bluuebunny/crossref_metadata_2025 huggingface dataset:
Extracellular matrix interactions during the in vivo degradation of collagen membranes in the rat skin: Immunohistochemical distribution of collagen types IV, V, and VI
Authors: Romanos G. E. , Hotz S. T. K. , Schröter‐Kermani C. , Strub J. R.
Published: December 2004

The role of collagen implant material (CIM) in periodontology is of considerable interest to the clinical dentist because of the capacity of connective tissue (CT) regeneration and partial prevention of epithelial cell migration onto the root surface. The aim of this study was to demonstrate alterations of the CT matrix after the use of CIM in subcutaneous pockets in the rat skin. We used 15 rats in this study. After sedation, two subcutaneous pockets (2 cm in length) were surgically made in the animals' backs. Collagen membranes were implanted in one of the two pockets (test site). The other pocket served as control. Then, 4, 7, 14, 21, and 28 days after implantation, the animals were sacrificed and biopsies preserved for histologic and immunohistochemical examination. Incubation with antibodies against CT matrix components (collagen type IV, V, and VI) were used for immunostaining. Histologically, the CIM was migrated by inflammatory cells in the first 7 days. Newly formed fibroblasts and blood vessels (BV) were present 14 days postimplantation. Collagen type IV was localized in the basement membranes of the epithelium, BV, and nerves. An increase in the BV amount was demonstrated around (and later in) the implant material. Collagen type V was found in a filament pattern of distribution and was inserted into the implant after 4 weeks of healing. Collagen type VI showed a microfibrillar pattern of distribution with a delayed formation in the graft mass. The data showed the alterations of the matrix after implantation of collagen type I membranes in the rat skin. Furthermore, the CIM promoted fibroblast adhesion, cellular incorporation of its mass, and the formation of different collagen‐type fibers. © 1995 John Wiley & Sons, Inc.
Articular cartilage repair using allogeneic perichondrocyteseeded biodegradable porous polylactic acid (PLA): A tissue‐engineering study
Authors: Chu Constance R. , Coutts Richard D. , Yoshioka Makoto , Harwood Frederick L. , Monosov Anna Z. , Amiel David
Published: December 2004

Efforts to expand treatment options for articular cartilage repair have increasingly focused on the implantation of cell‐polymer constructs. The purpose of this study is to determine the suitability of porous D,D‐L,L‐polylactic acid as a carrier for delivering repair cells obtained from rib perichondrium into full‐thickness articular cartilage defects. In vitro characterization of perichondrocyte‐polylactic acid composite grafts was combined with in vivo assessment of the early articular cartilage repair in a clinically relevant model. Using a fluorescent double‐stain protocol to visualize live and dead cells in situ, primary cells cultured from perichondrium were found to be capable of attaching to and surviving within a porous D,D‐L,L‐polylactic acid matrix. These perichondrocyte‐polylactic acid composite grafts were then implanted within osteochondral defects drilled into the left medial femoral condyles of 16 adult New Zealand white rabbits. Experimental animals were sacrificed 6 weeks after implantation and the repair tissue was evaluated grossly, histologically, and biochemically. Grossly, 96% (15/16) of the experimental animals demonstrated repairs consisting of a smooth, firm neocartilage which appeared similar in color and texture to the surrounding articular surface. Matrix staining for cartilaginous protein was seen surrounding chondrocyte‐like cells in the cartilage regions of the repair. Cellular alignment was found to be related to scaffold architecture. These results suggest that scaffolds composed of porous D,D‐L,L‐polylactic acid support the growth of cartilaginous repair tissue and are compatible with both in vitro and in vivo survival of chondrogenic cells. © 1995 John Wiley & Sons, Inc.
Rheological enhancement of mechanically activated α‐tricalcium phosphate cements
Authors: Gbureck U. , Spatz K. , Thull R. , Barralet J. E.
Published: December 2004

