bluuebunny/crossref_metadata_2025 huggingface dataset:
Occluding Junctions in MDCK Cells: Modulation of Transepithelial Permeability by the Cytoskeleton
Authors: Meza Isaura , Sabanero Myrna , Stefani Enrique , Cereijido Marcelino
Published: December 2004

In MDCK cell monolayers the opening and resealing of occluding junctions can be induced by removal and restoration of calcium to the external medium. The overall changes in permeability of the occluding junctions in the monolayer can be monitored by the drop and recovery of the total transepithelial electrical resistance. We have investigated the effects of cytochalasin B (CB) on this process. When CB is added to sealed monolayers there is a gradual drop in the electrical resistance across the monolayer. This drop is accompanied by a slow disorganization of the microfilament pattern of these cells, including a disturbance of a ring of cortical microfilaments that is normally associated with the junctions. Cells in open monolayers treated with CB will not reseal and have an altered filament distribution. These cells do not have a continuous cortical ring.

We have used a voltage scanning technique that uses a microelectrode to measure the resistance at selected points along the junction which surrounds a single cell. In untreated, closed monolayers, the junction is heterogeneous with alternating points of high and low conductance. In closed monolayers treated with CB, although there are low conductance points, we have observed an increased frequency of high conductance points that correlates with the change in the overall conductance. The frequency of high conductance points along the junction and the overall conductance both increase with time of exposure to CB.

In an effort to understand the molecular basis for the permeability changes induced by EGTA and CB, we have looked for differences in the protein components of the cell membranes of open, closed, and CB‐treated MDCK monolayers. This was done by radioiodinating the surface membrane proteins under control and experimental conditions that bring about permeability changes. No significant differences in the labeled protein patterns were found under these conditions. These results suggest that the permeability changes involve only a structural rearrangement of membrane components. In addition we have observed that about 36% of the surface label remains bound to the insoluble cytoskeletons obtained from cells in control and experimental conditions that alter the permeability of the tight junctions. The iodinated proteins attached to the CS include polypeptides with Mr of ≥ 120K daltons as well as peptides with Mr = 56K, 50K, 36K, and 18K daltons.
Inhibition by Glucocorticoids of Endocytosis in a Macrophage‐Like Cell Line
Authors: Miller Steven C. , Melnykovych George
Published: December 2004

A macrophage‐like cell line (P388D1) has been used to demonstrate that glucocorticoids inhibit the fluid‐phase endocytosis of fluorescein‐labeled dextran (FITC‐dextran). Initial experiments demonstrated that the interaction of FITC‐dextran with cells had all the features of fluid‐phase uptake, ie, the amount taken up was proportional to the concentration in the medium, the uptake proceeded continuously with time and was blocked at 4°C. Dexamethasone (10−7M) had no effect on endocytosis until 11 hours after addition of the steroid, when it inhibited the uptake of FITC‐dextran by 35%. The amount of inhibition increased with longer exposure times to the hormone up to 50% after 22 hours. Although this effect on endocytosis was Observed prior to any effect on growth of the cells, endocytosis as well as cell proliferation were inhibited in a dose‐dependent fashion. A preliminary survey of selected steroids has established that the inhibition of endocytosis was restricted to steroids of the glucocorticoid class. The key experiments were also performed using horseradish peroxidase instead of FITC‐dextran with, essentially, identical results.
A Possible Role for Ligatin and the Phosphoglycoproteins It Binds in Calcium‐Dependent Retinal Cell Adhesion
Authors: Marchase Richard B. , Koro Lillian A. , Kelly Charles M. , McClay David R.
Published: December 2004

Ligatin is a filamentous plasma membrane protein that serves as a baseplate for the attachment of peripheral glycoproteins to the external cell surface. Ligatin can be released from intact, embryonic chick neural retinal cells by treatment with 20 mM Ca++ without adversely affecting their viability. α‐Glucose‐1‐phos phate is also effective in removing ligatin‐associated glycoproteins from intact cells. After either of these treatments, the retinal cells seem not to exhibit Ca++ ‐dependent adhesion for one another. It is thus suggested that ligatin in neural retina may serve as a baseplate for the attachment to the cell surface of glycoproteins active in Ca++‐dependent adhesion. The finding that Ca++ serves to protect Ca++‐dependent adhesion molecules from digestion by trypsin is discussed in relation to steric constraints on trypsin's accessibility to these adhesion molecules because of their possible binding to arrayed ligatin filaments.
Spectrin Domains: Proteolytic Susceptibility as a Probe of Protein Structure
Authors: Speicher David W. , Marchesi Vincent T.
Published: December 2004

Mild treatment of human erythrocyte spectrin with trypsin produces discrete intermediate‐sized peptides. The effects of buffer composition, enzyme–substrate ratio, temperature, and other experimental parameters on the resulting peptide pattern have been examined. Spectrin is capable of regaining its proteolytic resistance after NaDodSO4‐induced denaturation, permitting the use of isolated subunits to study spectrin structure and function. Tryptic digestion of isolated subunits also has greatly facilitated the identification of the subunit origin of the intermediate‐sized peptides. Isolated subunits could also be recombined to form functional units similar but not identical to the native dimeric form of the molecule. Spectrin apparently is composed of numerous large protease‐resistant regions or domains connected by small protease sensitive segments. The structural integrity and accessibility of these sites is minimally affected by oligomeric state or proteolytic digestion conditions. The similarities of sizes, isoelectric points, and amino acid compositions of many intermediate‐size peptides from areas of both subunits suggest that at least part of spectrin's structure may have evolved via replication of a single gene. A possible structural repeat of approximately 50,000 daltons is hypothesized.
Inhibition of Neoplastic Cell Growth by Quiescent Cells Is Mediated by Serum Concentration and cAMP Phosphodiesterase Inhibitors
Authors: Bertram John S. , Bertram Barbara B. , Janik Przemyslaw
Published: December 2004

