bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative kinetics of phosphomannosyl receptor‐mediated pinocytosis of fibroblast secretion acid hydrolases and glycopeptides prepared from them
Authors: Fischer H. David , Creek Kim E. , Strisciuglio Pietro , Sly William S.
Published: December 2004

In a previous report we demonstrated that phosphorylated oligosaccharides isolated from acid hydrolases were subject to pinocytosis by phosphomannosyl receptors present on the cell surface of human fibroblasts [9]. However, limiting quantities of oligosaccharides precluded detailed comparison of the kinetics of pinocytosis of these phosphorylated oligosaccharides to those of the acid hydrolases from which they were derived. In this report we present studies comparing the kinetics of pinocytosis of acid hydrolases from NH4Cl‐induced fibroblast secretions with those of concanavalin A‐binding glycopeptides prepared from them by pronase digestion. The uptake of both secretion acid hydrolases and 125I‐labeled glycopeptides was linear for at least 3 hr, saturable, inhibited competitively by mannose 6‐phosphate, and destroyed by prior treatment of the ligand with alkaline phosphatase. The inhibition constants of excess unlabeled glycopeptide for the uptake of 125I‐labeled glycopeptides (Ki of 1.5 × 10−6 M) and for the uptake of secretion acid hydrolases (Ki of 2.2 × 10−6 M) were remarkably similar. Furthermore, the Ki for mannose 6‐phosphate inhibition of pinocytosis of glycopeptide uptake (3 × 10−5 M) compares closely to that previously determined for the pinocytosis of intact “high‐uptake” acid hydrolases (3–6 × 10−5 M).

“High‐uptake” fractions of both ligands were prepared and quantified by affinity chromatography on immobilized phosphomannosyl receptors purified from bovine liver. Only 10% of the concanavalin A‐binding glycopeptides bound to the immobilized phosphomannosyl receptors, while 80% of the acid hydrolases from which they were prepared bound and were eluted with 10 mM mannose 6‐phosphate. However, the fraction of each type of ligand that binds to the immobilized phosphomannosyl receptors accounts for all the uptake activity of that ligand. The pinocytosis rates (% of added ligand internalized/mg protein/hr) of the “high‐uptake” fraction of both intact acid hydrolase (12%/mg/hr) and glycopeptide (6%/mg/hr) differed by only twofold. The apparent Kuprake for both ligands was of the same order of magnitude. The similarity in the kinetics of pinocytosis of the secreted acid hydrolases and of the phosphomannose‐bearing glycopeptides prepared from them suggests that the structural information which confers high‐affinity binding to the phosphomannosyl receptor is contained in the glycopeptide units themselves. No additional information from the intact protein backbone appears essential for phosphomannosyl receptor‐mediated pinocytosis.
Rotaviruses code for two types of glycoprotein precursors
Authors: Ericson Brad L. , Petrie Betty L. , Graham David Y. , Mason Bruce B. , Estes Mary K.
Published: December 2004

Rotaviruses are nonenveloped viruses that code for two glycoproteins: a structural glycoprotein (VP7) and a nonstructural glycoprotein (NS29). The precursor to VP7 (37K) was shown to contain a 1.5K cleavable signal sequence. The 37K precursor was authentically processed (signal sequence cleaved and the polypeptide “core” glycosylated) when synthesized in a cell‐free system supplemented with dog pancreatic microsomes. Similar experiments were performed with the nonstructural glycoprotein precursor (20K); however, the 20K precursor contained an integral (noncleavable) signal sequence. Both precursors were inserted into membranes cotranslationally and both glycosylated products underwent post‐translational oligosaccharide processing. The results suggest a morphogenetic scheme for the simian rotavirus SA11.
A simian virus 40‐encoded protein of Mr 74,000 daltons is structurally related to the capsid proteins of the virus
Authors: Krippl Bernd , Dreiseikelmann Brigitte , Werchau Hermann
Published: December 2004

We have demonstrated the synthesis of a 74,000‐dalton protein (74K protein) in African green monkey kidney cells infected with simian virus (SV)40. The 74K protein was detected late during the lytic cycle. Its synthesis was inhibited by arabinosyl cytosine as was the synthesis of the capsid proteins. Monospecific antibodies raised against VP1 and VP3 precipitated the structural proteins and the 74K protein. The 74K protein was not found in purified virions. Tryptic peptide analysis demonstrated that the 74K protein shares methionine‐ and serine‐containing peptides with VP1 and VP3 and thus is structurally related to the capsid proteins.
Cell cycle and cyclic AMP‐dependent phosphorylation of plasma membrane proteins p14 and p24: Defects in smooth surface transformed cells
Authors: Boman Bruce M. , Zschunke Michael A. , Scott Robert E.
Published: December 2004

