions, cell density did not significantly inhibit pinocytosis rate. When confluent quiescent cultures in 2.5% serum were exposed to 10% serum, the resulting round of DNA synthesis was accompanied by enhanced pinocytosis per cell, even though the cells were incontact with one another. Furthermore, in a SV40‐viral transformed 3T3 cell line, both the growth fraction and the pinocytosis rate per cell remained unchanged over a wide range of culture densities. These studies indicate that density‐dependent inhibition of pinocytosis in 3T3 cells appears to be secondary to growth‐inhibition rather than to any direct physical effects of cell–cell contact.
Expression of red cell membrane proteins in erythroid precursor cells
Authors: Yurchenco Peter D. , Furthmayr Heinz
Published: May 2005

Specific antibodies to human glycophorin A and spectrin were used to study the expression of these membrane proteins in normal and pathologic human bone marrow. In immunofluorescence experiments spectrin and glycophorin A are found in 50–60% of the nucleated cells in normal bone marrow. These two proteins are expressed at all stages of red cell differentiation and can be traced at least to the earliest morphologically recognizable nucleated red cell precursor, the proerythroblast; the two proteins are specific for cells of the red cell series and are not found to be expressed in lymphocytic, granulocytic cells or platelets. These conclusions were drawn from studies on bone marrow in patients with a temporary block in erythropoiesis at the level of stem cells or of the pronormoblast. Bone marrow from these individuals either lacked all nucleated cells stainable for glycophorin A and spectrin or contained only pronormoblasts. Similar findings were obtained on spleen cells from mice which were made severely anemic by multiple injections with N‐acetyl‐phenylhydrazine. Antibodies to a sialoglycoprotein isolated from mouse red cell membranes stain 70–80% of all cells in the spleen of anemic animals, while only 1–2% of such cells are seen in the spleen of normal animals. Spectrin and glycophorin A could be labeled metabolically and isolated using specific antibodies. The human tumor cell line K562 expresses both membrane proteins, but induction experiments with various agents thus far have failed to change their expression.
Activation of T lymphocytes by the Fc portion of immunoglobulin
Authors: Thoman Marilyn L. , Morgan Edward L. , Weigle William O.
Published: May 2005

T lymphocytes are stimulated to release T‐cell‐replacing factors in response to Fc fragments of human IgG. Lyt 1+23− T cells are directly triggered to factor production by Fc subfragments, derived from intact Fc fragments by macrophage‐dependent enzymatic cleavage. These factor(s) replace T cell function in two Fc‐mediated immune responses; induction of polyclonal antibody synthesis, and potentiation of anti‐SRBC responses.
Long‐term maintenance of “cloned” human PLT cells in TCGF with LCL cells as a feeder layer
Authors: Hank Jacquelyn A. , Inouye Hiroo , Guy Lynn A. , Alter Barbara J. , Bach Fritz H.
Published: May 2005

The long‐term maintenance of T cells “cloned” by limiting dilution in TCGF was enhanced by the use of irradiated autologous lymphoblastoid cell line (LCL) cells as well as irradiated LCL cells of the individual to which the T cells were originally primed. It was possible to obtain more than 1 × 1012 cells from a “clone” seeded at one cell per well. Some of the clones tested express primed LD‐typing activity.
Divalent cation dependent ATPase activities of red blood cell membranes: Influence of the oxidation of membrane thiol groups close to each other
Authors: Scutari G. , Ballestrin G. , Covaz A. L.
Published: May 2005

An Mg2+‐dependent low ATPase activity can be detected in erythrocyte “white membranes,” in addition to that of the well known (Ca2+ + Mg2+)‐ATPase. The thiol oxidizing agent diamide affects both activities. The oxidation of neighboring thiols seems to leave the mechanism of the (Ca2+ + Mg2+)‐ATPase amplification system evoked by Ca2+ largely unaffected. The perturbation caused by diamide in the membranes seems to affect primarily a step of the ATP hydrolysis mechanism that is common to both ATPase activities. The effectiveness of diamide seems to be the same when either Ca2+ and Mg2+, or Mg2+ alone are present during the reagent action. Reduction of disulfide bonds by DTE after diamide treatment restores the (Ca2+ + Mg2+)‐ATPase activity but is unable to take the Mg2+‐ATPase activity back to the original level.