Most biocements are two‐ or three‐component acid‐based systems with large differences in the component particle sizes, which occurs by virtue of the differing processing routes. This work aimed to improve injectability and strength of a single reactive component cement, that is, mechanically activated α‐tricalcium phosphate (TCP)‐based cement by adding 13–33 wt % of several fine‐particle‐sized (d50 of 0.5–1.1 μm) fillers [dicalcium phosphate anhydrous (DCPA), titanium dioxide (TiO2), and calcium carbonate] to the monomodal α‐TCP matrix (d50 = 9.8 μm). A high ζ‐potential was measured for all particles in trisodium citrate solution. The fraction of α‐TCP cement “injected” through an 800‐μm hypodermic needle was found to be only 35% at a powder‐to‐liquid ratio of 3.5 g/mL. In contrast, the use of fillers decreased cement viscosity to a point, where complete injectability could be obtained. Mechanistically, these additives disrupted α‐TCP particle packing yet decreased the interparticle spacing by a factor of ∼5.5 such that the electrostatic repulsion effect was enhanced. A strength improvement was found when DCPA and TiO2 were used as fillers despite the lower degree of conversion of these cements. Compressive strengths of precompacted cement samples increased from 70 MPa for unfilled α‐TCP cement to 140 (110) MPa for 23 wt % DCPA (or TiO2) fillers as a result of porosity reduction. Strength improvement for more clinically relevant uncompacted cements was achieved by higher powder‐to‐liquid ratio mixes for filled cements such that maximum strengths of 90 MPa were obtained for 23 wt % DCPA filler compared with 50 MPa for single‐component α‐TCP cement. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 73B: 1–6, 2005
New infusion device for trans‐tissue, sustained local delivery of anticancer agent to surgically resected tissue: Potential use for suppression of local recurrence of pancreatic cancer
Authors: Manabe Tatsuya , Okino Hidenobu , Maeyama Ryo , Mizumoto Kazuhiro , Tanaka Masao , Matsuda Takehisa
Published: December 2004

Local recurrence is a major cause of death of patients who have undergone resection for pancreatic cancer. To reduce the incidence of local recurrence, a new drug‐infusion device, which is fixed on resected tissues immediately after surgery, was devised for trans‐tissue, sustained local delivery. The drug‐infusion device proposed here has the following functions: tight adhesion of the device on resected tissues, sustained drug infusion to the tissue, drug reloading, and easy removal from the body after use. The fabricated prototype experimental device, described here, was a thin infusion pouch made of a thin elastomer film (segmented polyurethane), which is connected to an elastomeric tube for drug reloading. The suppressive effect of the device delivering the anticancer agent, gemcitabine (GEM), was examined using subcutaneous tumor‐bearing athymic mouse. A low‐dose, local sustained GEM delivery using the device significantly suppressed the tumor growth and regrowth after surgery. The preliminary model experimental result appears to provide a promising therapeutic procedure for increased survival rate of the patients undergoing surgery for pancreatic cancer. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 73B: 203–207, 2005
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

An 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

The 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

A 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

Spectrin, 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

Synexin 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

We 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

Epinephrine 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

Regulation 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

We 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

Ligatin, 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

Reports 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

Hamster 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

We 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

Actin 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

Antiserum 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

Membrane 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

Phosphatidylcholine 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.
Preparation of rat monoclonal antibodies to epitopes encoded by the viral oncogene (v‐fms) of McDonough feline sarcoma virus
Authors: Anderson Soni J. , Furth Mark E. , Wolff Linda , Ruscetti Sandra K. , Sherr Charles J.
Published: December 2004

The McDonough strain of feline sarcoma virus (SM‐FeSV) contains a viral oncogene, v‐fms, transduced from cat cellular genetic sequences designated c‐fms. Monoclonal antibodies reactive to antigenic determinants encoded by v‐fms were prepared by immunizing rats with live, syngeneic SM‐FeSV‐transformed cells, and fusing splenic lymphocytes from a tumor‐bearing animal with cultured rat myeloma cells. Culture supernatants from hybrids producing antibodies to epitopes encoded by v‐fms were identified by immunoprecipitation of radiolabeled polypeptides from SM‐FeSV‐transformed mink cells. Four positive hybrids were cloned twice in soft agar, established as stable lines, and grown in defined serum‐free medium to facilitate purification of homogeneous antibodies. The monoclonal antibodies were used to assay SM‐FeSV‐specific products by “immunoblotting” of elcctrophoretically separated proteins, and by fixed‐cell immunofluorescence.
Genetic recombination in avian retroviruses
Authors: Skalka A. M. , Boone L. , Junghans R. , Luk D.
Published: December 2004