Two proteins which are localized to the cytoplasmic surface of the plasma membrane, p14 and p24, undergo cyclic AMP‐dependent phosphorylation in rapidly growing nontransformed murine embryo cells. In this cell system, growth arrest in the G1 phase of the cell cycle induced by growth factor deprivation is associated with the reversible loss in ability to phosphorylate these substrates. By contrast Simian virus 40 and methylcholanthrene transformed cells show both defective G1 growth control and defects in their ability to phosphorylate p14 and p24 under all tested growth conditions. These data suggested a correlation between defects in the physophorylation of p14 and p24 and defects in the ability of transformed cells to G1 growth arrest. The results of the current studies by contrast show that 3T3 T proadipocytcs which have been transformed by the smooth surface tumorigenesis method show different characteristics. They retain the ability to G1 growth arrest in serum‐deficient medium. They show cyclic AMP‐dependent phosphorylation of p14 and p24 during exponential growth. They do not, however, down regulate p14 and p24 phosphorylation in association with G1 growth arrest. These observations suggest that neoplastic transformation is not necessarily associated with absolute defects in the ability to phosphorylate p14 and p24. Rather, the results of the current study suggest that the inability to modulate the cyclic AMP‐dependent phosphorylation of plasma membrane p14 and p24 proteins during the G1 phase of the cell cycle may be more tightly associated with neoplastic transformation.
A model for the structure of fumarate reductase in the cytoplasmic membrane of escherichia coli
Authors: Weiner Joel H. , Lemire Bernard D. , Jones Robert W. , Anderson Wayne F. , Scraba Douglas G.
Published: December 2004

By a recombinant DNA approach we have prepared Escherichia coli cytoplasmic membranes that are highly enriched in the terminal electron transfer enzyme fumarate reductase. This enzyme is composed of four nonidentical subunits in equal molar ratio. A 69,000‐dalton covalent flavin‐containing subunit and a 27,000‐dalton nonheme iron‐containing subunit make up a membrane extrinsic catalytic domain. Two very hydrophobic subunits of 15,000 and 13,000 daltons make up the hydrophobic membrane anchor domain. Electron microscopy of negatively stained membranes shows a characteristic knob‐and‐stalk‐type structure composed of the catalytic domain. The anchor polypeptides have been analyzed for hydrophobic segments and α‐helical content and a model for their organization within the lipid bilayer is presented. The results reviewed in this paper suggest a model for the fumarate reductase complex in the cytoplasmic membrane
Light regulation of photosynthetic membrane structure, organization, and function
Authors: Melis Anastasios
Published: December 2004

The light environment during plant growth determines the structural and functional properties of higher plant chloroplasts, thus revealing a dynamically regulated developmental system. Pisum sativum plants growing under intermittent illumination showed chloroplasts with fully functional photosystem (PS) II and PSI reaction centers that lacked the peripheral chlorophyll (Chi) a/b and Chl a light‐harvesting complexes (LHC), respectively. The results suggest a light flux differential threshold regulation in the biosynthesis of the photosystem core and peripheral antenna complexes. Sun‐adapted species and plants growing under far‐red‐depleted illumination showed grana stacks composed of few (3–5) thylakoids connected with long intergrana (stroma) thylakoids. They had a PSII/PSI reaction center ratio in the range 1.3–1.9. Shade‐adapted species and plants growing under far‐red‐enrichcd illumination showed large grana stacks composed of several thylakoids, often extending across the entire chloroplast body, and short intergrana stroma thylakoids. They had a higher PSII/PSI reaction center ratio, in the range of 2.2–4.0. Thus, the relative extent of grana and stroma thylakoid formation corresponds with the relative amounts of PSII and PSI in the chloroplast, respectively. The structural and functional adaptation of the photosynthetic membrane system in response to the quality of illumination involves mainly a control on the rate of PSII and PSI complex biosynthesis.
The detergent solubility properties of a malarial (Plasmodium knowlesi) variant antigen expressed on the surface of infected erythrocytes
Authors: Howard Russell J. , Barnwell John W.
Published: December 2004

Four detergents have been compared for identification of the Plasmodium knowlesi variant antigen on infected erythrocytes by immunoprecipitation analysis. Erythrocytes infected with late trophozoite and schizont forms of cloned asexual parasites were labeled by lactoperoxidase‐catalyzed radioiodination and extracted either with the anionic detergents sodium dodecyl sulfate (SDS) or cholate, the neutral detergent Triton X‐100, or the zwitterion 3‐[(3‐cholamidopropyl)di‐methylammonio]‐1‐propane sulfonate (CHAPS). After addition of Triton X‐100 to SDS and cholate extracts, parallel immunoprecipitations of the four extracts were performed using rhesus monkey antisera of defined agglutinability. Identical results were obtained with clone Pkl(A+ ), which has 125I‐variant antigens of Mr 210,000 and 190,000, and with clone Pkl(B+)l+, which hasvariant antigens of Mr 200,000–205,000. SDS yielded maximal levels of immunoprecipitated 125I‐variant antigens. Variant‐specific immunoprecipitation was detected in some experiments with Triton X‐100 and cholic acid but with significantly lower recovery than with SDS. CHAPS extraction did not yield the variant antigens on immunoprecipitation. The variant antigens could also be identified in Triton X‐100‐insoluble material by subsequent extraction with SDS, indicating that failure to recover these proteins in the Triton X‐100‐soluble fraction is due to failure of this detergent to extract the variant antigens rather than to degradation during extraction. We suggest that the 125I‐variant antigens either have a structure that renders them intrinsically insoluble in Triton X‐100, cholate, or CHAPS, or that they are associated in some way with host cell membrane components that also resist solubilization by these detergents.
Detergent dissociation of bovine liver phosphomannosyl binding protein
Authors: Mitchell Diane C. , Maler Thomas , Jourdian George W.
Published: December 2004

We have reported previously the isolation and partial characterization of a 215‐kilodalton (Kd) phosphomannosyl binding protein from bovine liver membranes [3,9]. In the present studies evidence is presented that the binding protein is an aggregate. Four N‐terminal amino acids were detected, and the complex could be dissociated into subunits.

