bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhanced human osteoblast cell adhesion and proliferation on 316 LS stainless steel by means of CO2 laser surface treatment
Authors: Hao L. , Lawrence J. , Phua Y. F. , Chian K. S. , Lim G. C. , Zheng H. Y. 
Published: December 2004

AbstractAn effective and novel technique for improving the biocompatibility of a biograde 316 LS stainless steel through the application of CO2 laser treatment to modify the surface properties of the material is described herein. Different surface properties, such as surface roughness, surface oxygen content, and surface energy for CO2 laser‐treated 316 LS stainless steel, untreated, and mechanically roughened samples were analyzed, and their effects on the wettability characteristics of the material were studied. It was found that modification of the wettability characteristics of the 316 LS stainless steel following CO2 laser treatment was achieved. This improvement was identified as being mainly due to the change in the polar component of the surface energy. One‐day cell adhesion tests showed that cells not only adhered and spread better, but also grew faster on the CO2 laser‐treated sample than on either the untreated or mechanically roughened sample. Further, compared with the untreated sample, MTT cell proliferation analysis revealed that the mechanically roughed surface resulted in a slight enhancement, and CO2 laser treatment brought about a significant increase in cell proliferation. An increase in the wettability of the 316 LS stainless steel was observed to positively correlate with the cell proliferation. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 73B: 148–156, 2005
Deposition of Basement Membrane Proteins in Attachment and Neurite Formation of Cultured Murine C‐1300 Neuroblastoma Cells
Authors: Alitalo K. , Kurkinen M. , Virtanen I. , Mellström K. , Vaheri A. 
Published: December 2004

AbstractThe deposition of the basement membrane glycoproteins, laminin, fibronectin, and type IV procollagen was studied by indirect immunofluorescence microscopy during the attachment and differentiation of murine C‐1300 neuroblastoma cells. A typical cytoplasmic perinuclear staining for the basement membrane antigens was seen both in undifferentiated and differentiated cells. Freshly seeded suspended cells lacked surface fluorescence but in two hours after plating, distinct punctate laminin deposits became discernible on the ventral surface of the cells. Notably, in sparsely seeded undifferentiated cultures, the cell‐associated extracellular laminin deposits could only be detected under the primary attaching cells, whereas daughter cells in clonal cell colonies lacked such fluorescence. In cultures induced to neurite formation with dibutyryl cyclic AMP, laminin deposition was also detected in association with the growing cytoplasmic extensions. No distinct differences were found between the secreted proteins of cultures of differentiated and nondifferentiated neuroblastoma cells, but the patterns of fucosylation of high‐molecular weight proteins in the two cultures were markedly different.We conclude that cultured neuroblastoma cells both synthesize, secrete and deposit laminin. The distribution of laminin during neuroblastoma cell attachment and neurite extension suggests that this glycoprotein may be involved in cell–to–substratum interactions in C‐1300 cell cultures.
Separation of Glycopeptides by High Performance Liquid Chromatography
Authors: Rosner Marsha Rich , Robbins Phillips W. 
Published: December 2004

AbstractA method is presented for separation of tryptic glycopeptides‐containing oligosaccharides of the N‐asparagine‐linked type. High performance liquid Chromatography (HPLC) of glycopeptides on a C18 reverse‐phase system eluted with a gradient of 0%–50% acetonitrile in 0.1 M NaPO4 pH 2.2 resolves the two major glycosylation sites from the envelope glycoprotein (G) of vesicular stomatitis virus. Glycopeptides containing N‐linked oligosaccharides of the complex type coelute with those containing N‐linked oligosaccharides of the neutral, high mannose type, indicating that separation is based upon peptide rather than carbohydrate composition. The contribution of the carbohydrate component to glycopeptide elution, as determined by cleavage of the high mannose oligosaccharides with endo‐β‐Nacetylglucosaminidase H, is that of a significant, but minor, decrease in peptide retention time. Comparison of the tryptic glycopeptide profiles of G isolated from both wild type and mutant strains of VSV illustrates the rapid, reproducible, and quantitative nature of the technique. Through HPLC analysis of appropriately treated glycopeptides, it is possible to explore both the nature and extent of glycosylation at individual sites in glycoproteins in a single step.
The Molecular Basis for Membrane – Cytoskeleton Association in Human Erythrocytes
Authors: Bennett Vann 
Published: December 2004