The hypothesis is discussed that the redox state of one (or more than one) couple of SH close to each other and possibly connected to the active site, may be an important factor in optimizing the efficiency of Ca action on the (Ca2+ + Mg2+)‐ATPase.
Modulation of EGF binding and action by succinylated concanavalin A in fibroblast cell cultures
Authors: Ballmer Kurt , Burger Max M.
Published: May 2005

The role of the binding of succinylated concanavalin A to tissue culture cells in influencing epidermal growth factor (EGF)‐mediated cell proliferation has been studied. Succinylated concanavalin A dramatically reduces the stimulation of 3T6 cells by EGF in Dulbecco's modified Eagle's medium (DME) containing insulin and vitamin B12 as additional growth factors, but no serum. Furthermore, binding studies using 125I‐labeled EGF have shown that the binding of EGF to the cell surface is reduced upon addition of succinylated concanavalin A.
Restricted infectivity of ecotropic type C retroviruses in mouse teratocarcinoma cells: Studies on viral DNA intermediates
Authors: Yang Wen K. , d'Auriol Luc , Yang Den‐Mei , Kiggans James O. , Ou Chin‐yih , Périès Jorge , Emanoil‐Ravicovitch Rodica
Published: May 2005

Replication of Gross strain N‐tropic type C retrovirus was markedly restricted in a pluripotential undifferentiated embryonal cell line (PCC4) of murine teratocarcinoma, whereas the same virus could cause productive infection in a myoblast‐derived differentiated line (PCD1) of the same tumor origin. To investigate the restriction mechanism, we compared the initial viral DNA formation in these two cell lines. Analyses by means of a modified Hirt extraction procedure and a modified Southern gel transfer method indicated that PCC4 and PCD1 cells supported the synthesis of viral DNA intermediates after inoculation of the Gross virus. In both cells, a linear DNA duplex (form III viral DNA) appeared at 4 hr, reached a maximal level at 8–9 hr, and declined rapidly thereafter, while two closed‐circular supercoiled DNA duplexes (form I viral DNA) showed their appearance, increase and decline in the 8–24 hr period. During the period from 34 to 78 hr after virus inoculation, another burst of viral DNA synthesis occurred in PCD1 cells, presumably due to secondary virus infection, while at this period both form III and form I viral DNAs became undetectable in PCC4 cells. The Hirt supernatant DNAs prepared from PCD1 and PCC4 cells 10 hr after virus inoculation were equally infectious for NIH3T3 cells in a DNA transfection assay. Both PCD1 and PCC4 cells were very poor recipients for DNA transfection, although one positive result with PCD1 cells might suggest a difference between the two cell types in this aspect. These results indicate that restriction of type C retrovirus in undifferentated embryonl carcinoma cells occurs at a step subsequent to formation and maturation of viral DNA intermediates.
Inhibition of adipose conversion in 3T3‐L2 cells by retinoic acid
Authors: Murray Thomas , Russell Thomas R.
Published: May 2005

The role of retiinoic acid in modulating the differentiation of 3T3‐L2 fibroblasts into adipocytes has been examined. Results indicate that the retinoid is capable of effectively inhibiting the degree of adipose conversion which is brought about by treatment of preadipocytes with 1‐methyl‐3‐isobutylxanthine plus dexamethasone. Morphological and enzymatic (fatty acid synthetase activity) expression of the adipose phenotype are both inhibited more than 90% by 10−6 M retinoic acid. The inhibition is concentration dependent with retinoic acid levels as low as 10−11 M capable of reducing adipose conversion by 20%. Retinoic acid must be administered simultaneously with the triggering agents to be effective. Exposure of nongrowing preadipocytes to retinoic acid does not alter the ability of the cells to differentiate in response to a subsequent treatment with methylisobutylxanthine plus dexamethasone. Further, the inhibition is reversible. Cultures in which methylisobutylxanthine plus dexamethasone triggered differentiation has been blocked by addition of retinoic acid (10−6M) will readily undergo adipose conversion in response to a second treatment with methylisobutylxanthinthine plus dexamethasone in the absence of the retioid. Similar inhibition of differentiation was found when cultures were treated with drugs in medium supplemented with either newborn calf serum or fetal calf serum. However, the extent to which methylisobutylxanthine plus dexamethasone are able to promote differentiation in these cells is considerably greater in medium containing fetal calf serum.
Inhibition of α‐bungarotoxin binding to acetylcholine receptors by antisera from animals with experimental autoimmune myasthenia gravis
Authors: Claudio Toni , Raftery Michael A.
Published: May 2005