We have demonstrated that confluent monolayers of the mouse fibroblast cell line C3H/10T1/2 (10T1/2) have the ability to cause reversible growth inhibition of cocultured transformed cells. This was first demonstrated for de novo transformed cells and later extended to established cell lines of proven oncogenicity in vivo. This growth inhibition could be increased by growing the 10T1/2 cells to high density in increasing concentrations of serum or by elevating intracellular concentrations of cAMP using inhibitors of phosphodiesterase (PDE). These manipulations, which in cocultures of nontransformed and transformed cells caused complete inhibition of tumor cell growth, had no effect on growth rate or saturation density of either ceil type when cultured alone, demonstrating the cooperative nature of this phenomenon. This cooperation could not be produced by transfer of culture medium, demonstrating the requirement for intimate cell contact. Inhibition of the formation of transformed foci of cells in these mixed cultures was accompanied by a decrease in the incorporation of labeled thymidine into these cultures; the kinetics of this inhibition and recovery suggested a rapidly reversible effect on cell cycle transit times. The potent inhibitor of cAMP PDE, Ro 20‐1724 induced dose dependent increases in intracellular cAMP in both nontransformed and in transformed cells. However, at a concentration of 10−4 M Ro 20‐1724, which inhibited tumor cell growth in mixed cultures, cAMP was elevated 30‐fold in nontransformed versus only 3‐fold in transformed cells.

The inhibitory effects of PDE inhibitors on tumor growth have been extended to an in vivo model system, utilizing Lewis lung carcinoma cells growing as metastases in the lungs of C57B1 mice. In these mice, inoculated intravenously with a single cell suspension of Lewis lung cells, the formation of lung metastases was dramatically decreased by the twice daily administration of either isobutylmethylxanthine or Ro 20‐1724; PDE inhibitors were shown to be active in vitro. The latter compound, which showed highest activity in vitro, was also substantially more potent in vivo as an inhibitor of lung tumor colony formation and doubled the life span of the tumor bearing animals. Cell cycle analysis of lung tumor colonies by the labeled mitosis method showed that both phosphodiesterase inhibitors caused a prolonged G1 phase in the cell cycle but failed to influence other phases. Although detailed analysis of host tissues is not complete, prolonged treatment with these drugs caused no statistically significant weight loss or changes in counts of red or white blood cells indicating a selective growth inhibition of transformed cells at these doses. Studies to determine the mechanism of the cellular communication and the nature of the signal are in progress.
A study of cAMP binding proteins on intact and disrupted sperm cells using 8‐azidoadenosine 3′,5′‐cyclic monophoshate
Authors: Schoff Patrick K. , Forrester Ian T. , Haley Boyd E. , Atherton Robert W.
Published: December 2004

The photoaffinity probe (32P)8‐N3 cAMP was used to label the cAMP binding proteins in washed ejaculated human sperm. Three saturable binding proteins were photolabeled in both intact and disrupted cells with apparent molecular weights of 55,000, 49,000 and 40,000 daltons corresponding to the regulatory subunits of type II and type I cAMP‐dependent protein kinase (cAMP‐PK) and to an endogenous proteolytic product of the regulatory subunits, respectively. Photoincorporation in the three proteins could be totally blocked by preincubating the cells with cAMP. Cell‐free seminal plasma was found to be free of detectable (12P) 8‐N3 cAMP‐binding proteins. The 8‐N3 cAMP was also effective in stimulating endogenous cAMP‐PK activity in intact and disrupted sperm. A substantial amount of (32P) 8‐N3 cAMP binding to types I and II regulatory subunits and cAMP‐PK activity was detected on washed intact cells, intact cells. Intact cell bound 1.80 pmol of (32P) 8‐N3 cAMP/mg protein and had cAMP‐PK activity of 824 units/108 cells. Disrupted cells bound 3.95 pmol (32P) 8‐N3 cAMP mg protein and had a cAMP‐PK activity of 2,206 units/108 cells. The data presented support the concept of two classes of cAMP receptors being differentially available to externally added (32P) 8‐N3 cAMP and proteases. Cellular membrane integrity and membrane sidedness are discussed as possible explanations for the observation reported.
Macromolecular synthesis and stability in growth‐arrested BALB/C‐3T3 cells
Authors: Wharton Walker , Pledger W. J.
Published: December 2004

Concentrations of methylglyoxal bis‐(guanylhydrazone) (mGBG) that inhibited serum‐stimulated BALB/c‐3T3 cells in late G1 caused a marked inhibition of 3H‐leucine incorporation during a 20‐min incubation. No decrease was observed in the incorporation of 3H‐uridine during a 20‐min incubation; however, the amount of acid‐insoluble 3H‐uridine in mGBG‐treated cultures was decreased when the incubation period was longer than 20 min. The amount of the decrease in the accumulation of incorporated 3H‐uridine was directly proportional to the length of the incorporation time. Between 10 and 12 h after quiescent BALB/C‐3T3 cells were serum‐stimulated in mGBG no additional 3H‐uridine was accumulated. The stability of the incorporated 3H‐uridine, as determined by acid‐insoluble radioactivity remaining after the addition of actinomycin D, was less in cells cultured in mGBG. Exogenous spermine or spermidine reversed the inhibition of 3H‐uridine accumulation in acid‐insoluble material produced by mGBG as well as the decrease in stability of the incorporated 3H‐uridine in acid‐insoluble material. The effects of mGBG on both the incorporation of 3H‐uridine and the stability of the incorporated 3H‐uridine can apparently be accounted for by an effect on ribosomal RNA.
Heterogeneity of 14S and 30S dynein ATPase activities with respect to activation by calmodulin
Authors: Blum Jacob J. , Hayes A. , Vanaman T. , Schachat F. H.
Published: December 2004

