bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanisms of lymphocyte‐mediated lysis
Authors: Joag Sanjay , Zychlinsky Arturo , Young John Ding‐E 
Published: December 2004

AbstractCytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use multiple mechanisms to destroy their target cells. Pore formation resulting in osmotic lysis of the target is one mechanism; the pore‐forming protein (perforin) responsible for this activity has been purified. Antigenically and functionally it resembles proteins of the membrane attack complex of complement. The other known mediators of cytotoxicity appear to be closely interrelated. Tumor necrosis factor (TNF), lymphotoxin (LT), and leukalexin are the three members of this group that have been purified, although their mechanisms of action are still unknown. CTLs fragment the DNA of target cells, as do TNF, LT, and leukalexin; this may be one of the mechanisms of action of these mediators. CTLs and NK cells do not self lyse. The basis of this phenomenon is unclear, although recent advances have shed some light on the problem.
Novel gene exon homologous to pancreatic phospholipase A2: Sequence and chromosomal mapping of both human genes
Authors: Seilhamer J. J. , Randall T. L. , Johnson L. K. , Heinzmann C. , Klisak I. , Sparkes R. S. , Lusis A. J. 
Published: December 2004

AbstractWe described previously the cloning and DNA sequence of the human gene encoding pancreatic phospholipase A2 [DNA 5, 519]. When pancreatic phospholipase A2 (PLA2) cDNA was used to screen a human genomic library, two classes of clones were obtained. One class encoded the pancreatic enzyme, and a second class encoded one exon of an apparently related PLA2. No additional PLA2 gene exons displayed sufficient homology to be detected by the probe. A homologous sequence in both rat and porcine genomic DNA was detected by DNA blot hybridization, and the corresponding gene fragments were cloned and sequenced. Within the deduced amino acid sequences, the presence of known functional residues along with the high degree of interspecies conservation suggests the genes encode a functional PLA2 enzyme form. The encoded sequence lacks Cys11, as do the “type II” viperid venom and other nonpancreatic mammalian PLA2 enzymes. The sequence is distinct from porcine intestinal PLA2 and appears not to be a direct homolog of the recently published rabbit ascites and rat platelet enzymes. Hybridization of DNA probes containing sequences from these genes to genomic DNA blots of mouse/human somatic cell hybrids permitted chromosomal assignment for both. The pancreatic gene mapped to human chromosome 12, and the homologous gene mapped to chromosome 1.
Developmentally regulated 75‐kilodalton protein expressed in LLC‐PK1 cultures is a component of the renal Na+/glucose cotransport system
Authors: Wu Jin‐Shyun Ruth , Lever Julia E. 
Published: December 2004

AbstractNa+/D‐glucose symport is a secondary active glucose transport mechanism expressed only in kidney proximal tubule and in small intestine. A monoclonal antibody that recognized the Na+/glucose symporter of pig renal brush border membranes also recognized a 75‐kD protein in apical membranes isolated from highly differentiated LLC‐PK1 cultures, an epithelial cell line of pig renal proximal tubule origin. The 75‐kD antigen was enriched from solubilized LLC‐PK1 apical membranes by means of high‐pressure liquid chromatography. The symporter antigen became apparent on the apical membrane surface after the development of a confluent monolayer in correlation with the expression of transport activity. Long‐term treatment of cultures with the differentiation inducer hexamethylene bisacetamide was accompanied by a dramatically increased expression of the symporter antigen as detected quantitatively by Western blot analysis and qualitatively by immunofluorescence staining. The number of symporter‐positive cells was dramatically increased after inducer treatment as predicted for differentiation‐regulated expression. These results identify a 75‐kD protein as a component of a developmentally regulated renal Na+/glucose symporter expressed in cell culture.
Characteristics of the binding of human C‐reactive protein (CRP) to laminin
Authors: Swanson Steven J. , McPeek Mary M. , Mortensen Richard F. 
Published: December 2004