The avian retroviruses—and probably other retroviruses as well—undergo a variety of recombinational events with relatively high efficiency. An understanding of the molecular basis of these events should provide insight into the important biological properties these agents exhibit when they become integrated into somatic or germ‐line host cells, when they exchange genetic information among themselves, or when they transduce host cell genes. In this article we review molecular models for homologous recombination, against a background of the other types of recombination events that arc typical of these viruses. It seems probable that the retroviruses will provide useful models for analysis of a variety of DNA rearrangements known to occur in eukaryotic cells.
Regulation of herpes simplex virus gene transcription in vitro
Authors: Beck Thomas W. , Millette Robert L.
Published: December 2004

We used partially purified RNA polymerase II from uninfected (Pol II) and from herpes simplex virus type 1 (HSV‐1) infected HEp‐2 cells (Pol II‐H) to transcribe HSV‐1 DNA in vitro. Gel electrophoretic analysis of the products produced from native HSV‐1 DNA yielded weight average chain lengths of 4.0 and 3.5 kb for the Pol II and Pol II‐H products, respectively. Blot hybridization analyses of the HSV DNA transcripts showed that both enzymes transcribed RNA from essentially all regions of the genome. However, Pol II preferentially transcribed regions coding for the immediate‐early or alpha mRNAs, whereas Pol II‐H preferentially copied regions coding for the early (β) and late (γ) gene products. Transcriptional analyses of the cloned HSV‐1 Bam HI‐Q fragment (containing the thymidine kinase (TK) gene) and its subfragments showed that (1) the major transcripts produced by Pol II‐H were distinctly different from those produced by Pol II; (2) Pol II and Pol II‐H utilized different promoters for the synthesis of major transcripts; (3) both enzymes produced three minor transcripts that were partially overlapping and in opposite direction to the TK gene; and (4) only Pol II‐H initiated transcription from the TK promoter. In contrast, both Pol II and Pol II‐H generated an identical set of transcripts from an adenovirus 2 early region DNA fragment. The sizes of the products suggest that RNA processing may be occurring in vitro. These results show that HSV‐1 infection alters the in vitro transcriptional specificity of RNA polymerase II and demonstrate that this system should be useful for studying in vitro the regulation of gene transcription.
Analysis of v‐mos encoded proteins in cells transformed by several related murine sarcoma viruses
Authors: Papkoff Jackie , Lai Mei H.‐T. , Hunter Tony
Published: December 2004

We have used antisera against synthetic peptides to identify and characterize a 37,000 dalton v‐mos encoded protein (p37mos) in cells transformed by M‐MuSV 124. p37mos, a phosphoprotein, comprises only about 0.0005% of total cellular protein in cell lines transformed by M‐MuSV 124. NIH 3T3 cells acutely infected with M‐MuSV 124, however, contain 30–100‐fold more p37mos. These elevated levels of p37mos correlate with striking morphological changes and cell death in the acutely infected cell population. Using the antipeptide antisera, we have extended the analysis of v‐mos proteins to include several other MuSV variants that contain a similar v‐mos gene to M‐MuSV 124. With the exception of P85, the gag‐mos fusion protein from ts110 MuSV, the v‐mos gene of these variants is expressed as a 35,000–37,000 dalton protein (size depending on the particular virus).
Epidermal growth factor stimulates fluid phase endocytosis in human fibroblasts through a signal generated at the cell surface
Authors: Wiley H. Steven , Cunningham Dennis D.
Published: December 2004

We have investigated the stimulation of fluid phase endocytosis by epidermal growth factor (EGF) in normal human fibroblasts using 125I‐labeled polyvinylpyrrolidone (125I‐PVP) as a fluid phase marker. We found that EGF initially induced a thereefold increase in the rate of 125I‐PVP uptake. This initial burst of fluid uptake terminated within 10 min. Thereafter, the rate of fluie uptake in EGF‐treated cells was approximately 40% higher than in control cells. To identify the cellular site of EGF action in stimulating fluid phase endocytosis, we examined the kinetics of the induction of this response as well as the kinetics of cell surface binding and internalization of 125I‐EGF. Although there was no detectable lag between binding of EGF to the cell surface and its internalization, the kinetics of the two processes were quite different. Significantly, the kinetics of induction of 125I‐PVP uptake matched the kinetics of binding of 125I‐EGF to its cell surface receptors, indicating that the signal for the increase in fluid phase endocytosis is generated at the cell surface. To determine if EGF‐stimulated fluid phase endocytosis was related to EGF‐stimulated endocytosis of its own receptor, we compared the EGF dose dependency and time course of the two processes. Although the stimulated endocytosis of the EGF receptor was not saturable with respect to the concentration of EGF used, the stimulation of fluid phase endocytosis was half maximal at an EGF concentration of 1 ng/ml and saturated at a concentration of 5 ng/ml. Also, the stimulation of fluid phase endocytosis was sevenfold greater initially after adding EGF than after a 30‐min continuous incubation with the hormone, whereas the enhanced clearance of the EGF receptor did not change during this time period. We conclude that the EGF‐stimulated increase in fluid phase endocytosis is not directly coupled to EGF‐stimulated endocytosis of its own receptor but instead to a separate signal generated at the cell surface.
Fibronectin is apparently not involved in species‐specific reaggregation of cells from the marine sponge geodia cydonium
Authors: Conrad J. , Diehl‐Seifert B. , Zahn R. K. , Uhlenbruck G. , Zimmermann E. , Müller W. E. G.
Published: December 2004