AbstractSpectrin, the major cytoskeletal protein in erythrocytes, is localized on the inner membrane surface in association with membrane‐spanning glycoproteins and with intramembrane particles. The presence of a specific, high‐affinity protein binding site for spectrin on the cytoplasmic surface of the membrane has been established by measurement of reassociation of spectrin with spectrin‐depleted inside‐out vesicles. A 72,000 Mr proteolytic fragment of this attachment protein has been purified, which bound to spectrin in solution and competed for reassociation of spectrin with vesicles. A 215,000 Mr polypeptide has been identified as the precursor of the spectrin‐binding fragment. The membrane attachment protein for spectrin was named ankyrin, and has been purified and characterized. Ankyrin has been demonstrated to be tightly associated in detergent extracts of vesicles with band 3, a major membrane‐spanning polypeptide, and to bind directly to a proteolytic fragment derived from the cytoplasmic domain of band 3. Ankyrin is thus an example of a protein that directly links a cytoplasmic structural protein to an integral membrane protein. The organization of the erythrocyte membrane has implications for more complex cell types since immunoreactive forms of ankyrin distinct from myosin or filamin have been detected by radioimmunoassay in a variety of cells and tissues. Indirect immunofluorescent staining of cultured cells reveals immunoreactive forms of ankyrin in a cytoplasmic meshwork and in a punctate distribution over nuclei. The staining changes dramatically during mitosis, with concentration of stain at the spindle poles in metaphase and intense staining of the cleavage furrow during cytokinesis.
Further Characterization of the Aggregation and Fusion of Chromaffin Granules by Synexin as a Model for Compound Exocytosis
Authors: Creutz Carl E. , Scott Janet H. , Pazoles Christopher J. , Pollard Harvey B. 
Published: December 2004

AbstractSynexin was isolated from bovine liver and found to aggregate adrenal chromaffin granules in the same Ca2+‐dependent manner as previously described for adrenal synexin. The chromaffin granule aggregating activity of liver synexin was blocked in vitro by the addition of an antibody prepared to the 47,000 molecular weight band extracted from an SDS gel of an adrenal medullary synexin preparation. Chromaffin granules aggregated by synexin fused when exposed to cis‐unsaturated fatty acids at concentrations comparable to those released from phospholipids by stimulated secretory cells. The synexin‐induced aggregation reaction was blocked by Erythrosin B, a common food coloring, and by the phenothiazine antipsychotics trifluoperazine and promethazine. The aggregation and fusion of chromaffin granules thus appears to be a useful model system for studying synexin from diverse tissues and for testing pharmacologically or texicologically active substances for effects on secretory systems.
Metabolism of Benzo(a)pyrene by Human Epithelial and Fibroblastic Cells: Metabolite Patterns and DNA Adduct Formation
Authors: Bartley Jack , Bartholomew James C. , Stampfer Martha R. 
Published: December 2004