Conditions are described for an assay that allows the percent inhibition of α‐bungarotoxin binding to acetylcholine receptors by antisera and monovalent antigen‐binding fragments of antibody molecules (Fab) to be determined. Anti‐Torpedo californica acetylcholine‐receptor antisera, prepared in New Zealand White rabbits and Lewis rats, were tested for the ability to inhibit [125I]‐α‐bungarotoxin binding to membrane‐associated and detergent‐solubilized T californica acetylcholine receptors. Similar inhibition studies were performed using rabbit antisera and antigen‐binding fragments prepared against each of the four acetylcholine receptor subunits. Antisera and antigen‐binding fragments prepared against intact receptor could inhibit a maximum of 50% of the α‐bungarotoxin binding to solubilized receptor. The results using monovalent antigen‐binding fragments indicated that the inhibition was not due to antibody‐mediated aggregation of receptor molecules. Rabbits and rats immunized with receptor denatured by sodium dodecyl sulfate all produced antisera that could bind to nondenatured receptor, but none of these animals developed experimental autoimmune myasthenia gravis. These results suggest that the antigenic determinants present on acetylcholine receptors responsible for induction of experimental auto‐immune myasthenia gravis are lost with sodium dodecyl sulfate denaturation. A strong correlation was also observed between the presence of experimental autoimmune myasthenia gravis in rats and rabbits and the ability of the antisera from these animals to inhibit 50% of α‐bungarotoxin binding to solubilized acetylcholine receptors.
In vitro maintenance of differentiation marker synthesis by subpopulations of mouse thymocytes
Authors: Rothenberg Ellen , Triglia Dennis
Published: May 2005

Mouse thymocyte populations enriched in functionally incompetent, “immature” cells on the one hand, or in competent “mature” cells on the other hand, express different steady‐state levels of certain surface antigens and marker enzymes. In the cases of the glycoproteins H‐2 (K and D), Qa, and TL, and the DNA polymerase terminal deoxynucleotidyl transferase (TdT), these levels reflect different rates of de novo synthesis in the two populations. Thus each population appears to manifest a characteristic pattern of synthetic rates for the various products relative to total protein synthesis. To investigate the maintenance of these patterns, enriched pools of “immature” and “mature” thymocytes were incubated in vitro for 24 h, and the rates of product synthesis before and after culture were compared. H‐2 synthesis, initially most rapid in the mature cells, continued to be made at the highest rate in this population. TdT synthesis, a characteristic activity of the immature cells, was not induced in the mature cells, but proceeded at an increased relative rate in the immature population. Therefore, the differences between the rates of H‐2 and TdT synthesis were stable properties of the two thymocyte populations. Another marker of immature cells, TL, did not continue to be produced in parallel with TdT. Rather, its synthesis was selectively curtailed in relation to the continuing protein synthesis in the immature cultures. This non‐coordinate regulation of TL and TdT production in immature thymocytes may be due to several mechanisms. These are discussed with regard to their implications for pathways of thymocyte maturation.
The hatching enzyme from xenopus laevis: Limited proteolysis of the fertilization envelope
Authors: Urch Umbert A. , Hedrick Jerry L.
Published: May 2005