Bovine liver membranes were extracted with the detergent, Zwittergent, in the presence of protease inhibitors. The extract was subjected to affinity chromatography on phosphomannan‐Sepharose 4B, and proteins with apparent Mr values of 215 and 57 Kd were eluted with mannose 6‐phosphate. As reported previously, extraction with Triton X‐100 yielded only the higher molecular weight material. When the binding protein was incubated at 4°C in the presence of Zwittergent TM 3‐14 the 215‐Kd form slowly dissociated into smaller subunits; after two months, the major species had an apparent Mr of 57 Kd. The subunits derived from the binding protein were recognized by antiserum raised against purified binding protein. Dissociation of the binding protein by Zwittergent was enhanced by incubation at 37°C, the presence of dithiothreitol, and low pH values. The subunit mixture enriched in the 57‐Kd subunit had a lowered ability to bind ligands containing the phosphomannosyl recognition marker. Binding was partially restored (>48% of the initial value) when dissociated receptor was back exchanged with Triton X‐100.
Stimulation by glutamine of the formation of N6‐hydroxylysine in a cell‐free extract from aerobacter aerogenes 62‐1
Authors: Jackson G. E. D. , Parniak M. A. , Murray G. J. , Viswanatha T.
Published: December 2004

Glutamine may serve as an activator and/or regulator of the N6‐hydroxylase (E.C. 1.14.99) of Aerobacter aerogenes 62‐1. Activation and stabilization of N6‐hydroxylase activity was observed both in vivo and in vitro. Growth in a glutamine‐supplemented medium resulted in (1) maximum N6‐hydroxylase activity at an earlier stage of growth and (2) higher N6‐hydroxylase activity and continued aerobactin synthesis into stationary phase. Storage of P2 in the presence of L‐glutamine (1 mM) significantly increased the lifetime of the labile N6‐hydroxylase activity. Inclusion of L‐glutamine in the incubation mixture typically resulted in a 2‐3‐fold activation of the hydroxylase activity. The stimulatory effect of glutamine was independent of and additive to the enhancement of N6‐hydroxylation by the active component(s) in the supernatant, S2 fraction. Glutamic acid‐γ‐semihydrazide activated slightly in the absence of glutamine but activation of the system by glutamine was decreased by this compound. Azaserine was shown to be an uncompetitive inhibitor with respect to lysine and this inhibition was not reversed by glutamine.
Characterization of thermotropic structural transitions of the erythrocyte membrane: A biochemical and electron‐paramagnetic resonance approach
Authors: Minetti Maurizio , Ceccarini Marina , Maria Anna , Di Stasi M.
Published: December 2004

The relationship between membrane structural properties and functions has been generally inferred from observed thermotropic phenomena. By the use of 16‐dinyloxyl stearic acid spin probe we investigated the red blood cell membrane components involved in three characteristic thermotropic structural transitions occurring at 8, 20, and 40°C. The transition at 8°C is removed by chymotrypsin treatment at the cytoplasmic membrane layer. The 20°C phase transition is unmodified after chymotrypsin treatment and occurs at 15°C after complete proteolysis of intramembranc chymolrypsin insensitive peptides. Liposomes from the total lipid extract of RBC show only one thermotropic transition at 15°C. The 40°C phase transition is absent in vesicles free of skeletal proteins, in vesicles obtained after RBC storage, and in low‐ionic‐strength resealed ghosts. Transitions at 8°C and 40°C appear to be due to the interactions of cytoplasmic exposed proteins with membrane, whereas the 20°C transition is intrinsic to the lipid component.
Amino acid pools in cultured muscle cells
Authors: Low Robert B. , Stirewalt William S. , Rittling Susan R. , Woodworth Robert C.
Published: December 2004

Compartmentalization of cellular amino acid pools occurs in cultures of cardiac and skeletal muscle cells, but the factors involved in this are not clear. We have further defined this problem by analyzing the intracellular free leucine and the transfer‐RNA‐(tRNA)‐bound leucine pool in cultures of skeletal and cardiac muscle incubated with 3H‐leucine in the presence and absence of serum and amino acids. Withdrawal of nitrogen substrates caused substantial changes in leucine pool relationships–in particular, a change in the degree to which intracellular free leucine and tRNA‐leucine were derived from the culture medium. In separate experiments, the validity of our tRNA measurements was confirmed by measurements of the specific activity of newly synthesized ferritin after iron induction. We discuss the implications of these findings with regard‐to factors involved in the control of amino acid flux through the cell, as well as with regard to design of experiments using isotopic amino acids to measure rates of amino acid utilization.
Oncogenes: Growth regulation and the papovaviruses polyoma and SV40
Authors: Smith Alan E.
Published: December 2004

Cellular oncogenes and their activated and retrovirus‐coded counterparts play an important role in cellular regulation. Here the relationship between such oncogenes and the genes coding for the transforming proteins of the papovaviruses, polyoma viruses, and simian virus 40 (SV40) is discussed. It is concluded that polyoma virus may transform established cells by a mechanism involving activation of a cellular oncogene product, whereas SV40 may transform by a mechanism involving a previously little studied cytoplasmic form of the transforming protein.
Phosphorylation of the solubilized insulin receptor by the gene product of the Rous sarcoma virus, pp60src
Authors: White Morris F. , Kahn C. Ronald , Werth Diane K. , Pastan Ira
Published: December 2004