AbstractWe demonstrate in cell culture that mammary epithelial cells from normal human breast specimens metabolize benzo(a)pyrene (BaP) and form adducts with the bases of their DNA more readily and at lower concentrations of BaP than do fibroblasts from the same specimens. BaP metabolism and adduct formation was determined in the same incubations with epithelial cells grown out in early passage from each of three specimens and with fibroblasts from one of these specimens. The metabolite pattern of the epithelial cells was indicative of preferential formation of 7, 8‐dihydrodiol‐9, 10‐dihydroepoxybenzo(a)pyrene the ultimate carcinogen. In contrast, fibroblasts formed mainly mono‐ and dihydroxide derivatives of BaP. The metabolite pattern from epithelial cells was compatible with the ease in which adducts between DNA and the diolepoxide of benzo(a)pyrene were formed. These results provide evidence that chemical carcinogens should be considered as possible factors in the induction of breast cancer in women.
Alpha‐Adrenergic Stimulation of Phosphatidylinositol Synthesis in Human Platelets as an Alpha‐2 Effect Secondary to Platelet Aggregation
Authors: Wallace Michael A. , Agarwal Kailash C. , Garcia‐Sainz J. Adolfo , Fain John N. 
Published: December 2004

AbstractEpinephrine and adenosine diphosphate (ADP) stimulated 3H‐glycerol uptake into phosphatidylinositol of human platelets. Yohimbine, an alpha‐2 adrenoceptor antagonist, markedly reduced epinephrine‐stimulated 3H‐glycerol uptake into phosphatidylinositol; while prazosin, an alpha‐1 antagonist, was without effect. Likewise, yohimbine, but not prazosin, blocked epinephrine‐induced platelet aggregation. Furthermore, clonidine, a specific agonist for alpha‐2 adrenoceptors, stimulated incorporation of‐3H‐glycerol into phosphatidylinositol and promoted platelet aggregation in the presence of low concentrations of ADP. These studies indicate that the effects of epinephrine on platelet aggregation and phosphatidylinositol synthesis are mediated through alpha‐2 adrenoceptors. Further, since the stimulation of phosphatidylinositol synthesis seen with epinephrine was also observed with ADP, this suggests that the increased 3H‐glycerol labeling is an indirect result of platelet aggregation.
Regulation of Lactose Transport by the Phosphoenolpyruvate‐Sugar Phosphotransferase System in Membrane Vesicles of Escherichia coli
Authors: Dills Steven S. , Schmidt Mary R. , Saier Milton H. 
Published: December 2004

AbstractRegulation of lactose uptake by the phosphoenolpyruvate‐sugar phosphotransferase system (PTS) has been demonstrated in membrane vesicles of Escherichia coli strain ML308‐225. Substrates of the phosphotransferase system inhibited D‐lactate energized uptake of lactose but did not inhibit uptake of either L‐alanine or L‐proline. This inhibition was reversed by intravesicular (but not extravesicular) phosphoenolpyruvate. Lactose uptake was also inhibited by enzyme IIIglc preparations that were shocked into the vesicles, and this inhibition was reversed by phosphoenolpyruvate. Intravesicular HPr and enzyme I stimulated methyl α‐glucoside uptake but did not inhibit or stimulate lactose accumulation. Vesicles maintained at 0°C for several days partially lost 1) the ability to take up lactose, 2) the ability to accumulate PTS substrates, and 3) PTS‐mediated regulation. Phosphoenolpyruvate addition restored all of these activities. These results support a mechanism in which the relative proportions of phosphorylated and nonphosphorylated forms of a phosphotransferase constituent regulate the activity of the lactose permcase.
A Common Role for Target Cell Histocompatibility Antigens in Both Nonspecific and Specific T Lymphocyte‐Mediated Cytolysis
Authors: Berke Gideon , Hu Valerie , McVey Ella , Clark William 
Published: December 2004

