bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interaction of very low density lipoprotein with chicken oocyte membranes
Authors: Krumins Solveig A. , Roth Thomas F. 
Published: December 2004

AbstractThe interaction of hen 125I‐VLDL (very low density lipoprotein) with chicken oocyte membranes was characterized using a rapid sedimentation assay. Equilibrium and kinetic studies showed an apparent dissociation constant (Kd) 8.7–9.1 × 10‐8 M or 43.5–45.5 μg VLDL protein/ml. Binding capacity was 2.0 μg VLDL protein/mg membrane homogenate protein. The apparent rate constants were, k1 = 2.4 × l05 M‐1min‐1 and k2 = 2.1 × 10‐2 min‐1. Specific binding required the presence of divalent cations. Whereas binding was completely re‐stored after treatment with EDTA by the addition of Mn++, only 60% of binding was restored using Ca++.
Differential effects of polyamines on rat thyroid protein kinase activities
Authors: Levasseur Steven , Henricks Lorraine , Poleck Thomas , Friedman Yochanan , Burke Gerald 
Published: December 2004

AbstractOrnithine decarboxylase, the rate‐limiting enzyme in polyamine biosynthesis, has been shown to be regulated in thyroid by thyrotropin both in vivo and in vitro. Little, however, is known of the role of polyamines in thyroid cell function. Since studies in other tissues suggest that polyamines may influence protein phosphorylation, we studied the effect of the polyamines on various protein kinase activities in rat thyroid. Putrescine, spermidine, and spermine inhibit cyclic‐AMP‐dependent histone H1 kinase activity when measured in the cytosol fraction of rat thyroid; this effect is largely reproduced by NaCl concentrations of equivalent ionic strength. Both spermidine and spermine effect a 1.6–2.4‐fold increase in cytosolic cyclic‐AMP‐independent (messenger‐independent) casein kinase activity; stimulation by both polyamines is maximal at 5mM. A similar profile of stimulation is observed for messenger‐independent casein kinase activity in crude nuclear preparations. Sodium chloride fails to stimulate both cytosolic and nuclear messenger‐independent casein kinase activities at ionic strength equivalent to the spermine concentrations used. Spermine, but not putrescine, spermidine, or sodium chloride, inhibits calcium/phospholipid‐dependent protein kinase C activity in cytosol extracts partially purified by DEAE chromatography. These findings suggest that regulation of protein kinase(s) by polyamines may represent a proximal locus (i) of action of thyrotropin‐regulated ornithine decarboxylase activity in thyroid.
Tubulin‐colchicine complex (TC) inhibits microtubule depolymerization by a capping reaction exerted preferentially at the minus end
Authors: Bergen Lawrence G. , Borisy Gary G. 
Published: December 2004

AbstractThe effects of colchicine and tubulin‐colchicine complex (TC) on microtubule depolymerization were studied using the axoneme‐subunit system described previously [Bergen LG, Borisy GG; J Cell Biol 84:141–150, 1980]. This system allows the independent analysis of the polymerization kinetics at both the plus and minus ends of a microtubule. Depolymerization was induced by isothermal dilution with 10 volumes of an experimental solution containing colchicine, TC, or buffer alone. Colchicine alone (5–100 μM) blocked depolymerization at the minus end, whereas depolymerization at the plus end occurred at almost control rates. A similar effect was produced by TC (0.4 : 1–1 : 1 molar ratio to free tubulin). High molar ratios of TC to tubulin (10:1) blocked depolymerization at both plus and minus ends, and intermediate molar ratios of TC:T allowed depolymerization of the plus ends but at attenuated rates. The blockage was not readily reversible; TC‐affected ends neither shortened upon dilution nor grew longer upon incubation with additional tubulin. We conclude that TC at suprastoichiometric ratios to tubulin inhibits microtubule depolymerization by a capping reaction and that this effect is exerted preferentially at the minus end.
Spectrin involvement in a 40°C structural transition of the red blood cell membrane
Authors: Minetti Maurizio , Ceccarini Marina , Di Stasi Anna Maria M. , Petrucci Tamara C. , Marchesi Vincent T. 
Published: December 2004