Demembranated cilia of Tetrahymena pyriformis were extracted with KCl or Tris‐EDTA and the crude dyneins from each resolved by sucrose density gradient sedimentation into 14S‐K, 30S‐K, 14S‐E and 30S‐E dyneins, respectively. The calmodulin activation ratio (ATPase activity in presence of added calmodulin/ATPase activity in absence of added calmodulin) did not vary across the 30S dynein fractions regardless of the method of extraction nor did it vary across the 14S‐E region. However, in going from the “heavy” fractions to the “light” fractions of the 14S‐K region, it increased markedly.

The concentration of calmodulin required for half‐maximal activation did not differ appreciably in the “light” versus the “heavy” fractions of the 14S‐K region, suggesting that the affinity for calmodulin does not vary in these fractions. SDS‐polyacrylamide gel electrophoresis studies showed the presence of several polypeptides that varied in a systematic fashion across the 14S‐K region and hence may be involved in regulating the sensitivity of 14S‐K dynein ATPase activity to added calmodulin.
Protein‐dependent lipid lateral phase separation as a mechanism of human erythrocyte ghost resealing
Authors: Minetti Maurizio , Ceccarini Marina
Published: December 2004

The hypothesis of a correlation between a 10°–20°C lipid phase transition and the resealing process of human erythrocyte membrane has been investigated. The conditions required to reseal human erythrocyte ghosts have been studied by measuring the amount of fluorescein‐labeled dextran (FD) that is trapped into the membrane.

Temperature per se was sufficient to induce membrane resealing: (1) at 5 mM sodium phosphate, pH 7.8 (5P8), resealing began at 12°C; (2) at salt concentrations above 8 mM sodium phosphate, it occurred at lower temperature; and (3) in isotonic saline was detected just above 5°C.

The removal of peripheral membrane proteins from unsealed membranes by chymotrypsin at 0°C in 5P8 was followed by membrane resealing.

This seems to imply that the presence of proteins is necessary to maintain the membrane unsealed. Protein‐induced lateral phase separation of lipids may be a reasonable mechanism for the observed phenomena. In fact, the permeability of phosphatidylserine‐phosphatidylcholine mixed liposomes to FD is modified by lipid lateral phase separation induced by pH or poly‐L‐lysine.

Electron spin resonance studies of membrane fluidity by a spin labeled stearic acid showed a fluidity break around 11°C, which may be due to a gel–liquid phase transition. Fluidity changes are abolished by chymotrypsin treatment.

It is suggested that a lateral phase separation is responsible for the permeability of open ghosts to FD. Accordingly, disruption of phase separation apparently produces membrane reconstitution. In this respect peripheral proteins and particularly the spectrin‐actin network, may play a major role in membrane resealing.
Detection of a complex of SV40 large tumor antigen and 53K cellular protein on the surface of SV40‐transformed mouse cells
Authors: Santos Myriam , Butel Janet S.
Published: December 2004

The possible interaction between simian virus 40 (SV40) large tumor antigen (T‐ag) and cellular proteins in the plasma membrane of SV40‐transformed mouse cells was investigated. The presence of SV40 T‐ag, 53,000 (53K) cellular protein, and histocompatibility (H‐2) antigens on the surface of SV40‐transformed cells was demonstrated by immunofluorescence. The use of lactoperoxidase‐catalyzed cell surface iodination and a differential immunoprecipitation technique established that large T‐ag is associated with the 53K host‐coded protein on the surface of the transformed cells. In contrast, no detergent‐stable complex between large T‐ag and H‐2 antigens was detected. Both labeled T‐ag and 53K protein were coprecipitated from surface‐iodinated SV40‐transformed cells by monoclonal antibodies directed against either the viral or the cellular protein. Based on the unique antigenic sites recognized by the anti‐T monoclonal antibodies, it appears that both the carboxy and amino termini of the T‐ag polypeptide are exposed on the surface of SV40‐transformed mouse cells. The nature of the association between surface T‐ag and 53K protein, as well as that between the molecular complex and the plasma membrane, remains to be determined. The possible effect of the surface‐associated T‐ag/53K complex on cellular proliferation is considered.
Parameters for crystal growth of ribosomal subunits
Authors: Yonath A. , Müssig J. , Wittmann H. G.
Published: December 2004

A systematic analysis of the parameters that control the crystal growth of the large subunit of ribosomes from B stearothermophilus has been carried out. Several parameters have been identified and classified according to their significance. It was found that only biologically active particles can crystallize and that the critical period for the crystallization process is the first few days, during which changes in the volume and content of the crystallization drop are of importance for both nucleation and crystal growth. Consequently, an experimental procedure for fine control of these variables has been developed. As a result of these studies, the reproducibility of crystal formation was increased, and larger and more stable crystals have been obtained.
Biochemical and morphological properties of bovine erythrocyte membrane glycoproteins
Authors: Fletcher M. A. , Brunschwig J. P. , Lo H. , Caldwell K. E. , Lo T. M.
Published: December 2004