AbstractWe have investigated the role of target cell major histocompatibility complex antigens (MHC‐Ag) in nonspecific lectin‐dependent lymphocyte‐mediated cytolysis (LDCC). In contrast to previous reports, we provide evidence that in LDCC the lectin Concanavalin A (Con A) does not mediate lysis by simply bridging cytotoxic T lymphocytes (CTL) and targets via cell surface sugars or by activating the lytic function of CTLs attached to targets via the lectin. Lysis occurs when target cells are pretreated with lectin, but not when CTL are pretreated. Moreover, when CTL populations are used as both aggressors and targets, and only one is pretreated with lectin, lysis occurs only in the direction of the pretreated CTL target. We have observed that in LDCC, as in specific CTL‐mediated killing, target recognition proceeds through interaction of CTL receptors (distinct from sugar moieties) and target cell surface determinants perhaps modified by, but distinct from, the lectin itself. We present evidence that the target determinants recognized in LDCC are MHC‐Ag: 1) Cells that display reduced amounts of MHC‐Ag are poor targets in LDCC; 2) removal of MHC‐Ag by papain renders targets refractory to LDCC, however susceptibility is regained upon regeneration of MHC‐Ag; and 3) antisera to target cell MHC‐Ag block LDCC. The latter finding is also observed in oxidation‐dependent CTL‐mediated cytotoxicity. Involvement of MHC proteins in both specific and nonspecific CTL‐mediated lysis reconciles an apparent fundamental distinction between these two processes and suggests a possible role for MHC proteins in a postrecognition step(s) leading to lysis.
Brain Ligatin: A Membrane Lectin That Binds Acetylcholinesterase
Authors: Gaston S.M. , Marchase Richard B. , Jakoi E.R. 
Published: December 2004

AbstractLigatin, a lectin that recognizes phosphorylated sugars, has been demonstrated in mammalian tissues to bind specific hydrolases to cell surfaces. Ligatin exists as a filament that can be released from membranes still complexed with its bound hydrolases by treatment of membrane preparations with CaCl2 and/or pH 8.0. The ligatin‐hydrolase complexes subsequently can be dissociated with ethyleneglycol‐bis(β‐amino‐ethyl ether) N, N′‐tetraacetic acid, resulting in a concurrent depolymerization of the ligatin filament. From membrane preparations of cerebrum, this procedure solubilized ligatin and a membrane‐bound acetylcholinesterase (EC 3.1.1.7). Binding of the cosolubilized acetylcholinesterase to ligatin could be demonstrated in vitro by affinity chromatography using the immobilized lectin. Ligatin‐hydrolase complexes have been shown to be dissociated by specific phosphorylated sugars (mannose 6‐phosphate and glucose 1‐phosphate). These sugars were also effective in eluting bound brain acetylcholinesterase from ligatin affinity columns. Analysis of labeled glycitols produced by tritiated borohydride reduction confirmed the presence of phosphorylated sugars on the ligatin‐cosolubilized material from brain.
The Polymeric State of Actin in the Human Erythrocyte Cytoskeleton
Authors: Atkinson Mark A.L. , Morrow Jon S. , Marchesi Vincent T. 
Published: December 2004

AbstractReports on the polymeric state of actin in the red cell have been diverse. We have used phalloidin to stabilize the actin in erythrocyte ghosts prior to extraction in low ionic strength media. A mild proteolytic digestion and Sepharose 4B gel filtration enable an F‐actin polymer to be isolated in pure form [1]. Detailed size analysis of this polymer in a range of experiments suggests that actin exists in the erythrocyte principally as a polymer of 100 nm length composed of 30 monomers in a double helical chain 15 monomers long with an estimated molecular weight of 1.3 × 106 daltons.
Benzo(a)pyrene‐Binding Proteins of Hamster Embryo Cell Nuclei: Comparison of Nuclear Isolation Procedures
Authors: MacLeod Michael C. , Mansfield Betty K. , Selkirk James K. 
Published: December 2004

AbstractHamster embryo cells metabolize benzo(a)pyrene to derivatives that covalently modify nuclear macromolecules including proteins. Not all proteins are modified to the same extent nor by the same metabolites. In particular, a protein of apparent molecular weight 32,000 is highly modified by derivatives of trans‐9,10‐dihydro‐9,10‐dihydroxy B(a)P. This protein is shown here to be preferentially lost from nuclei during purification by centrifugation through high molarity sucrose solutions followed by osmotic shock. It does not appear to be a cytoplasmic contaminant, but shares many properties of an abundant protein from Xenopus laevis oocytes, nucleoplasmin.
Modulation of epidermal growth factor‐dependent protein phosphorylation in cell membrane preparations by receptor down regulation
Authors: Fernandez‐Pol J. A. 
Published: December 2004