Both the insulin receptor and the gene product of the Rous sarcoma virus, pp60src, are protein kinases which phosphorylate themselves and other proteins on tyrosinc residues. Addition of the solubilized insulin receptor to purified pp60src increased the phosphorylation of the β‐subunit of the insulin receptor. Phosphorylation of the insulin receptor by pp60src occurred both in the absence and presence of insulin but did not alter the insulin dose response for autophosphorylation of the receptor. Increasing concentrations of pp60src increased the phosphorylation of the receptor and at high concentrations equaled the maximal effect produced by insulin. Our observations suggest a possible mechanism by which the metabolically regulated insulin receptor tyrosine kinase could be altered by other tyrosine kinases such as that associated with pp60src. Further studies will be required to determine if the insulin receptor is phosphorylated by pp60src in Rous sarcoma virus‐infected cells.
Studies on the biosynthesis of cartilage proteoglycan in a model system of cultured chondrocytes from the Swarm rat chondrosarcoma
Authors: Kimura James H. , Lohmander L. Stefan , Hascall Vincent C.
Published: December 2004

Biosynthesis of cartilage proteoglycan was examined in a model system of cultured chondrocytes from a transplantable rat chondrosarcoma. Extensive modification with the addition of chondroitin sulfate glycosaminoglycan, N‐linkcd oligosac‐charide, and O‐linked oliogosaccharide is required to convert a newly synthesized core protein precursor into a proteoglycan. Kinetic analyses revealed the presence of a large pool of core protein precursor (t1/2 ∼ 90 min) awaiting completion into proteoglycan. The large t1/2 of this pool allowed kinetic labeling experiments with a variety of radioactive precursors to distinguish between early biosynthetic events associated primarily with the rough endoplasmic reticulum from late events associated primarily with the Golgi apparatus. The results of a series of experiments indicated that the addition of N‐linked oligosaccharide chains occurs early in the biosynthetic process in association with the rough endoplasmic reticulum, whereas the initiation and completion of O‐linked oligosaccharides occurs much later, at about the same time as chondroitin sulfate synthesis. This also indicated that keratan sulfate chains, when present in the completed molecule, are added in the Golgi apparatus, as they are probably built on oligosaccharide primers closely related to the O‐oligosaccharide chains. Furthermore, when 3H‐glucose was used as the precursor, the entry of label into xylose, the linkage sugar between the core protein and the chondroitin sulfate chain, was found to occur within 5 min of the entry of label into galactose and galactosamine in the remainder of the chondroitin sulfate chain. This indicated that the initiation and completion of the chondroitin sulfate chain occurs late in the pathway probably entirely in the Golgi apparatus. Thus, proteoglycan synthesis can be described as occurring in two stages in this system, translation and N‐glycosylation of a core protein precursor which has a long half‐life in the rough endoplasmic reticulum, followed by extensive rapid modification in the Golgi complex in which the majority of glycosaminoglycan and oligosaccharide chains are added to the core protein precursor with subsequent rapid secretion into the extracellular matrix.
Metabolic properties of an azaguanine‐resistant variant of Chinese hamster ovary cells (azarts) with normal levels of hypoxanthine‐guanine phosphoribosyltransferase activity
Authors: Plagemann Peter G. W. , Wohlhueter Robert M.
Published: December 2004

Azarts Chinese hamster ovary cells were 20 to 50 times more resistant to 8‐amaguanine and 50 to 10 times more resistant to both 6‐thioguanine and 6‐mercaptopurine than wild‐type cells. Resistance correlated with a failure of azarts cells to incorporate 8‐amaguanine into the nucleotide pool and into nucleic‐acids. The uptake of hypoxanthine and guanine, on the other hand, was about the same in both types of cells and the hypoxanthine‐guanine phosphoribosyltransferase of the azarts cells as measured in cell lysates was unaltered both in concentration and kinetic properties with hypoxanthine as well as 8‐azaguanine as substrate. Plasma membrane permeability to 8‐azaguanine and the regulation of intracellular pH were also not altered in azarts cells and there was no significant degradation of 8‐azaguanine or azaguanine nucleotides. We conclude therefore that in azarts cells the phosphoribosylation of 8‐azaguanine per se is specifically blocked but that this effect is abolished upon cell lysis.
Biological activities of laminin
Authors: Kleinman Hynda K. , Cannon Frances B. , Laurie Gordon W. , Hassell John R. , Aumailley Monique , Terranova Victor P. , Martin George R. , DuBois‐Dalcq Monique
Published: December 2004

Laminin is a multifunctional protein with diverse biological activities. Like fibronectin, it can influence cell adhesion, growth, morphology, differentiation, migration, and agglutination as well as the assembly of the extracellular matrix. Laminin primarily affects cells of epithelial origin, and the response varies depending on the cell. Because most differentiated cells are difficult to maintain in culture, laminin may be an important supplement in studies on cell differentiation in vitro.
Anti‐idiotypic antibody identifies the cellular receptor of reovirus type 3
Authors: Gaulton Glen , Co Man Sung , Greene Mark I.
Published: December 2004