The major and minor sialoglycoproteins of the bovine erythrocyte have been solubilized and extensively purified. A comparison of composition revealed that the major glycoprotein had 77% carbohydrate and 23% peptide, and the minor one had 27% carbohydrate and 73% peptide. Molar ratios of sugars were related, however, the major glycoprotein had twice as much galactose and sialic acid as did the minor glycoprotein. Molecular weights, estimated from retardation coefficients of mobility in sodium dodecyl sulfate gel electrophoresis, were 55,000 for the major glycoprotein and 34,000 for the minor glycoprotein. The glycoproteins were studied by electron microscopy before and after delipidation and after ultracentrifugation. The major glycoprotein, prior to delipidation, formed large micelles. After delipidation, the major glycoprotein could not be visualized suggesting that it did not form aggregates in aqueous solution. The minor glycoprotein was visualized as rather uniform spherical aggregates (62 Å average diameter) which tended to form short chains and small clumps. These characteristic aggregates were seen both before and after delipidation. After ultracentrifugation, fixation and sectioning both glycoproteins appeared to have formed microcrystalline arrays with average periodicity of 49 Å.
The antigenic structure of the acetylcholine receptor from torpedo californica
Authors: Gullick W. J. , Lindstrom J. M.
Published: December 2004

The immunological structure of the acetylcholine receptor (AChR) from the electric organ of Torpedo californica was studied using a large number of monoclonal antibodies which were initially selected for their abilities to bind to intact AChRs. The monoclonal antibodies were tested for their ability to bind to denatured AChR subunits labeled with 125I. Antibodies derived from rats immunized with individual denatured subunits or a mixture of subunits of Torpedo AChR reacted well in the assay. A much smaller proportion of antibodies derived from rats immunized with native Torpedo AChR or native AChR from Electrophorus electricus electric organ, bovine muscle, or human muscle reacted with denatured subunits of Torpedo AChR. Many monoclonal antibodies reacted with more than one subunit, but they always reacted best with the subunit used for immunization.

Those monoclonal antibodies that bound to intact subunits were mapped more precisely by their ability to bind characteristic fragments of each subunit generated by proteolysis with Staphylococcal V8 protease. These fragments were analyzed by SDS polyacrylamide gel electrophoresis, and monoclonal antibodies that precipitated the same fragment pattern were placed in groups. By this method, we define a minimum of 28 determinants on Torpedo AChR.
Modulation of the neuronal binding of the β subunit of nerve growth factor (NGF) by the α‐NGF subunit
Authors: Woodruff Nathaniel R. , Neet Kenneth E.
Published: December 2004

The effect of the α subunit of the 7S‐NGF on the binding of β‐NGF to its two classes of sites on target cells has been studied. The presence of μM concentrations of α‐NGF causes the displacement of 125I‐β‐NGF from one class of sites on dissociated dorsal root ganglia neurons from stage E9 chicken embryos. At O.1 nM 125I‐β‐NGF, increasing α‐NGF concentrations produce a monotonic displacement curve with half‐maximal displacement occurring at 10 μM α‐NGF. The affinity and number of sites of the 125I‐β‐NGF displaced by α‐NGF are similar to those of β‐NGF that binds to the higher affinity (site I) receptors. The binding to the lower affinity class of sites (site II) is not affected‐by concentrations of α‐NGF up to 30 μM. This modulation of 125I‐β‐NGF binding does not occur with equivalent concentrations of serum albumin. No detectable neuronal binding of 125I‐β‐NGF was found, suggesting that the mechanism does not involve direct competition for receptor sites. The dissociation constant for the α‐β complex is in the μM range, and formation of this complex in solution can thus compete with the process of 125I‐β‐NGF binding to neurons. A model accounting for these observations includes binding of the α‐β complex to the lower affinity but not to the higher affinity sites. We conclude that there are differences in the specificity of the two classes of receptors.
Multiple specific binding sites for purified glucocorticoid receptors on mammary tumor virus DNA
Authors: Payvar Farhang , Firestone Gary L. , Ross Susan R. , Chandler Vicki L. , Wrange Örjan , Carlstedt‐Duke Jan , Gustafsson Jan‐åke , Yamamoto Keith R.
Published: December 2004

Glucocorticoid hormones selectively stimulate the rate of transcription of integrated mammary tumor virus (MTV) sequences in infected rat hepatoma cells. Using two independent assays, we find that purified rat liver glucocorticoid receptor protein binds specifically to at least four widely separated regions on pure MTV proviral DNA. One of these specific binding domains, which itself contains at least two distinct receptor binding sites, resides within a fragment of viral DNA that maps 110–449 bp upstream of the promoter for MTV RNA synthesis. Three other binding domains lie downstream of the promoter and within the MTV primary transcription unit. Restriction fragments bearing separate binding domains have been introduced into cultured cells; transformants have been recovered in which the introduced fragments arc expressed under glucocorticoid control. Thus, it appears that this assay will be useful for assessing the biological significance of the receptor binding sites detected in vitro.
Cells transformed by a wide variety of agents express higher abundance levels of some cellular RNA species
Authors: Schutzbank T. , Levine A. J.
Published: December 2004