Experiments were carried out to test the hypothesis that fibronectin is involved in reaggregation of dissociated sponge cells. Cells from the siliceous sponge Geodia cydonium were extracted with urea to solubilize fibronectin from cells of higher multicellular organisms. The crude extract was further fractionated by DNA, heparin, and collagen affinity chromatography; they were termed Geodia fibronectinlike fractions. The fibronectinlike fractions contained a series of proteins with molecular weights different from that of the genuine fibronectin. The Geodia fibronectinlike fractions did not react with antiserum, produced against human fibronectin, under formation of a precipitin line. Using this antiserum the sponge cells could not be specifically labeled with FITC‐anti‐IgG antiserum. Radioimmunoprecipitation experiments revealed that the Geodia fractions contain—if at all—0.1% fibronectin or fibronectinlike protein at the most. In the crucial experiments it was shown that the Geodia fibronectinlike fractions, human fibronectin, and antifibronectin antiserum exerted no influence on adhesion of Geodia cells either in the absence or in the presence of the soluble aggregation factor. Based on these findings, we conclude that fibronectin is apparently not present on Geodia cells and does not play a role in aggregation of this biological system.
Activation of the glucocorticoid–receptor complex
Authors: Schmidt Thomas J. , Barnett Carol A. , Litwack Gerald
Published: December 2004

A crucial step in the interaction of glucocorticoids with target cells is the activation step, which involves a conformational change in the cytoplasmic glucocorticoid–receptor protein complexes and facilitates their binding to the cell nucleus. Activation can be quantified by measuring the ability of glucocorticoid‐receptor complexes to bind to polyanions, such as DNA‐cellulose, and unactivated complexes can be separated from activated complexes by rapid ion exchange chromatography using diethylaminoethyl (DEAE)‐Sephadex or DEAE‐cellulose. Activation occurs in vivo under physiological conditions and the rate of activation of cytoplasmic glucocorticoid–receptor complexes can be enhanced in vitro by physical manipulations (elevated temperature, increased ionic strength, dilution). In vitro studies suggest that activation is a regulated process and a low molecular weight component termed modulator, which has been identified in rat hepatic cytosol, inhibits activation. Additional studies employing phosphatase inhibitors, such as molybdate, and purified calf intestinal alkaline phosphatase suggest that either the receptor protein or a regulatory component is dephosphorylated during activation. Results obtained with specific chemical probes suggest that activation results in the exposure of basic amino acid residues consisting minimally of lysine, arginine, and histidine. Pyridoxal 5′‐phosphate, a specific probe for lysine residues, exerts dual effects on glucocorticoid‐receptor complexes, since it stimulates the rate of activation and also inhibits the binding of previously activated complexes to nuclei or DNA‐cellulose. The ability of 1,10‐phenanthroline, a metal chelator, to inhibit the DNA‐cellulose binding of activated complexes suggests that a metal ion(s) located at or near the DNA binding site may become exposed as a consequence of activation. Collectively, the results of these various experiments suggest that activation is a regulated biochemical phenomenon with physiological significance.
Nucleotide sequence and organization of the transforming region and large terminal redundancies (LTR) of avian myeloblastosis virus (AMV)
Authors: Papas Takis S. , Rushlow Keith E. , Watson Dennis K. , Lautenberger James A. , Perbal Bernard , Baluda Marcel E. , Reddy E. Premkumar
Published: December 2004