AbstractProteins involved in a structural transition detected in red blood cell membranes at 40°C by spin labeling methods have been investigated. Antibodies specific for spectrin, band 3, and protein 4.1 have been used as specific probes to modify membrane thermotropic properties. Spectrin seems to be involved in a 40°C transition detected in ghosts by both a stearic acid spin label (16‐doxyl stearic) and a sulfhydryl‐specific maleimide analogue spin label. Circular dichroism and maleimide spin labeling studies of purified spectrin show a slow unfolding of the protein structure starting at 25‐30°C and a massive transition with an onset temperature of 48 and 40°C, respectively. This thermotropic behavior of spectrin could be the process that modifies membrane physicochemical properties above 40°C that are detected by the stearic acid spin label. The transition detected by the stearic acid spin label was modified both by antispectrin antibodies and anti‐4.1 protein antibodies, but not by antibodies specific for the cytoplasmic domain of band 3. These results suggest an involvement of protein 4.1 in regulating spectrin unfolding at the membrane level. A selective inhibition of the transition detected by the maleimide spin label has been obtained with a monoclonal antispectrin antibody at 1 : 1 molar ratio. The involvement in this transition of a localised spectrin domain(s) containing few exposed sulfhydryl groups is proposed.
Hydrophobicity and amphiphilicity in protein structure
Authors: Eisenberg David , Wilcox William , McLachlan Andrew D. 
Published: December 2004

AbstractThe importance of the hydrophobic interaction in stabilizing native protein structure has long been appreciated. However, more than other component forces, this one has resisted quantitative description. We present two approximate methods of assessing the hydrophobic component to the free energy of protein folding. Both are expressed in terms of what can be called hydrophobic moments of the protein. The first method is intended to yield an approximate value for the hydrophobic energy. This energy is calculated from a set of atomic coordinates in terms of the hydrophobicity (or 0th hydrophobic moment) of each amino acid residue and its accessibility or lack of it to aqueous solvent. The second method considers the first moment of the hydrophobicity of a group of residues, the hydrophobic moment. Segments of secondary structure in folded proteins tend to have hydrophobic moments that oppose each other. For example, α‐helices on the protein surface tend to have one hydrophobic face and one hydrophilic face, with the hydrophilic face out towards the solvent. This pattern of organization is often apparent from a computer model of the protein that shows the magnitude and direction of the hydrophobic moment of each segment of secondary structure. Examples are given for the incorrectly folded structures of Novotný et al [J Mol Biol 177:787, 1984] and for the correct structures to which they correspond.
Relationship between protease activity and a sialoglycopeptide inhibitor isolated from bovine brain
Authors: Sharifi B. G. , Bascom C. C. , Fattaey H. , Nash S. , Johnson T. C. 
Published: December 2004

AbstractWe have recently described the isolation and purification to homogeneity of a new Sialoglycopeptide from bovine brain cell surfaces that reversibly inhibits protein synthesis and DNA synthesis of normal but not transformed cells. Active inhibitory preparations, however, were shown to contain a protease activity that was not lost upon purification. Several experiments were performed to establish the relationship between the proteolytic activity of the Sialoglycopeptide and the biological inhibitory activity. Both the protease activity and inhibitory activity were stable at pH 6–8 but were reduced or completely destroyed below pH 4 and above pH 9. Acid inactivation was reversible and upon dialysis, both the biological inhibitory and protease activities were regained. Deglycosylation and CNBr cleavage indicated that the polypeptide backbone, rather than carbohydrate moiety, played an important role in the protease and biological inhibitory activities. Furthermore, chemical modification of amino and tyrosine groups indicated that both residues are essential for both activities. Thus, the biological inhibitory activity and protease activity are very closely related and most likely reside with the same polypeptide sequence.
Association of DNA with the nuclear lamina in Ehrlich ascites tumor cells
Authors: Krachmarov Chavdar , Iovcheva Christina , Hancock Ronald , Dessev George 
Published: December 2004