Hatching in the amphibian Xenopus laevis involves release of an embryo‐secreted hatching enzyme, a protease, which weakens the envelope surrounding the embryo. The envelope is not totally solubilized, which infers that only selected envelope components are hydrolyzed by the enzyme. The susceptibility of the glycoprotein components composing the envelope to hydrolysis by the hatching enzyme was investigated. Isolated envelopes in various physical states, ie, particulate and solubilized, were treated with the hatching enzyme, and the resulting envelope hydrolysis products were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibility of the envelope components to proteolysis was not a function of the state of the envelope. The envelope components most susceptible to proteolysis were the 125K and 11 8K components followed by the 60K and 71 – 77K components. These components are minor constituents of the envelope. The major constituents, 33K and 40K, were relatively resistant to hydrolysis by the hatching enzyme. From these observations, we infer that the envelope components hydrolyzed are components that link or bind together the major structural elements of the envelope, eg, the 33K and 40K components. Selective destruction of the components required for maintaining the structural integrity of the envelope, eg, the “nuts and bolts” of the structure, permits a weakening of the envelope that allows the embryo to hatch without having to destroy totally (hydrolyze) the envelope.
Effect of exogenous fatty acids on growth, membrane fluidity, and phospholipid fatty acid composition in yeast
Authors: Esfahani Mojtaba , Kucirka Elise M. , Timmons Frank X. , Tyagi Somdev , Lord Arthur E. , Henry Susan A.
Published: May 2005

The growth response of a double‐mutant fatty acid auxotroph of yeast Saccharomyces cerevisiae to exogenous saturated fatty acids of a homologous series from 12:0 to 16:0, each supplied with oleate, linoleate, linolenate, or cis‐Δ11‐ eicosenoate, cannot be explained in terms of the efficiency of incorporation of the fatty acids into phospholipids or alteration of membrane fluidity. There is, however, a negative correlation between growth and levels of 12:0 plus 13:0 in phospholipids, as well as a positive correlation between growth and levels of 14:0, 1 5:0, and 1 6:0. We, therefore, conclude that the predominant factor in these phospholipid fatty acyl chain modifications is maintenance of an optimal concentration of C14:0 through C16:0 in phospholipids of this organism.
The effects of dipalmitoyl phosphatidyl choline on the precipitation of native fibrils and segment‐long‐spacing aggregates from collagen solution
Authors: McKenzie James C. , Belton John C. , Klein Robert M.
Published: May 2005

The effect of dipalmitoyl phosphatidyl choline (DPPC), the major phospholipid component of pulmonary surfactant, on the precipitation of collagen in the form of native fibrils and segment‐long‐spacing (SLS) aggregates was studied in vitro. The effects of DPPC on both phases of collagen fibrillogenesis were analyzed spectrophotometrically, and alterations in the morphology of precipitated fibrils and SLS aggregates were ascertained by transmission electron microscopy (TEM). Low concentrations of DPPC inhibited the growth phase of fibrillogenesis, while higher concentrations were required to inhibit nucleation. Both the meshwork density and mean width of precipitated fibrils were altered by DPPC, as was the size of SLS aggregates. Segment‐long‐spacing aggregates prepared from pepsin‐treated collagen were inhibited to a greater degree than SLS aggregates prepared from untreated collagen, indicating that the pepsin‐susceptible residues of the telopeptide extensions of tropocollagen molecules stabilize SLS aggregates against the effects of DPPC. Based on these results and the inhibition of the growth phase at lower concentrations than those which inhibited the nucleation phase of fibrillogenesis, it was concluded that the primary mechanism of DPPC inhibition is electrostatic interference between the positively charged phospholipid molecules and the net positive charge of collagen. It is proposed that pathological conditions involving the pulmonary epithelium may allow interaction between surfactant and collagen, which could further weaken the interstitial connective tissue.
Protease‐insensitive sea urchin embryo cell adhesions become protease sensitive in the presence of azide or cytochalasin B
Authors: Bertolini Donald R. , Watanabe Michiko , Turner Robert S.
Published: May 2005

To understand the nature of the cell adhesions that must be modified during sea urchin embryo primary mesenchyme formation, we are studying the adhesive components of the hatched blastula stage embryo of Strongylocentrotus purpuratus. Pronase treatment conditions have been defined that leave the cells intact and able to recover from the effects of the protease upon its removal. Under these conditions, adhesion of the cells to tissue culture plates is totally eliminated, but cell‐cell adhesion formation is only partially inhibited. Analysis of iodinated cell surface proteins indicates that most are affected by thepronase. Further studies of pronase effects found that sodium azide‐treated cells are slightly adhesive and that pronase treatment of azidc‐treated cells totally eliminates cell‐cell adhesions.
Phosphoproteins in dictyostelium discoideum
Authors: Coffman D. S. , Leichtling Ben H. , Rickenberg H. V.
Published: May 2005