Avian mycloblastosis virus (AMV) is a replication‐defective acute leukemia virus, requiring a helper virus to provide the viral proteins essential for synthesis of new infectious virus. The genome of the AMV has undergone a sequence substitution in which a portion of the region normally coding for the “env” protein has been replaced by chicken cellular sequences. These latter sequences are essential for the transforming activity of the virus. We have determined the complete nucleotide sequence of this region. Examination of the AMV oncogenic sequence revealed an open reading frame starting with the initiation codon ATG within the acquired cellular sequences and terminating with the triplet TAG at a point 33 nucleotides into helper viral sequences to the right of helper‐viral‐cellular junction. The stretch of 795 nucleotides would code for a protein of 265 amino acids with a molecular weight of 30,000 daltons. The eleven amino acids at the carboxy terminus of such a protein would be derived from the env gene of helper virus.
Ultrastructural and biochemical identification of con A receptors in the desmosome
Authors: Shida Hisato , Gorbsky Gary , Shida Mariko , Steinberg Malcolm S.
Published: December 2004

Correlated ultrastructural and biochemical methods were used to identify and localize Concanavalin A (Con A) receptors in the desmosomes of bovine epidermis. Specific carbohydrate residues were labeled with ferritin‐Con A in thin sections of tissue embedded in a hydrophilic resin. Quantitative mapping of ferritin distribution in labeled desmosomes revealed that Con A receptors are localized in the intercellular zone and concentrated along the desmosomal midline or central dense stratum. Labeling was almost entirely absent when sections were treated with ferritin‐Con A in the presence of 0.1 M α‐methyl mannoside, a hapten‐inhibitor of Con A. “Whole” desmosomes and desmosomal intercellular regions (desmosomal “cores”) were purified from bovine muzzle epidermis. Sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis reveals a limited number of major desmosomal protein constituents. Certain of these are glycoproteins and are greatly enriched in the core fraction. Almost all the desmosomal glycoproteins are intensely labeled when electrophoretic gels of whole desmosome or core fractions are exposed to fluorescent Concanavalin A.
A monoclonal antibody to the human epidermal growth factor receptor
Authors: Waterfield Michael D. , Mayes Elaine L. V. , Stroobant Paul , Bennet Paul L. P. , Young Susan , Goodfellow Peter N. , Banting George S. , Ozanne Bradford
Published: December 2004

A monoclonal antibody of the IgG class, EGFR1, has been isolated using cells of the epidermoid carcinoma line A431 as immunogen. The A431 antigen recognized by EGFR1 has an apparent molecular weight of approximately 175,000, is a cell‐surface molecule which can be specifically cross‐linked to EGF, exhibits an EGF‐stimulated protein kinase activity, binds to EGFR1 in a number of human cell lines to a degree which parallels EGF binding, and shows EGF‐dependent internalization in A431 cells and human fibroblasts. We therefore conclude that EGFR1 is directed against an antigenic site on the human EGF receptor. EGFR1 is not mitogenic for human fibroblasts and does not inhibit EGF binding under a variety of assay conditions. The characterization of EGFR1 has allowed the unambiguous assignment of the structural gene for the human EGF receptor to chromosome 7. Preliminary results suggest that a convenient method for isolating a range of anti‐EGF receptor monoclonal antibodies can be developed, based on a hybridoma supernatant screening assay in which positive supernatants bind selectively to a human‐mouse cell hybrid containing human chromosome 7.
Butyrate‐induced histone hyperacetylation in human and mouse cells: estimation of putative sites of histone acetylation in vivo
Authors: Pantazis Panagiotis , Bonner William M.
Published: December 2004

Human and mouse cells in culture were treated with various concentrations of sodium butyrate. Acid‐extracted histones of control and butyrate‐treated cells were analyzed by two‐dimensional gel electrophoresis. All core histones of the control cells contained modified forms. All core histones of the butyrate‐treated cells were hyperacetylated. Depending on the number of acetylation sites per molecule, each histone or histone variant exhibited a characteristic number of acetylated forms. This number was the same for each histone common in human and mouse cells treated with butyrate. Histones 2A.1, 2A.2, and 2A.X have two sites of inner acetylation; 2A.Z has 3; 2B's have 5; and each one of the H3 variants as well as H4 have 4.
Ecdysteroid binding activity in embryos of drosophila melanogaster
Authors: Osterbur David L. , Yund Mary Alice
Published: December 2004

Ecdysteroid binding proteins have been found in nuclei of Drosophila melanogaster embryos. Comparison of results derived from Scatchard analysis, analogue binding competition, and sucrose gradient centrifugation has revealed no significant differences between the properties of the putative embryonic receptor and those of the receptor found in imaginal disks or Kc cells.