AbstractWe have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single‐stranded DNA and pulse‐labeled DNA show higher binding efficiencies than native uniformly labeled DNA. When mixing occurs in 2 M NaCl, complex formation is inhibited.When nuclei are digested with DNAse I under conditions that favor chromatin condensation, DNA associated with matrices subsequently prepared from such nuclei is markedly enriched in satellite DNA. If digestion is carried out with DNAse II while nuclei are decondensed in EDTA, no enrichment in satellite DNA is observed.Preparations of purified, high‐molecular weight, double‐stranded DNA contain variable amounts of fast‐sedimenting aggregates, which are insoluble in 2 M NaCl but are dispersed by DNA fragmentation or denaturation.These results point at some artifacts inherent in studies of DNA bound to residual nuclear structures in vivo and suggest conditions expected to avoid these artifacts.Further, using controlled digestion with DNAse II, we have studied the in vivo association of DNA with nuclear lamina isolated from Ehrlich ascites tumor cells. In the course of DNA fragmentation from above 50 kbp to about 20 kbp average size, the following events were observed. The DNA of high molecular weight (much longer than 50 kbp) behaved as if tightly bound to the nuclear lamina, as judged by sedimentation in sucrose and metrizamide density gradients, electron microscopy, and retention on glass fiber filters. As the size of DNA decreased, it was progressively detached from the nuclear lamina, and at about 20 kbp average, length practically all DNA was released. The last 1–4% of DNA, although cosedimenting with the nuclear lamina in sucrose gradients, behaved as free DNA, banding at 1.14 g/cm3 in metrizamide density gradients and showing less than 4% retention on filters.At no stage of digestion did the DNA cosedimenting with nuclear lamina show changes in satellite DNA content relative to that of total DNA or enrichment in newly replicated DNA.It was shown, however, that digestion of nuclear lamina‐DNA complex with EcoRI or Hae III led to the formation of DNA‐protein aggregates, which banded at 1.35 g/cm3 in high salt containing metrizamide density gradients and which were strongly enriched in satellite DNA.These results argue against the existence of direct tight bonds between DNA and nuclear lamina in vivo but demonstrate that such bonds can be generated under certain conditions in vitro.
Antipeptide antibodies directed against the carboxy‐terminal region of SV40 structural proteins VP2 and VP3
Authors: Streckert Hans‐Jürgen , Brüssow Harald , Sure Klaus , Werchau Hermann 
Published: December 2004

AbstractRabbits were immunized with a synthetic heptapeptide of the sequence Arg‐Asn‐Arg‐Ser‐Ser‐Arg‐Ser corresponding to the carboxy‐terminal region of the SV40 viral proteins VP2 and VP3. The raised antibodies recognize the viral proteins in enzyme‐linked immunosorbent (ELISA) and Western blot assay. Specificity of the antibodies were confirmed by competition experiments. The antibodies recognize VP2 and VP3 in infected cells by immunofluorescence and in subcellular fractions by ELISA. No interaction with virions was observed.
Differential expression of metastasis‐associated cell surface glycoproteins and mRNA in a murine large cell lymphoma
Authors: Nicolson Garth L. , LaBiche Ronald A. , Frazier Marsha L. , Blick Mark , Tressler Robert J. , Reading Christopher L. , Irimura Tatsuro , Rotter Varda 
Published: December 2004

AbstractA metastatic variant cell subline of the Abelson virus‐transformed murine large lymphoma/lymphosarcoma RAW 117 has been selected in vivo ten times for liver colonization. Highly metastatic subline RAW117‐H10 forms greater than 200 times as many gross surface liver tumor nodules as the parental line RAW117‐P. Analysis of cellular proteins and glycoproteins indicates reduced expression of murine Moloney leukemia virus‐associated p15, p30, and gp70, and increased expression of a sialoglycoprotein, gp150, in the highly metastatic H10 cells. Northern analyses of oncogene expression suggested that mRNA of various oncogenes was expressed equally or not expressed in the RAW117 cells of differing metastatic potential. Differential gene expression was examined using a cDNA library of 17,600 clones established from poly A+ mRNA isolated from H10 cells. The cDNA library was screened by the colony hybridization technique using probes made from both RAW117‐P and ‐H10 cells. Approximately 99.5% of these cDNA clones were expressed identically in P and H10 cells. Of the few differentially expressed cDNA clones (approx. 150/17,600), one‐half of these were identified as Moloney leukemia virus sequences in a separate probing with a radiolabeled Moloney leukemia virus probe. The remainder of the differentially expressed mRNA detected by colony hybridization of the cDNA library were expressed at higher levels (approx. 1/6) or lower levels (approx. 1/3) in the highly metastatic H10 cells.
Partial purification of microsomal signal peptidase from hen oviduct
Authors: Baker R. Keith , Bentivoglio Gian Paolo , Lively Mark O. 
Published: December 2004