A cDNA‐cloned library was prepared from mRNA synthesized by SV40‐transformed mouse cells. Eleven cDNA clones were selected based on their ability to hybridize higher levels of mRNA in SV40‐transformed 3T3 cells than in 3T3 cells. These cDNA clones were employed to screen the steady‐state levels of cytoplasmic RNAs in a wide variety of viral (SV40, polyoma, adenovirus, and Rous sarcoma virus) and nonviral (methylcholanthrene, embryonal carcinoma) transformed cell lines. Two of the cDNA clones—A17 and 104—detected greater than 40–100‐fold higher levels of mRNA in all the transformed cell lines tested when compared to nontransformed cells (3T3, C3HEF). The levels of mRNA complementary to these two cDNAs were regulated in a temperature‐sensitive fashion (87–100‐fold) in both SV40tsA‐ and RSV ts‐src‐transformed murine cell lines. These two cDNA clones detected greater than 100‐fold, higher levels of complementary RNA derived from SV40 tumor tissue than in normal mouse liver. RNA species complementary to cDNA clones A17 or 104 were not detected in either actively growing nontransformed cells or in serum‐stimulated 3T3 cells. The abundance levels of mRNAs detected by these two cDNA clones appear to be regulated 100‐fold or greater by the transformed state, independent of the transforming agent. The higher levels of these RNA species detected in transformed mouse cells appear not to be solely regulated by the state of growth of nontransformed cells.
Sequence analysis and in vitro transcription of portions of the epstein‐barr virus genome
Authors: Deininger P. L. , Bankier A. , Farrell P. , Baer R. , Barrell B.
Published: December 2004

The 17,180 base‐pair Eco‐RI‐C fragment of Epstein‐Barr virus has been sequenced in its entirety. This same fragment has also been analyzed for RNA polymerase II promoters, which are active in a soluble in vitro assay. These data are compared to the availability of predicted open reading frames and other potential nucleotide signals associated with transcription. In addition, the DNA sequence of a number of previously undetected repeated DNA sequences from this and several nearby regions of the viral genome are reported.
Characterization of viral genomes in the liver and serum of chimpanzee long‐term hepatitis B virus carriers: A possible role for supercoiled HBV‐DNA in persistent HBV infection
Authors: Ruiz‐Opazo Nelson , Chakraborty Prasanta R. , Shafritz David A.
Published: December 2004

In chimpanzee hepatitis B virus (HBV) carriers, the molecular mechanism for viral persistence has been examined by analyzing the properties of viral DNA molecules in liver and serum. Two extrachromosomal HBV‐DNA molecules migrating on Southern blots at 4.0 kb and 2.3 kb were observed in chimpanzee liver DNA. There was no evidence for integration of HBV sequences into the host genome. The HBV‐DNA molecule which migrated at 4.0 kb position represents a full‐length “nicked,” relaxed circular form, and the DNA molecules migrating at 2.3 kb position represents a supercoiled form of the HBV genome. Evidence for supercoiled HBV‐DNA in serum was obtained by production of the relaxed circular intermediate upon digestion of Dane particle DNA with specific nucleases S1 and Bal 31. A possible role of these two extrachromosomal HBV‐DNA molecules in the biology of hepatitis B virus infection and the mechanism for viral persistence arc discussed.
Biologic activity of anti‐thyrotropin anti‐idiotypic antibody
Authors: Farid Nadir R. , Brioñes‐Urbina Rosario , Nazrul‐Islam M.
Published: December 2004

We raised an antihuman thyrotropin anti‐idiotypic antibody and showed that it was active at the thyrotropin receptor. Thus this antibody inhibited 125I b‐TSH binding to thyroid plasma membranes, stimulated adenylate cyclase activity through a guanyl nucleotide‐dependent mechanism, increased radioiodide entry rate into isolated porcine thyroid follicular cells, and induced such cultured cells to organize into follicles. All these parameters are typical of thyrotropin action. This work raises the possibility that thyroid stimulating antibodies that cause the hyperthyroidism of Graves disease may be, at least in some patients, anti‐thyrotropin anti‐idiotypic antibodies. It also offers a novel method whereby antireceptor antibodies used in the isolation and characterization of the receptor may be raised from ligands.
A basement membrane‐associated glycoprotein from skeletal muscle
Authors: Marton Linda S. G. , Arnason Barry G. W.
Published: December 2004

We have isolated a major glycoprotein that appears to be associated with rat skeletal muscle basement membrane. We determined that the glycoprotein was part of the muscle cell surface complex when we found it to be enriched in preparations of muscle ghosts. We isolate the glycoprotein from homogenized muscle preextracted with 4 M and 8 M urea. It elutes as a major component in the presence of 8 M urea/50 mM 2‐mercaptoethanol. Its apparent molecular weight on sodium dodecyl sulfate gels is 130,000. Amino acid analysis indicates that it is not a collagen but that it does contain small amounts of hydroxyproline and hydroxylysine. There may be collagenous domains in the glycoprotein molecule, for it is cleaved into three fragments by purified bacterial collagenase. Immunoperoxidase staining confirms that the 130,000‐dalton protein is localized at the surface of adult skeletal muscle cells. It is probably a general basement membrane‐associated glycoprotein because we found material immunologically cross‐reactive with the muscle glycoprotein in basement membrane regions of kidney, liver, brain, and small intestine. We have shown the glycoprotein to be distinct from fibronectin, laminin, and types I, III, IV, and V collagens in enzyme‐linked immunosorbent assays.
Biochemical characterization of transformation‐specific proteins of acute avian leukemia and sarcoma viruses
Authors: Moelling K. , Owada M. K. , Greiser‐Wilke I. , Bunte T. , Donner P.
Published: December 2004