The binding and subsequent infectivity of reovirus to target cells are mediated by interaction with specific cell surface viral receptors. To gain a more detailed understanding of the biochemistry of the reovirus receptor and the cellular consequences of viral attachment, we have studied the binding of type 3 reovirus (Dearing strain) in a quantitative manner utilizing an antiidiotypic antibody probe. A syngeneic monoclonal antiidiotypic antibody (87.92.6) was prepared by immunization with hybridoma cells which secrete an antireovirus hemagglutinin‐specific antibody. This antiidiotypic antibody was previously shown to specifically recognize the cell surface receptor for reovirus type 3. In this report, we demonstrate that antiidiotype mimicked reovirus tropism in binding to murine thymomas; antiidiotype inhibited the binding of reovirus to specific targets, but not the binding of anti‐H‐2; and cross linking of receptor‐bound antiidiotype by antiimmunoglobulin induced patching, but not capping of reovirus receptors. Utilizing radiolabeled antiidiotype, we next quantitate the number of reovirus receptors on Rl.l and YAC thymoma cells and, finally, report on the preliminary identification of the reovirus receptor as a 67,000‐Da membrane glycoprotein.
Receptors for insulin and epidermal growth factor: Interaction with organomercurial agarose
Authors: Aglio Linda S. , Maturo Joseph M. , Hollenberg Morley D.
Published: December 2004

The receptor for both insulin and epidermal growth factor (EGF) from human placental membranes, after crosslink labeling with 125I‐labeled insulin and EGF, can be adsorbed to an organomercurial‐agarose derivative (Affi‐Gel 501) and can be recovered from the gel by elution with dithiothreitol (DTT). Pretreatment of crosslink‐labeled membranes with N‐ethylmaleimide (NEM) blocks the ability of the receptor to react with the organomercurial column. NEM also abolishes the protein kinase activity of both receptors. Under appropriate conditions, insulin can promote the reaction of the insulin receptor with the organomercurial‐agarose derivative. For both the insulin and EGF receptors, our results provide an avenue for the isolation of the sulfhydryl‐containing receptor domains that may play a role in the control of receptor function.
Structure of the murine anion exchange protein
Authors: Kopito Ron R. , Lodish Harvey F.
Published: December 2004

A full‐length clone encoding the mouse erythrocyte anion exchange protein band 3, has been isolated from a cDNA library using an antibody against the mature erythrocyte protein. The complete nucleotide sequence has been determined. Substantial homology is evident between the deduced murine amino acid sequence and published sequences of fragments of human band 3 protein. The amino‐terminal 420 and the carboxy‐terminal 32 residues constitute polar, soluble domains, while the intervening 475 amino acids are likely to be intimately associated with the lipid bilayer. Hydrophobic analysis of this sequence, together with structural studies on the human protein, suggests the possibility of at least 12 membrane spans, predicting that both the amino‐ and carboxy‐termini are intracellular.
The design, synthesis, and characterization of tight‐binding inhibitors of calmodulin
Authors: DeGrado William F. , Prendergast Frank G. , Wolfe Henry R. , Cox Jos A.
Published: December 2004

Based on a consideration of the probable structure of calmodulin and sonic natural peptides known to interact with it, two calmodulin‐binding peptides were designed. These peptides bind to calmodulin in helical conformations and are capable of forming electrostatic and hydrophobic interactions with calmodulin. Their dissocation constants for binding (⩽ 210 and 400 pM) place them as the tightest‐binding inhibitors of calmodulin thus far reported. The study of the interactions of these and similar peptides with calmodulin will provide valuable insights into the mechanisms whereby calmodulin binds to target enzymes, and it also serves as an excellent model system for exploring the physical chemistry of protein‐protein interaction.
Analysis of stable and unstable viral forms in SV40‐infected human keratinocytes
Authors: Naimski Piotr , Steinberg Mark L.
Published: December 2004

We analyzed the slate of the genomic DNA of the papovavirus SV40 in human keratinocytes as viral‐infected cells gradually acquired a transformed phenotype over time. Initially, the vast majority of the viral DNA is maintained either in a full‐length supercoiled form or as truncated subgenomic fragments with little evidence of integration. However, analyses of clonal populations revealed great heterogeneity and instability of the viral DNA, and we were able to isolate one clonal subpopulation in which integrated forms of the virus appeared to predominate. Similarly, uncloned populations eventually ceased production of the “free” viral DNA after several years in culture and instead came to display tandemly repeated SV40 copies at a single host integration site. Interestingly, Bgl II digestion of host DNA generated restriction fragments containing the integrated SV40 DNA, which were of differing sizes in cultures at the 144th vs the 163rd serial passage suggesting modification or rearrangement of sequences at or near the integration site. Host sequences flanking the integrated viral DNA at the 163rd serial passage have been isolated on restriction fragments generated by Eco RI, Bam HI, and Hpa II digestion. These analyses suggest that the integrated virus is linearized near the Bgl I site and contains a large deletion in the SV40 early region at one of the viral‐host junctions.
Inhibition of ornithine decarboxylase activity and cell growth by diamines: A comparison between the effects of two homologs, 1,3‐diaminopropane and 1,4‐diaminobutane (putrescine)
Authors: Linden Margareta , Oredsson Stina M. , Anehus Siw , Heby Olle
Published: December 2004