AbstractSignal peptidase has been purified approximately 600‐fold from hen oviduct microsomes. Treatment of microsomes with ice‐cold sodium carbonate at pH 11.5 removes soluble and extrinsic membrane proteins prior to solubilization of signal peptidase with Nonidet P‐40. After dialysis to pH 8.2, the solubilized enzyme is chromatographed on diethylaminoethyl cellulose at pH 8.2. More than 90% of contaminating proteins bind to the column while signal peptidase and endogenous phospholipid are eluted in the column void volume. Enzyme activity subsequently binds to carboxymethyl cellulose at pH 5.8 and is eluted by approximately 100 to 200 mM NaCl during a NaCl gradient. Polypeptides present in partially purified hen oviduct signal peptidase have relative molecular masses ranging from 54 kD to less than 11 kD with major bands at 29, 23, 22, 19, 18 and 13 kD. The purified peptidase requires phospholipid for activity and is maximally active in the presence of 2 mg/ml phosphatidylcholine.
Effects of monensin on vesicular transport pathways in the perfused rat liver
Authors: Kloppel Thomas M. , Brown William R. , Reichen Juerg 
Published: December 2004

AbstractIn the rat hepatoctye, the internalization and degradation of asialoglycoproteins and the secretion of plasma and biliary proteins require specific intracellular sorting of vesicles. To aid in the biochemical characterization of these different vesicular pathways, we examined the effects of the ionophore monensin on the uptake and degradation of 125I‐asialoorosomucoid (ASOR) and on the secretion of plasma and biliary proteins by the in situ perfused rat liver. In control livers, 77% of injected 125I‐ASOR was extracted on first pass; 93% of the extracted radioactivity was released back into the circulation (totally degraded and some intact ASOR was found); and approximately 2% was recovered in the bile, some of which was intact. Monensin treatment decreased first pass uptake of 125I‐ASOR to 57% and abruptly blocked the release of radioactivity into the perfusate and the bile. When hepatic proteins were biosynthetically labeled with 3H‐leucine, monensin treatment dramatically reduced and delayed the secretion of newly synthesized proteins into both the perfusate and the bile. In contrast with control livers, in which secretion of protein into the perfusate preceded secretion of protein into the bile, TCA‐precipitable 3H‐protein appeared in bile about 20 min before TCA‐precipitable 3H‐protein appeared in the perfusate in monensin‐treated livers. Thus, monensin treatment in the perfused liver blocked the degradation of asialoglycoproteins and inhibited the secretion of plasma proteins but had less effect on biliary protein secretion. These data document physiologic effects of monensin in an intact organ and suggest that biochemical distinctions between different vesicular pathways exist in the rat hepatocyte.
Molecular cloning of a tumor promoter‐inducible mRNA found in JB6 mouse epidermal cells: Induction is stable at high, but not at low, cell densities
Authors: Smith James H. , Denhardt David T. 
Published: December 2004

AbstractFrom the mouse JB6 epidermal cell line C122 we have isolated a cDNA clone representing a 1.6‐kilobase mRNA, called 2ar, that exhibits a biphasic induction in response to 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). The first phase of induction in subconfluent cells is transient, peaking at 6 h after the addition of TPA and returning to noninduced levels by 24 h. When the cells reach plateau density, in the continued presence of TPA, this mRNA is reinduced and remains so upon continued exposure to the tumor promoter. Serum and certain growth factors also induce 2ar mRNA in serum‐deprived quiescent fibroblasts. In vitro nuclear “run‐on” transcription experiments indicate that the induction of 2ar mRNA is controlled at the transcriptional level.
Structure of amplified DNA, analyzed by pulsed field gradient gel electrophoresis
Authors: Borst P. , Van Der Bliek A. M. , Van Der Velde‐Koerts T. , Hes E. 
Published: December 2004