The biological and biochemical properties of the transformation‐specific proteins of three avian oncornaviruses with different oncogenic potentials were compared, namely the gag‐myc protein of the avian myelocytomatosis virus MC29, the gag‐erb A protein of the avian erythroblastosis virus AEV, and the gag‐fps protein of Fujinami sarcoma virus FSV. These oncogenes were analyzed in transformed fibroblasts that expressed only the transforming proteins but showed no virus replication. Monoclonal antibodies against the viral structural protein p19, which is the N‐terminus of the proteins, were used for indirect immunofluorescence, for immunoprecipitation of the proteins from subcellular fractions, and for immunoaffinity column chromatography. With this last method a 3000‐fold purification of the proteins was obtained. By indirect immunofluorescence it was shown that the gag‐myc protein was located in the nucleus, and bound to DNA after purification. The gag‐erb A protein was not nuclear but probably located in the cytoplasm and did not bind to DNA after purification. Neither of the two proteins exhibited protein kinase activity. In contrast, the gag‐fps protein did not bind to DNA but showed protein kinase activity after purification. It was not located in the nucleus either.
Binding of isolated 3T3 surface membranes to growing 3T3 cells and their effect on cell growth
Authors: Lieberman Michael A. , Keller‐McGandy Christine E. , Woolsey Thomas A. , Glaser Luis
Published: December 2004

We have quantitated by autoradiography the binding of [125I]labeled 3T3 plasma membrane fragments to 3T3 cells growing on the surface of plastic dishes; ie, the same conditions in which these membranes specifically arrest the growth of 3T3 cells early in the G1 phase of the cell cycle. We have been able to demonstrate that binding of membranes to cells is coincidental with the expression of the growth inhibitory activity of protein(s) present in the membrane fragments. Treatments that reduce binding (heat denaturation of the membranes or culture in the presence of high scrum) also reduce growth inhibitory activity. [125I]labeled membranes bound to cells are located primarily on the cell surface (as determined by electron microscope autoradiography) and are exchangeable with unlabeled membranes. We conclude that binding of membranes to cells is necessary but may not be sufficient for the expression of the growth inhibitory activity of these membranes. This approach provides information not only on the average level of binding of membranes to cells, but also provides a quantitative assessment of the variation of the level of membrane to cell binding between different cells in the population.
Characteristics of plasma membrane isolated from a mouse T lymphoma line: comparison after nitrogen cavitation, shearing, detergent treatment, and microvesiculation
Authors: Culvenor Janetta G. , Mandel Thomas E. , Whitelaw Alison , Ferber Ernst
Published: December 2004

Plasma membrane was isolated from the mouse T lymphoma cell line WEHI‐22 using four different methods of cell disruption followed by centrifugal fractionation. Disruption by nitrogen cavitation or by shearing with a cell pump produced plasma membrane vesicles of similar buoyant density (1.10 g/ml) and morphological appearance. Few C‐type virus particles were present. Cell disruption with 2% Tween‐40 produced membrane vesicles of similar morphology but lower density (1.09 g/ml). All of the above preparations resulted in vesicles with aggregated intramembranous particles after freeze fracture. Microvesiculation with a sublytic concentration of a lysophosphatidylcholine analog (ET‐12‐H) (0.0032% w/v) produced small membrane vesicles which could be isolated without differential centrifugation. However, these had a slightly higher density than vesicles prepared by cavitation or shearing and were co ntaminated by virus particles. Unlike the other preparations, vesicles prepared with ET‐I2‐H had dispersed intramembranous particles. The enzyme γ‐glutamyl transferase was enriched from 20‐ to 45‐fold in the membrane preparations and proved a suitable plasma membrane marker for these cells whose 5′‐nucleotidase content is very low.
Transformation of MMC‐E epithelial cells by acute 3611‐MSV: inhibition of collagen synthesis and induction of novel polypeptides
Authors: Keski‐Oja Jorma , Rapp Ulf R. , Vaheri Antti
Published: December 2004

Mouse embryo epithelial cells MMC‐E were transformed by novel fibrosarcoma‐inducing murine sarcoma virus 3611 ‐MSV. The cells were analyzed for the production and deposition of pericellular glycoproteins by immunofluorescence and by radioactive metabolic and cell surface labeling techniques followed by analysis in polyacrylamide gels and fluorography. The pericellular fibronectin matrix was lost, but unlike in virus‐transformed fibroblastic cells, the production of fibronectin was not affected. The major differences detected were decrease in collagen production and initiation of synthesis of two major glycoproteins with Mr 58,000 and 60,000. Cell surface carbohydrate labeling indicated that after 3611‐MSV transformation the cells expressed Mr 100,000 and 68,000 polypeptides. The present and previous results show that viral transformation of epithelial cells induces different transformed phenotypes that are associated with distinct alterations in pericellular glycoproteins.
The fate of the N‐formyl‐chemotactic peptide receptor in stimulated human granulocytes: subcellular fractionation studies
Authors: Jesaitis Algirdas J. , Naemura Joseph R. , Painter Richard G. , Schmitt Manfred , Sklar Larry A. , Cochrane Charles G.
Published: December 2004