The phosphoproteins of Dictyostelium discoideum were compared at different stages of development by polyacrylamide gel electrophoresis. Certain phosphoproteins of vegetative amoebae were conserved while others appeared and disappeared during development. Four major phosphoproteins with apparent subunit molecular weights of 50,000, 47,000, 38,000, and 34,000 disappeared precociously in response to exogenous cAMP. Two membranal phosphoproteins, with apparent subunit molecular weights of 80,000 and 81,000, appeared precociously in response to added cAMP. One of these phosphoproteins, molecular weight of 80,000, has been identified tentatively as the “contact site A” glycoprotein. Another membranal protein, with apparent subunit molecular weight of 42,000, unaffected in its appearance by cAMP, has been identified tentatively as phosphoactin.
Isolation of a high molecular weight glycoconjugate derived from the surface of S purpuratus eggs that is implicated in sperm adhesion
Authors: Glabe Charles G. , Lennarz William J.
Published: May 2005

Sea urchin sperm–egg adhesion is mediated by bindin, a sperm surface protein that has lectin‐like activity. Bindin agglutinates eggs, and this interaction has been shown to be inhibited by glycopeptides released from the egg surface by protease treatment. In this study, we report the purification and properties of such an egg surface glycoconjugate that may be involved in sperm adhesion. The glycoconjugate was partially purified by gel filtration and affinity chromatography on bindin particles. Upon gel filtration on Sepharose CL 4‐B, the glycoconjugate elutes near the void volume, suggesting that it has a molecular weight in excess of one million. In addition, we have found that the egg surface glycoconjugate agglutinates bindin particles, indicating that it is multivalent. Carbohydrate analysis indicates that the glycoconjugate is composed primarily of fucosc, xylose, galactose, and glucose. This purified egg surface component is the most potent inhibitor of bindin‐mediated egg agglutination yet described.
Microfilament reorganization in normal and cytochalasin B treated adherent thrombocytes
Authors: Taylor Richard G. , Lewis Jon C.
Published: May 2005

Thrombocyte adhesion following activation by a Formvar surface involves a morphologic transition resulting in a fully spread cell. Correlative SEM and whole mount TEM were used to study the cytoskeletal alterations that accompany changes in surface morphology during adhesion. Following initial adhesion, thrombocytes extend slender pseudopods containing longitudinally oriented bundles of filaments that are 13–22 nm in diameter. Concomitant with pseudopod extension, a cytoplasmic hyalomere, consisting of a dense filamentous network, extends between the pseudopods and ultimately results in a fully spread cell. Treatment of thromboyctes with cytochalasin B (10−5 M) caused clumping of the hyalomere filament network and retraction of the hyalomere. Examination of partially retracted cells revealed that pseudopod filament bundles were continuous with the contracting filamentous network. It is concluded that pseudopod filament bundles and cytoplasmic hyalomere filaments are interconvertible and that their organizational relationship changes in accordance with gross morphologic changes.
The dynamic state of liver gap junctions
Authors: Yancey S. Barbara , Nicholson Bruce J. , Revel Jean‐Paul
Published: May 2005

By the use of a simple, rapid method for the isolation of gap junctions from small amounts of rat liver (2–3 g), we have followed the incorporation of the radiolabeled amino acid precursors 3H‐leucine and 35S‐methionine into the gap junction protein. In timed studies with 35S‐methionine as precursor, the specific activity in the protein is maximal by 4 h after a single injection of 300 μCi/100 g body weight. From the decay in the specific activity with time after a single injection, the gap junction protein has an apparent half‐life of about 19 h. Because of problems of reutilization of radiolabeled amino acid with 35S‐methionine as precursor, this apparent halflife probably overestimates the true half‐life and indicates a surprisingly rapid turnover of the gap junction protein. This short half‐life suggests that, in rat liver, the gap junctions may be very responsive to alterations in physiological demands.
An immunochemical approach for the analysis of membrane protein alterations in Ca2+‐loaded human erythrocytes
Authors: Bjerrum O.J. , Hawkins M. , Swanson P. , Griffin M. , Lorand L.
Published: May 2005