AbstractWe have reported previously [6] that epidermal growth factor (EGF)‐induced down regulation of EGF receptors in normal rat kidney (NRK) cells results in a selective decrease in the in vitro EGF‐dependent 32P‐phosphorylation of two membrane phosphoproteins of Mr I70K and Mr I50K. In this report, we further characterized the modulation of 32P‐phosphorylation of the 170K‐ and 150K‐dalton proteins by down regulation with EGF in NRK cells.While EGF binding to its receptors was a necessary condition to induce loss of EGF‐dependent phosphorylation of the 170K‐ and 150K‐dalton proteins, it was not sufficient. Thus, reduction in the temperature of the incubation of cells with EGF from 37°C to 4°C abolished the loss of EGF‐dependent phosphorylation of the 170K‐ and 150K‐dalton membrane proteins. When EGF was removed from the medium the EGF‐dependent phosphorylation of the 170K‐ and l50K‐dalton proteins was quickly replenished; by 3 hr one‐half of the “down regulated” phosphorylation was restored. All EGF‐dependent phosphorylating capacity of the 170K‐ and l50K‐dalton protein bands returned by 6 hr after removal of the growth factor. The loss of EGF‐dependent phosphorylation of the 170K‐ and I50K‐dalton proteins occurred at physiological EGF concentrations (0.25–25 ng/ml) that span the concentration range which is mitogenic for NRK cells. Exposure of confluent nondividing NRK cells to 1 ng/ml EGF, followed by incubation for 5 hr at 37°C. led to a 50% reduction in the EGF‐dependent phosphorylation of the 170K‐ and 150K‐dalton proteins. Maximal reduction (∼95%) in the EGF‐dependent phosphorylation of the 170K‐ and 150K‐dalton proteins was noted with 10 ng/ml EGF for 5 hr. The EGF‐induced loss of EGF‐dependent phosphorylation was specific: several other growth factors did not produce phosphorylation loss of the 170K‐
In vitro labeling of proteins by reductive methylation: Application to proteins involved in supramolecular structures
Authors: Heacock C. S. , Bernstein B. W. , Duhaiman A. S. , Amorese D. A. , Bamburg J. R. 
Published: December 2004

AbstractActin and tropomyosin, purified from both muscle and brain, and α‐actinin, purified from muscle, have been labeled in vitro by reductive methylation to specific activities of greater than 105 dpm/μg protein. Actin so modified bound DNase I and polymerized identically to unmodified actin. Furthermore, the spectral properties of actin did not change after labeling. The interactions of labeled tropomyosin and α‐actinin with F‐actin were nearly identical to those of the unmodified proteins. These modified proteins comigrated with their unmodified counterparts in both SDS‐containing polyacrylamide gels and isoelectric focusing gels. The labeled actin was quantitatively extracted from SDS‐containing polyacrylamide gels (yield > 98% of radioactivity applied demonstrating that all of the radioactivity was protein bound. The reductive methylation procedure worked well at pH 8.0–8.5 in either pyrophosphate buffer or Bicine buffer using formaldehyde with [3H]‐sodium borohydride as the reducing agent. The procedure could also be performed at pH 7.0 in phosphate buffer using [14C]‐formaldehyde with sodium cyanoborohydride as the reducing agent. Proteins so labeled are ideal for use in quantitative experiments involving protein‐protein interactions.
Cross‐reactivity of antibodies against synthetic peptides
Authors: Walter Gernot , Werchau Hermann 
Published: December 2004