AbstractPulsed field gradient electrophoresis allows the separation of large DNA molecules up to 2,000 kilobases (kb) in length and has the potential to close the resolution gap between standard electrophoresis of DNA molecules (smaller than 50 kb) and standard cytogenetics (larger than 2,000 kb). We have analysed the amplified DNA in four cell lines containing double minute chromosomes (DMs) and two lines containing homogeneously staining regions. The cells were immobilized in agarose blocks, lysed, deproteinized, and the liberated DNA was digested in situ with various restriction endonucleases. Following electrophoretic separation by pulsed field gel electrophoresis, the DNA in the gel was analysed by Southern blotting with appropriate probes for the amplified DNA. We find that the DNA in intact DMs is larger than 1,500 kb. Our results are also compatible with the notion that the DNA in DMs is circular, but this remains to be proven. The amplified segment of wild‐type DNA covers more than 550 kb in all lines and possibly up to 2,500 kb in some. We confirm that the repeat unit is heterogeneous in some of the amplicons. In two cell lines, however, with low degrees of gene amplification, we find no evidence for heterogeneity of the repeats up to 750 (Y1‐DM) and 800 kb (3T6‐R50), respectively. We propose that amplicons start out long and homogeneous and that the heterogeneity in the repeat arises through truncation during further amplification events in which cells with shorter repeats have a selective advantage. Even if the repeats are heterogeneous, however, pulsed field gradient gels can be useful to establish linkage of genes over relatively short chromosomal distances (up to 1,000 kb). We discuss some of the promises and pitfalls of pulsed field gel electrophoresis in the analysis of amplified DNA.
Changes in surface glycopeptides after malignant transformation of rat liver cells and during the regression of hepatoma cells
Authors: Chaumeton Brigitte , Saunier Brigitte , Nato Farida , Goulut Chantal , Bourrillon Roland 
Published: December 2004

AbstractNormal liver cells, Zajdela's hepatoma cells, and regressing hepatoma cells were metabolically labeled with either radioactive glucosamine or mannose. Glycopeptides obtained by exhaustive pronase digestion of these cells were compared after fractionation by gel filtration on Bio‐Gel P‐6.Chemical analysis, affinity chromatography on immobilized lectins, alkaline treatment, and susceptibility toward endo‐β‐N‐acetylglucosaminidase and tunica‐mycin revealed dramatic changes in the glycopeptide patterns of transformed cells during the recovery of normal phenotype.The most prominent feature was the presence on the surface of hepatoma cells of a large glycopeptide, which was absent from normal liver cells and disappeared almost completely during the regression of hepatoma cells. This large glycopeptide had a Mr of 70,000, contained essentially O‐glycosidically linked glycan chains, and did not result from a hypersialylation.N‐glycosidically linked glycopeptides, high‐mannose, and complex‐type oligosaccharides were present in distinct proportions according to the differentiation state. Transformation of liver cells led to a reduction of high‐mannose type oligosaccharides and an increase in the degree of branching of complex‐type oligosaccharides. In addition, “bisected” glycopeptides were present only on hepatoma cells. The pattern of N‐linked glycopeptides of normal liver cells was recovered during the regression of hepatoma cells.The origin of glycopeptide differences between normal and transformed cells and the evidence of a relation between carbohydrate changes, in particular the appearance of a large glycopeptide, and tumorigenicity are discussed.
Estrogen‐induced lysosomal proteases secreted by breast cancer cells: A role in carcinogenesis?
Authors: Rochefort Henri , Capony Françoise , Garcia Marcel , Cavaillès Vincent , Freiss Gilles , Chambon Monique , Morisset Muriel , Vignon Françoise 
Published: December 2004

AbstractIn an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis, metastatic human breast cancer cell lines (MCF7, ZR75‐1) were found to secrete a 52,000 dalton (52K) protein under estrogen stimulation. Following its purification to homogeneity, the 52K protein was identified as a secreted procathepsin‐D‐like aspartyl protease bearing man‐nose‐6‐phosphate signals. This precursor display an in vitro autocrine mitogenic activity on estrogen‐deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its autoactivation. The total protease (52K + 48K and 34K) was detected and assayed by monoclonal antibodies and was found to be highly concentrated in proliferative and cystic mastopathies. In breast cancer, its cytosolic concentration appears to be correlated more to tumor invasiveness than to hormone responsiveness. The mRNA of the 52K protease accumulates rapidly following estradiol treatment, as was shown by Northern blot analysis with cloned cDNA. The 52K cathepsin‐D‐like protease is the first example of a lysosomal protease induced by estrogens in cancer cells. Results obtained using different approaches suggest that two cysteinyl cathepsins are also related to cell transformation and invasiveness. It has been proposed that cathepsin‐B is involved in breast cancer and metastatic melanoma, and its regulation by estrogen has been shown in the rat uterus. Cathepsin‐L corresponds to the major excreted protein (MEP) whose synthesis and secretion are markedly increased by transformation of NIH 3T3 cells with Ki ras and are regulated by several growth factors. In addition to secreted autocrine growth factors and to other proteases (plasminogen activator, collagenase), lysosomal cathepsins may therefore play an important role in the process of tumor growth and invasion as long as their precursor is secreted abundantly.