An increase in the intracellular concentration of Ca2+ in human erythrocytes results in the formation of γ‐glutamyl‐ϵ‐lysine cross‐linked membrane protein polymers. Following solubilization of the membranes with SDS, these polymers can be isolated on a Lubrol‐containing sucrose gradient. Immunoelectrophoresis of the polymeric material with a polyspecific rabbit antibody against human ghosts gave rise to a single, but heterogeneous, precipitate. The polymer was amphiphilic and, on addition to Triton‐solubilized erythrocyte membrane proteins, it coprecipitated with spectrin. When the antighost antibody was absorbed with the polymer prior to cross immunoelectrophoresis of normal erythrocyte membrane proteins, the precipitates of glycophorin, acetylcholinesterase, and hemoglobin were normal, whereas the anti‐body liters against band 3 protein, spectrin, and ankyrin became reduced. Furthermore, a rabbit antibody raised against the isolated human polymer reacted selectively with the same three membrane proteins. No reactions occurred with lysate proteins.
Modulation of one of three murine bone marrow stromal cell lines to adipose cells by serum and insulin
Authors: Anderson R.W. , Mann S.L. , Crouse D.A. , Sharp J.G.
Published: May 2005

Adipose cells have been recognized as an integral component of the bone marrow hematopoietic microenvironment in vivo and as an essential cell type required for in vitro maintenance of stem cells. Four stromal cell lines obtained from the adherent cell population of murine bone marrow cultures have been enriched and purified by multiple trypsinizations. We noted that these cell lines exhibited an accumulation of vacuoles of lipid, the extent of which varied be‐tween cell lines in response to a change from medium containing 10% fetal calf serum to medium containing 20% horse serum. The lipid was lost when the cell lines were transferred back into the medium supplemented with fetal calf serum. In light of the reported lipogenic and antilipolytic effects of insulin on fibroblasts and adipocytes, we investigated the ability of insulin to induce adipocyte transformation of these bone marrow stromal cell populations. Three cell lines were exposed to bovine insulin at concentrations ranging from 10−9 to 10−6 M. All three cell lines responded to the insulin by accumulating lipid, but the extent of accumulation and the insulin concentration at which maximum lipid content was attained were population specific. One cell line (MC1) responded fully at physiological levels of insulin (10−9 M), whereas the other two showed lipid accumulation only at pharmacological concentrations. The initial growth of MC1 was inhibited in the presence of 10−9 M insulin which is compatible with the observed differentiation to adipocytes. The growth of MC3 was unaltered in the presence of physiological concentrations of insulin, whereas that of MC4 was accelerated. Grafts of organ cultures of the cell lines under the kidney capsule of syngeneic mice developed specific characteristics rep‐resentative of the different cell lines. In particular, the majority of the grafts of MC1 consisted primarily of fat cells which were not observed in the grafts of MC3 and MC4. These data strongly suggest that these cell lines comprise cells with different potentialities and that the MC1 line represents a preadipocyte stromal cell of bone marrow.
Neuronal differentiation in cultures of weaver (wv) mutant mouse cerebellum
Authors: Willinger Marian , Margolis David M. , Sidman Richard L.
Published: May 2005