AbstractAntiserum against the synthetic peptide Lys‐Arg‐Ser‐Arg‐His‐Phe, corresponding to the carboxy terminus of polyoma virus medium tumor antigen (medium T antigen), immunoprecipitates a protein of 36,000 daltons from polyoma virus‐infected and uninfected cell extracts treated with the sulfhydryl group reagent N‐ethyl‐maleimide. This protein appears to share an antigenic determinant with medium T antigen that is normally buried inside the protein or covered up by another protein or cellular structure. The two‐dimensional tryptic fingerprints of the 36K protein and of medium T antigen are apparently unrelated to each other. Antiserum against the octapeptide Ac‐Met‐Asp‐Lys‐Val‐Leu‐Asn‐Arg‐Tyr, including the amino‐terminal heptapeptide sequence of the simian virus 40 (SV40) large tumor (T) and small T antigens, cross‐reacts with polyoma virus large T antigen, which has an identical amino‐terminal heptapeptide sequence except that Lys is replaced by Arg and Asn by Ser. The problem of cross‐reactivities of antipeptide sera is discussed.
Quantitative composition and characterization of the proteins in membrane vesicles released from erythrocytes by dimyristoylphosphatidylcholine. A membrane system without cytoskeleton
Authors: Weitz M. , Bjerrum O. J. , Ott P. , Brodbeck U. 
Published: December 2004

AbstractMembrane vesicles were prepared by incubation of human erythrocytes with dimyristoylphosphatidylcholine [3] and isolated by isopycnic centrifugation on Dextran density gradients. Protein analyses were carried out with crossed immunoelectrophoresis and dodecylsufate polyacrylamide gel electrophoresis. The right‐side‐out‐oriented membrane vesicles contained membrane and cytoplasmic proteins of the erythrocyte but lacked cytoskeletal components. Comparison of proteins in vesicles and erythrocyte membranes showed that acetylcholinesterase was enriched two to six times in the vesicles relative to both membrane‐spanning proteins, band 3, and glycophorin. Two further, hitherto unidentified, sialic acid‐containing membrane antigens were found in the vesicles. Both faced the outside of the membranes and were enriched two to seven times.Ankyrin was not present in the membrane vesicles and spectrin could not be detected by dodecylsulfate polyacrylamide gel electrophoresis. We suggest that the redistribution of proteins in the vesicles reflects differences in their interactions with other membrane components and their relative mobility within the erythrocyte membrane.
Transbilayer movement of various phosphatidylcholine species in intact human erythrocytes
Authors: van Meer Gerrit , Op den Kamp Jos A. F. 
Published: December 2004

AbstractPhosphatidylcholine specific phospholipid exchange protein was used to introduce (14C)‐labeled phosphatidylcholine of different fatty acyl compositions into the intact human erythrocyte. Hydrolysis by a combination of phospholipase A2 and sphingomyelinase was applied to prove that originally all newly introduced phosphatidylcholine resided in the outer monolayer. Subsequently the erythrocytes were reincubated at 37°C, and redistribution of the introduced (14C)phosphatidylcholine was monitored by applying the combination of phospholipases after different times of incubation. In the situation where 20% of the native erythrocyte phosphatidylcholine had been replaced by phosphatidylcholine from (14C)choline‐labeled rat liver microsomal membranes, a slow translocation of the (14C)microsomal phosphatidylcholine was found, with a half‐time of transbilayer equilibration of 10.8 hr. Furthermore, the transbilayer movement of probe amounts of (14C)dipalmitoyl‐phosphatidylcholine, (14C)egg phosphatidylcholine and (14C)soybean phosphatidylcholine was studied under conditions whereby the fatty acyl composition of the bulk erythrocyte phosphatidylcholine remained unchanged. In correlation to the increasing unsaturation of the probe, half‐times for the transbilayer equilibration were calculated to be 26.9, 12.8, and 8.1 hr, respectively.