The ornithine decarboxylase (ODC) activity of Ehrlich ascites tumor cells was almost completely inhibited by treatment with either putrescine (10 mM) or 1,3‐diaminopropane (5 mM). 1,3‐Diaminopropane treatment eradicated the cellular content of putrescine and reduced that of spermidine and spermine. Putrescine treatment caused a dramatic increase in cellular putrescine content and a temporary decrease in spermidine and spermine content. Despite the fact that 1,3‐diamino‐propane and putrescine inhibited the ODC activity more effectively than did α‐difluoromethylornithine (DFMO), an enzyme‐activated irreversible inhibitor of ODC, they were considerably less antiproliferative in action. However, as compared to DFMO the diamines were less effective in reducing the total polyamine (putrescine + spermidine + spermine) content of the cells.
Posttranslational phosphorylation of specific chromosomal proteins and transcription of hnRNA genes in isolated nuclei: Retention of in vivo sensitivity to 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB)
Authors: Holst Mikael , Egyházi Endre
Published: December 2004

The rapidly turning over phosphorylation of specific nuclear nonhistone proteins, especially 42‐, 33‐, and 30‐kDa polypeptides, and its relation to the transcriptional activity of hnRNA genes was investigated in isolated nuclei from salivary gland cells of Chironomus tentans. Incubation conditions promoting the phosphorylation of nonhistone proteins as well as the transcriptional activity of RNA polymerase II were established. The pattern of 32P incorporation into the nonhistone proteins found in isolated nuclei resembled that obtained in experiments with intact cells, and the endogenous RNA polymerase II retained its ability to reinitiate the transcription under in vitro assay conditions. In addition, the in vivo sensitivity of the phosphorylation of 42‐, 33‐, and 30‐kDa polypeptides, like the sensitivity of the initiation of hnRNA transcription to 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB), were preserved in the nuclear preparation. The experimental data taken together provide further support for the idea that the activation of hnRNA genes is causally related to the phosphorylation of specific nonhistone proteins.
The leucine binding proteins of Escherichia coli as models for studying the relationships between protein structure and function
Authors: Antonucci Tammy K. , Landick Robert , Oxender Dale L.
Published: December 2004

The genes encoding the leucine binding proteins in E coli have been cloned and their DNA sequences have been determined. One of the binding proteins (LIV‐BP) binds leucine, isoleucine, valine, threonine, and alanine, whereas the oilier (LS‐BP) binds only the D‐ and L‐isomers of leucine. These proteins bind their solutes as they enter the periplasm, then interact with three membrane components, livH, livG, and livM, to achieve the translocation of the solute across the bacterial cell membrane. Another feature of the binding proteins is that they must be secreted into the periplasmic space where they carry out their function. The amino acid sequence of the two binding proteins is 80% homologous, indicating that they arc the products of an ancestral gene duplication. Because of these characteristics of the leucine binding proteins, we are using them as models for studying the relationships between protein structure and function.
Suramin binds to platelet‐derived growth factor and inhibits its biological activity
Authors: Hosang Markus
Published: December 2004

The polyanion suramin was recently found to inhibit binding of 125I‐PDGF (platelet‐derived growth factor) to Balb/e 3T3 cell membranes. Cultured Swiss 3T3 cells were used to investigate the mode of action of suramin and to monitor its effect on the biological activity of PDGF. Evidence is presented that suramin inhibits cellular binding of PDGF by binding to PDGF itself, thereby preventing it from binding to its cell surface receptor: First, while suramin inhibited I‐PDGF binding with a hail maximum inhibition concentration of ∼60 μM or 90 μg/ml in a simultaneous competition assay, it was inactive in a sequential radioreciptor assay, in which an inhibitor is expected to be active if it interacts with the receptor (even with relatively low affinity) but to be inactive if it interacts with PDGF. Second, suramin prevented immunoprecipitation of 125I‐PDGF in a dose‐dependent manner, with a half maximum effective concentration of ∼50 μM. Furthermore, suramin efficiently dissociated 125I‐PDGF bound to its cell surface receptor, whereas unlabeled PDGF even in large excess was virtually inactive. This is also in line with the proposed direct interaction between PDGF and suramin, since such an interaction can he envisaged to induce a conformational change in the PDGF‐receptor complex, resulting in an increased off‐rate of the complex. Reduced 125I‐PDGF binding in the presence of suramin correlated directly with a suramin dose‐dependent inhibition of PDGF‐induced incorporation of 3H‐thymidine into quiescent Swiss 3T3 cells and of the proliferation of these cells.

These observations suggest that suramin, by complexing with PDGF, renders this mitogen unable to bind to its physiological receptor and, thereby, prevents PDGF‐initiated biological activities.
Quantitative relationship between initiation of hepatocarcinogenesis and induction of altered cell islands
Authors: Kaufmann William K. , Mackenzie Susan A. , Rahija Richard J. , Kaufman David G.
Published: December 2004