Experiments were performed to examine how human granulocytes, stimulated by N‐formyl‐chemotactic peptides, process the N‐formyl peptide receptor. One percent of the surface N‐formyl‐chemotactic peptide receptors of purified human granulocytes were covalently, specifically, and radioactively labeled at 4°C using the photochemically reactive N‐formyl‐chemotactic hexapeptide CHO‐Nle‐Leu‐Phe‐Nle‐[l25I] Tyr‐N°(6‐(4′‐azido‐2′‐nitrophenyl‐amino)hexanoyl)‐Lys. After incubation in the presence of 500 nM of N‐formyl‐Met‐Leu‐Phe at 37°C, the cells were lysed and fractionated by isopycnic surcrose density gradient sedimentation. Receptor‐associated radioactivity cosedimented with plasma membrane in fractions from cells kept at 4°C or incubated at 37°C for 2 min or less. Fractionation of cells incubated at 37°C for longer times revealed that the radioactivity sedi‐mented to lower densities coincident with Golgi markers and the site of noncovalently bound and internalized formyl‐chemotactic peptide. To follow the redistribution of unoccupied receptors, human granulocytes were stimulated with 500 nM N‐formyl‐Met‐Leu‐Phe at 37°C for 5 min, washed, lysed by N2 cavitation, and fractionated by rate zonal sucrose density gradient sedimentation. Compared to unstimulated controls the specific binding of N‐formyl‐Met‐Leu‐[3H]Phe decreased 76% ± 9% in plasma membrane fractions. N‐formyl‐Met‐Leu‐[3H]Phe‐binding activity associated with an intracellular pool cosedimenting with specific granules remained unchanged. Approximately 20% of the activity lost in the plasma membrane could be accounted for by a redistribution of specific N‐formyl‐Met‐Leu‐Phe binding to fractions enriched in azurophil granules. We conclude that the receptor is the carrier in the internalization of the N‐formyl‐chemotactic peptides to a Golgi‐enriched fraction and hypothesize that after a short residency in this fraction, the receptor may dissociate from the ligand and pass onto a fraction consedimenting with dense granules.
Photoaffinity labeling of the N‐formyl peptide receptor of human polymorphonuclear leukocytes
Authors: Painter Richard G. , Schmitt Manfred , Jesaitis Algirdas J. , Sklar Larry A. , Preissner Klaus , Cochrane Charles G.
Published: December 2004

Quantitative analysis of ligand‐occupied receptor interactions with elements of the cytoskeleton and with intracellular compartments requires a sensitive and simple method of identifying the receptor‐ligand complex in living cells. Toward this goal, we have prepared a photoactivatable arylazide derivative of the chemotactic peptide N‐formyl‐Nle‐Leu‐Phe‐Nle‐Tyr‐Lys, which can be radiolabeled to high specific activity with 125I. This derivative was biologically active as judged by its ability to elicit superoxide anion production by human PMNL at nanomolar concentrations (ED50 ∼ 0.7 nM). When incubated at 0°C with whole PMNL, radioactive ligand became specifically and saturably associated with a 60‐70,000‐dalton species (as assessed by SDS‐PAGE) after exposure to UV light. Addition of 10‐100‐fold excess of unlabeled parent or unlabeled azidopeptide derivative completely blocked uptake into this species. Approximately 20‐40% of the available surface receptor‐binding sites were covalently labeled under these conditions. Subcellular fractionation of the labeled cells on sucrose gradients after homogenization showed that the labeled species was primarily associated with plasma membrane‐rich fractions. The labeled receptor could be completely solubilized with Triton X‐100 in a form which eluted as a single species with a Stoke's radius of less than 50 Å on Sepharose 4B columns. In addition, the solubilized receptor‐ligand complex bound specifically to wheat germ agglutinin, indicating that it is probably a glycoprotein. The ability to label the receptor in living PMNL with a high efficiency should facilitate the study of receptor dynamics and receptor physiochemical properties in this system.
Receptor‐bound thrombin is not internalized through coated pits in mouse embryo cells
Authors: Bergmann John S. , Carney Darrell H.
Published: December 2004

The localization of thrombin receptors on mouse embryo (ME) cells was examined using electron microscope (EM) immunocytological techniques. ME cells were fixed with formaldehyde, prior to thrombin binding, and thrombin visualized on cell surfaces using affinity‐purified antithrombin rabbit antibody and colloidal gold labeled anti‐rabbit IgG. Colloidal gold particles were found in clusters on the surface of cells incubated with thrombin. There were approximately seven particles per cluster observed in thin sections with cluster diameters ranging from 70 to 200 nm. These clusters were not observed on cells incubated without thrombin. The total number of particles present on cells incubated with and without thrombin indicate that the colloidal gold labeling is approximately 98% specific for thrombin. Only four colloidal gold particles out of approximately 1,200 were associated with coated pits. Thus the thrombin receptor clusters do not appear to associate with coated membrane regions. To determine whether receptor‐bound thrombin was internalized by receptor‐mediated endocytosis, ME cells were incubated with 125I‐thrombin and examined using EM autoradiography and the trypsin sensitivity of 125I‐thrombin which was associated with the cells. In two types of experiments, where thrombin was incubated with cells at 4°C and the temperature increased to 37°C and where initial incubation was at 37°C, the receptor‐directed specific internalization proceeded at approximately the same rate as nonspecific internalization. These studies indicate that thrombin that binds to its receptors on ME cells is not rapidly internalized by receptor‐mediated endocytosis.
Organization and expression of hepatitis B sequences cloned from hepatocellular carcinoma tissue DNA
Authors: Dejean Anne , Carloni Guido , Bréchot Christian , Tiollais Pierre , Wain‐Hobson Simon
Published: December 2004

We have constructed a phage λ library of liver DNA fragments from West African patient who died of liver failure due to advanced hepatocellular carcinoma. Four hepatitis B virus (HBV) DNA‐carrying recombinants have been isolated, one clone (λÍ22) being analyzed in greatest detail. It contains approximately 3.8 kb of HBV DNA without detectable deletions or rearrangements. One site of integration lies close to the nick in free viral DNA. The restriction map of the HBV sequences is close to those published for the ay subtype. Coconvection of mouse Ltk−cells with λÍ22 and cloned: thymidine kinase gene results in the expression of gene S and the excretion of hepatitis B surface antigen (HBsAg) particles into the culture supernatant.
DNA methylation, retroviruses, and embryogenesis
Authors: Jaenisch Rudolf , Harbers Klaus , Jähner Detlev , Stewart Colin , Stuhlmann Heidi
Published: December 2004