In the present study we report for the first time a weaver (wv) gene dose effect on neuron survival and neurite formation in vitro. Dissociated cerebellar cells from postnatal 7‐ and 8‐day‐old normal ( + / + ), heterozygous weaver ( + /wv) and homozygous weaver (wv/wv) mice were cultured as monolayers on poly‐L‐lysine coated glass. Cell death occurred rapidly in wv/wv cultures. Cell counts showed that less than 20% of the total neurons and neuronal precursors (identified by “birthday” radiolabeling techniques) survived by Day 3. Cell death was less extensive in + /wv cultures with 65% of the total neurons and 80% of the precursors surviving by Day 3. In contrast to wv/wv cultures, younger neurons survive better than the total population in + /wv cultures. The impairment of neurite formation over the first week is also proportional to the number of mutant genes as shown by quantitation of (a) the percentage of cells with neurites; (b) the percentage of cells with neurites of a given length class with time; (c) the lengths of the longest processes formed per cell. The mean longest neurite lengths obtained by computer digitization at 6 days in vitro were 41.8, 26.8, and 9.0 μm for + / +, + /wv, and wv/wv granule cells, respectively.
Unscheduled synthesis of DNA and poly(ADP‐ribose) in human fibroblasts following DNA damage
Authors: McCurry Lynette Salter , Jacobson Myron K.
Published: May 2005

Unscheduled DNA synthesis has been measured in human fibroblasts under conditons of reduced rates of conversion of NAD to poly(ADP‐ribose). Cells heterozygous for the xeroderma pigmentosum genotype showed normal rates of UV induced unscheduled DNA synthesis under conditions in which the rate of poly(ADP‐ribose) synthesis was one‐half the rate of normal cells. The addition of theophylline, a potent inhibitor of poly(ADP‐ribose) polymerase, to the culture medium of normal cells blocked over 90% of the conversion of NAD to poly(ADP‐ribose) following treatment with UV or N‐methyl‐N′‐nitro‐N‐nitro‐soguanidine but did not affect the rate of unscheduled DNA synthesis.
Quantitation of changes in cell surface determinants during skeletal muscle cell differentiation using monospecific antibody
Authors: Grove Barbara Kay , Schwartz Gary , Stockdale Frank E.
Published: May 2005

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate anti‐genie changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS‐1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty‐one clones were obtained which produced antibodies specific for myogenic cells. Thirty‐five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.
Structural analysis of fibronectin with monoclonal antibodies
Authors: Atherton Blair T. , Taylor Deena M. , Hynes Richard O.
Published: May 2005

The reactivity of six monoclonal antibodies with fragments of fibronectin produced with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease is described. All these antibodies reacted with fragments derived from the C‐terminal one‐third of fibronectin. This region probably contains sites for the binding of fibronectin to cells, and to heparin and may also contain active sites for the reattachment, spreading, and alignment of transformed cells. Analysis of the reactivities of different sets of proteolytic fragments with the antibodies and with other ligands (eg. heparin) allows one to determine overlaps between the fragments and to locate the positions of the different binding sites for antibodies and ligands. One of the antibodies has allowed us to identify a site of structural difference between cellular and plasma fibronectins from hamsters. The site recognized by this antibody is located near to, but not at, the C‐terminal end and docs not involve carbohydrate groups. Because of its internal location in fibronectin, this difference suggests that there are probably different genes for cellular and plasma fibronectin. These monoclonal antibodies should be useful for further probing the functions present in the C‐terminal regions of fibronectin and for determining their locations.
Immunological analysis of a glycoprotein (contact sites A) involved in intercellular adhesion of dictyostelium discoideum
Authors: Murray Ben A. , Yee Lisa D. , Loomis William F.
Published: May 2005

We have prepared antisera in rabbits to the “contact sites A” glycoprotein (gp80) purified from Dictyostelium discoideum. IgG isolated from these anti‐sera reacts with a number of different proteins in D discoideum lysates, as analyzed by immune precipitation and by antibody staining of gel electropherograms transferred to nitrocellulose. Blocking experiments indicate that this cross‐reactivity reflects the presence of common antigeneic determinants on gp80 and other cellular proteins, rather than the presence of extraneous antibodies in the antisera. The spectrum of reactive proteins is different a: different stages of development. In particular, gp80 itself is synthesized only for a restricted period during the cell aggregation phase. The protein persists throughout development and can be detected in spores. Anti‐gp80 Fab fragments bind to the surface of developing D discoideum cells and specifically block their developmentally regulated adhesion. After absorption with vegetative cells, the IgG stains only gp80 and (to a lesser extent) one other band in lysates of aggregation‐competent cells. The absorbed antibodies also can block adhesion. Several proteins that appear late in development also arc stained by the absorbed IgG.