We have quantified the initiation of hepatocytic neoplasms and the induction of altered cell islands in regenerating livers of rats given a single treatment with one of three carcinogens before or during the peak of DNA synthesis after partial hepatectomy. For up to 20 wk after treating livers during the peak of DNA synthesis with methyl(acetoxymethy1)nitrosamine (DMN‐Ac), hepatocytic neoplasms were not seen. Thereafter, in rats fed the liver tumor promoter, phenobar‐bital, neoplasms emerged continuously so that by 60 wk after initiation, livers held an average of 5.5 neoplasms. Islands of cellular alteration, identified by their abnormal retention of glycogen on fasting, also appeared to emerge continuously between 20 and 60 wk after initiation. By 60 wk, promoted livers contained about 10,000 islands. In DMN‐Ac‐initiated, phenobarbital‐promoted livers, neoplasms and islands maintained a constant numerical relationship over time with about 1,450 islands emerging for every neoplasm that emerged. This ratio of islands to neoplasms differed according to the type of carcinogen used to initiate hepatocar‐cinogenesis and depending on whether promotion with phenobarbital was included. In livers initiated with DMN‐Ac but not promoted with phenobarbital, the ratio of islands to neoplasms was about 7,750: 1. In livers initiated by treatment with (±)‐7α,8β‐dihydroxy‐9β,10β‐epoxy‐7,8,9,10‐tetrahydrobenzo[a]pyrene at the peak of DNA synthesis and then promoted with phenobarbital, the ratio of islands to neoplasms was 7,200: 1. In livers exposed to gamma rays at the peak of DNA synthesis in regenerating livers and promoted, no neoplasms were seen in our sample although islands could be enumerated. Evaluation of another group of rats irradiated during the prereplicative phase of regeneration revealed two neoplasms in nine treated livers and a ratio of islands to neoplasms of greater than 12,000: 1. Thus, when comparing livers treated once with carcinogen and then promoted, this ratio of islands to neoplasms differed considerably according to the carcinogen being tested. These results suggest that the induction of glycogen‐retaining hepatocyte islands may not be a quantitative measure of the initiation of hepatocarcinogenesis.
Urokinase‐type and tissue‐type plasminogen activators have different distributions in cultured bovine capillary endothelial cells
Authors: Moscatelli David
Published: December 2004

The cell extracts and conditioned medium from cultured bovine capillary endothelial (BCE) cells were examined to determine the types of plasminogen activator (PA) present in each of these two fractions. The fractions were first analyzed by fibrin autography after sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis. The cell extracts contained two species of PA of Mr 48,000 and 28,000. Multiple forms of PA were detected in the conditioned medium: variable amounts of the Mr 48,000 and 28,000 forms and a broad band of activity with Mr in the range of 67,000–93,000. The major fraction of the Mr 48,000 form was in the cell extract. Treatment of the cells with 12‐0‐tetradecanoyl phorbol‐13‐acetate or with a preparation containing angiogenic activity resulted in a proportionate increase in the levels of all forms. The Mr 48,000 form was demonstrated to be a urokinase‐like PA, since it was immunoprecipitated with antibodies to urokinase. When conditioned medium or cell extracts from biosynthetically labelled BCE cells were incubated with antiserum to urokinase, the Mr 48,000 form was immunoprecipitated only from the cell extract. The Mr 67,000–93,000 forms were demonstrated to be tissue‐type PAs, since they were immunoprecipitated with antibodies to tissue PA. When the same conditioned medium or cell extracts were incubated with antiserum to tissue‐type PA, the Mr 67,000–93,000 forms were immunoprecipitated only from the conditioned medium. Therefore, BCE cells are able to produce both tissue‐type PA, which is primarily secreted, and urokinase‐type PA, which remains primarily cell associated.
Monitoring of individual human exposure to aflatoxins (AF) and N‐nitrosamines (NNO) by immunoassays
Authors: Wild C. P. , Umbenhauer D. , Chapot B. , Montesano R.
Published: December 2004

Highly sensitive immunoassays have been used to quantitate aflatoxins (AF) and N‐nitrosamines (NNO) in human body fluids and tissues, respectively. This approach was taken in order to quantitate environmental exposure to these agents at an individual level to facilitate the investigation of their role in the etiology of human cancer. In order to analyse AF in human urine, an immunopurification step has been developed by using AF‐specific antibody bound to AH‐Sepharose 4B gel in a small (4‐ml gel volume) affinity column prior to enzyme‐linked immunosorbent assay (ELISA). The ELISA can be used to quantitate aflatoxin B1 (AFB1) over the range 0.01 ng/ml to 10 ng/ml and the assay system has been validated by using human urine samples spiked with AFB1 over this concentration range. In addition, 29 urine samples from the Philippines have been analysed and found to contain a range of levels from zero to 4.25 ng/ml AFB1 equivalent with a mean of 0.875 ng/ml. This compared with a mean of 0.066 ng/ml AFB1 equivalent in samples from France.

Radioimmunoassay of O6‐methyldeoxyguanosine (O6‐medG) has been performed on human oesophageal and cardiac stomach mucosal DNA from tissue samples obtained during surgery in Linxian County, People's Republic of China, an area of high risk for both oesophageal and stomach cancer. Using the methodology described and having 1 mg of hydrolyzed DNA allows the detection of approximately 25 fmol O6medG per mg DNA. Of the 37 tissue samples analyzed from Linxian County, 17 samples had levels of O6‐medG ranging from 15 to 50 fmol/mg DNA, ten showed higher levels up to 160 fmol/mg DNA, and the remaining ten samples were below the limit of detection. For comparison, 12 tissue samples were obtained from hospitals in Europe and all showed levels below 45 fmol O6‐medG/mg DNA with seven below the limit of detection. All tissue samples from Linxian county showed normal levels of O6‐alkylguanine DNA alkyltransferase when compared to levels in other parts of the world.