By exposing preimplantation embryos to Moloney leukemia virus (M‐MuLV), we have previously derived substrains of mice designated as Mov‐1‐Mov‐13 which genetically transmit the virus from one generation to the next. In some of the substrains the inserted viral genome becomes activated at specific stages of embryogenesis and the available evidence suggests that these viral genomes are developmentally regulated. To investigate the effect of cellular differentiation on virus expression, M‐MuLV was introduced either into preimplantation or postimplantation mouse embryos or into embryonal carcinoma (EC) cells. Whereas preimplantation embryos or EC cells are not permissive for virus expression, efficient replication occurred in postimplantation embryos or in differentiated cell lines. The viral genomes introduced into early embryonal cells were highly methylated and noninfcctious when analyzed in the adult. In contrast, viral genomes introduced into postimplantation embryos or into differentiated cells remained unmethylated and were infectious in a transfection assay. These results demonstrate an efficient de novo methylation activity which appears to be involved in repression of genes introduced into pluripotent embryonal cells and which is not observed in cells of the postimplantation embryo or in differentiated cells in tissue culture.
Cholera toxin B‐subunit protects mammalian cells from ricin and abrin toxicity
Authors: Delfini Carlo , Sargiacomo Massimo , Amici Carla , Oberholtzer Goffredo , Tomasi Maurizio
Published: December 2004

The glycoproteins ricin and abrin intoxicate cells by inhibiting protein synthesis. Pretreatment of HeLa cells with cholera toxin partially protects them from ricin and abrin activity. The involvement in this phenomenon of the various effects of cholera toxin, namely, redistribution of membrane receptors elicited from protomer B and increasing cyclic AMP concentrations induced by protomer A, were studied. Substances able to enhance cyclic AMP concentrations do not affect ricin and abrin activity, while protomer B alone protects cells. In addition, the effects of several lectins on ricin or abrin toxicity were examined. Almost complete prevention of ricin or abrin activity was obtained using concanavalin A (Con A) and wheat germ agglutinin (WGA). Conversely, neither succinyl Con A nor Ulexeuropeus agglutinin (UEA) affected the cellular response. Both protomer B of cholera toxin and Con A did not alter the binding of ricin or abrin; they seem to protect cells by altering membrane structure.
Influence of growth conditions on the composition of the plasma membrane from yeast and on kinetic properties of two membrane functions
Authors: Stadtlander Klaus , Rade Sabine , Ahlers Jan
Published: December 2004

The influence of different growth conditions on the phospholipid composition and on two membrane functions, the Mg‐ATPase and the purine transport system, was investigated. Addition of cholinechloride to the growth medium led to a certain rise in the amount of phosphatidylcholine, whereas supplementation with ethanolamine resulted in a considerably higher portion of phosphatidylethanolamine. When yeast cells were cultured at lower temperatures we found more short‐chain fatty acids with a higher content of monounsaturated chains as compared to higher growth temperatures. Addition of paraquat, a herbicide which enhances lipid peroxidation by free radicals, reduced the amount of unsaturated fatty acids without influencing their chain length.

The altered membrane composition had no influence on the basic mechanism of interaction between ATPase, MgATP, and free Mg2+ ions. However, several kinetic constants such as Km, Vmax, Ka, and especially Ki were influenced to some extent. Whereas the affinity of the purine transport system to its substrate was not significantly changed by the growth conditions, an effect on Vnlax could be seen. Lower growth temperatures clearly led to higher maximal uptake velocities. The presence of paraquat during growth resulted in a considerable decrease of Vmax.
Lipolysis induction in adipocytes by a protein from tumor cells
Authors: Kitada Shinichi , Hays Esther F. , Mead James F. , Zabin Irving
Published: December 2004

Extracts of thymic lymphoma that are obtained from AKR mice and are kept in the cold for at least several days can induce lipolytic activity in rat adipocyte suspensions. Freshly prepared extracts have low activity but contain a low molecular weight material of less than 10,000 daltons that aggregates on standing in the cold and becomes active. Treatment of aged extracts with trypsin causes a loss in activity indicating that the active material is a protein. It has been obtained in partially purified form, is relatively heat stable, and is not a lipase.

Activity was also demonstrated in AKRXDBA/2 lymphoma (induced by AKR SL3‐3 virus) and in transplanted lymphomas from a Friend‐virus‐induced erythroleukemia cell line in DBA/2 mice, but was not detected in normal thymus, spleen, liver, or other tissues. The partially purified material produced a massive fat mobilization when injected into normal mice.
Ionic regulation of MEL cell commitment
Authors: Levenson Robert , Macara Ian , Cantley Lewis , Housman David
Published: December 2004

A key event in the initiation of the dimethyl sulfoxide (DMSO)‐induced program of murine erythroleukemia (MEL) cell differentiation is a rise in the level of cytoplasmic calcium ions. Our interest in the present study is whether other inducers of the terminal erythroid differentiation program also act via a calcium‐dependent pathway. Inhibition of calcium transport has been found to prevent the induction of MEL cell commitment by DMSO, butyric acid (BA), or hypoxanthine (HX). Enhancement of the calcium flux rate with A23187 or elevation of cytoplasmic calcium levels with FCCP stimulates the kinetics of commitment in response to all three inducers. These results suggest that of the inducers we have tested (DMSO, BA, and HX), all three act to initiate commitment via a common mechanism which involves modulation of cytoplasmic calcium levels.