The approaches described appear promising for assessing the role of AFB1 in the etiology of human liver cancer and of nitrosamines as possible causative agents in oesophageal or stomach cancer.
A set of stage‐dependent embryonic antigens expressed in cell cultures of BALB/c mouse embryos and in transformed cell lines
Authors: Gerhards Roswitha E. , Mehnert Franz E.
Published: December 2004

A rabbit antiserum (A2) directed against the detergent‐solubilized fraction of the simian virus 40‐transformed mouse embryo fibroblast cell line VLM detects common antigens in primary cell cultures from BALB/c mouse embryos and in transformed cell lines from various species. Positively reacting cell cultures show a set of polypeptides with molecular weight species p86, p74, p68, p46, p42, p40, and p35. As tested by Western blotting procedures, all immunoprecipitated proteins carry immunologically reactive determinants. By analysis with two‐dimensional gel electrophoresis, all precipitated polypeptides show charge heterogeneities. Concerning the two major members of the protein set, p40 consists of at least four subspecies with isoelectric points in the range of pH 6.2–6.8, whereas p35 is composed of two subspecies focusing between pH 6.4 and pH 7.2. By comparison of the two‐dimensional patterns of p35 of various transformed cell lines, a basic (pH 6.6–7.2) and an acidic (6.4–6.6) charge type of p35 could be observed. Comparative analyses of primary cell cultures from 12–16‐day mouse embryos show the immunoprecipitated set of polypeptides only in the 16‐day embryo cell cultures. After six further propagations, these cells express the immunoreactive proteins as strongly as the primary cell cultures. In embryonic, cell cultures of day 14 of gestation the expression of this set of antigens is induced only when cells are propagated at least six times. Under identical conditions these proteins could not be induced in cell cultures of 18‐day‐old mouse embryos. None of the polypeptides could be immunoprecipitated from primary mouse kidney cell cultures of 12‐day‐old mice even when the cultures were propagated at least‐15 times. This set of polypeptides is also present in simian virus 40‐transformed cells of hamster, rat, monkey, and human origin. These findings suggest that in simian virus 40‐transformed mouse cells, in addition to p53, the synthesis of other embryonic antigens is reactivated. The presence of the described set of polypeptides in polyoma virus‐transformed cells of rat and mouse origin and in cell lines derived from malignant human tumors might indicate common functions in metabolic patterns of transformed cells.
Assembly and dynamics of the actin filament system in nonmuscle cells
Authors: Pollard Thomas D.
Published: December 2004

Kinetic analysis has provided a detailed quantitative description of the mechanism of actin polymerization as well as the methods to analyze the mechanisms of action of actin‐binding proteins. In Acanthamoeba, five different proteins regulate the pool of monomers available for polymerization, cap the end of filaments, sever filaments, and cross‐link filaments. Remarkably, many of these interactions involve very‐low‐affinity bonds between the protein molecules.
In vivo biosynthesis of clathrin and other coated vesicle proteins from rat liver
Authors: Pierce L. Ross , Zurzolo Chiara , Edelhoch Harold
Published: December 2004

A biosynthetic study of rat liver coated vesicle (CV) proteins was undertaken by using in vivo labeling with L‐[35S]methionine. CVs were isolated and purified by using standard procedures and characterized by electron microscopy, sedimentation, and sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography, or by gel slicing and liquid scintillation counting. After 51/2 min of labeling (the earliest time examined), incorporation of radioactive clathrin heavy‐chain (180‐kD (kilodalton)) subunits as well as a 90‐kD CV‐associated protein into purified CVs was demonstrated. The level of labeled 180‐kD clathrin in coated vesicles increased rapidly during the First 2 hr of labeling and then continued to rise at a slower rate between 4 and 16 hr. This slow accumulation of labeled clathrin heavy chains in the CV pool may reflect early compartmental sequestration of a fraction of newly synthesized clathrin with delayed assembly into free CVs. By 16 hr of labeling, clathrin 180‐kD chains and the 90‐kD CV‐associated protein accounted for approximately 48 and 26%, respectively, of the radioactivity in all CV proteins. Two proteins of MWa 68 kD and 53 kD showed marked declines in cpm/unit protein between 30 min and 4 hr, raising the possibility that these species may, be transferred out of CVs during or after transport without loss of the other CV proteins. The possibility is also raised that clathrin heavy chains may be recycled during CV formation. Possible heterogeneity within individual CV preparations with respect to protein composition and derivation from both plasma membrane and Golgi regions are proposed.
Inhibition of mammalian collagenases by thiol‐containing peptides
Authors: Gray Robert D. , Miller Robert B. , Spatola Arno F.
Published: December 2004

The following thiol‐containing peptide analogues of the carboxyl side of the collagenase‐sensitive bond of collagen were synthesized and tested as inhibitors of collagenases partially purified from homogenates of rabbit V‐2 tumor and culture medium of pig synovium: HSCH2CH(CH3)CO‐Ala‐OEt (I), HSCH2CH‐(CH2Ph)CO‐Ala‐OEt (II), HSCH2CH[CH2CH(CH3)2]CO‐Ala‐OEt (III); HSCH2, CH‐[CH2CH(CH3)2]CO‐Ala‐Gly‐OEt (IV); HSCH2CH[CH2CH(CH3)2]CO‐Ala‐Gly‐Gln (V). The compounds are listed in order of their inhibitory potency when assayed with nonfibrillar‐acid‐soluble calfskin collagen at pH 7.6, 35°C. The best inhibitor (III) gave 50% inhibition between 1 and 4 μM. II was a competitive inhibitor with a Ki value of 75 μM. The enzymes preferred an isobutyl side chain at the 2‐carbon position, and, where tested (III, IV), did not discriminate strongly between stereoisomers at the chiral 2‐carbon. Increasing the length of the inhibitor did not markedly increase potency.