AbstractHuman CRP binds to the basement membrane protein laminin in vitro in a Ca2+‐dependent manner via the phosphorylcholine (PC) binding site of C‐reactive protein (CRP). The binding was saturable at a molar ratio of 4 (CRP/laminin). The specificity of the binding was shown by inhibition of binding of labeled CRP to laminin by unlabeled CRP, but not by human IgG. Specific binding was optimal in the presence of 5 mM Ca2+, but did not occur in the absence of Ca2+ or in the presence of EDTA. The binding of Ca2+ to CRP causes a conformational change in the molecule, which is required for binding to PC and to laminin. The PC binding site of CRP was implicated in the binding to laminin on the basis of inhibition by both soluble PC and anti‐idiotypic mAbs directed to the TEPC‐15 PC‐binding idiotype found on mouse antibodies to PC. In addition, mouse mAbs specific for the CRP PC binding site displayed decreased reactivity with CRP already bound to laminin. The binding of CRP to laminin provides a possible explanation for selective deposition of CRP at inflamed sites. The CRP‐laminin interaction may serve as a means of concentrating CRP at sites of tissue damage so that the CRP might function as a ligand for leukocytes, an event that will result in removal of necrotic tissue and cell debris.
The role of the idiotypic network in the induction of experimental systemic lupus erythematosus
Authors: Mozes Edna , Brocke Stefan , Shoenfeld Yehuda , Mendlovic Shlomo 
Published: December 2004

AbstractSystemic lupus erythematosus (SLE) has been induced in C3H.SW mice by their immunization with a human monoclonal anti‐DNA antibody that bears a common idiotype‐16/6 Id. Following immunization, high levels of murine anti‐16/6 and anti‐anti‐16/6 antibodies were detected in the sera of the immunized mice. Elevated titers of autoantibodies reacting with ssDNA, dsDNA, poly(I), poly(G), RNP, Ro, and La were also observed. The serological findings were associated with significant proteinuria, leukopenia, and elevated erythrocyte sedimentation rate. Immune complex deposition in the glomerular mesangium and sclerosis of the glomeruli were demonstrated. To study whether or not anti‐idiotypic antibodies are involved in the induction of the disease, a murine monoclonal antibody against the 16/6 Id was prepared and injected into C3H.SW mice. The anti‐16/6 Id antibody induced experimental SLE similarly to the 16/6 Id with an accelerated kidney pathology. A study performed on different mouse strains indicated that the susceptibility to the induction of SLE by the 16/6 Id is strain dependent and directly correlates to their ability to produce anti‐16/6 Id specific antibodies.
The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells
Authors: McGuire Paul G. , Seeds Nicholas W. 
Published: December 2004

AbstractLaminin and fibronectin are glycoproteins that influence cell behavior and mediate cell/substratum adhesion. We have examined the interaction of these macromolecules with the serine protease plasminogen activator (PA) in two types of extracellular matrices; one produced by the murine Engelbreth‐Holm‐Swarm (EHS) tumor (Matrigel™), and another by normal kidney epithelial cells in culture. Matrigel™ was found to contain significant quantities of tissue‐type PA (tPA). Two of the major components of Matrigel™, laminin and type IV collagen, were also examined. Tissue‐type PA was associated with purified preparations of laminin; however, it was not found associated with type IV collagen. Normal kidney epithelial cells in culture secrete large amounts of urokinase (UK) and deposit a subepithelial matrix containing both laminin and fibronectin. These matrix macromolecules were isolated from the deposited matrix by immunoprecipitation, examined by zymography, and found to contain UK. The potential role of this interaction in the mechanisms of cell migration and matrix remodeling is discussed.
Evidence of an auxin‐mediated phosphoinositide turnover and an inositol (1,4,5)trisphosphate effect on isolated membranes of Daucus carota L.
Authors: Zbell Bernd A. , Walter‐Back Cornelia , Bucher Hubert 
Published: December 2004

AbstractMicrosomal membranes from carrot suspension cells were phosphorylated in vitro with [γ‐32P]ATP. In the presence of submicromolar concentrations of the natural auxin indoleacetic acid (IAA), a rapid, but transient decrease of the [32P] label could be detected in the phospholipid extracts of the membranes. The phytohormone effect was not the result of an inhibition of the lipid phosphorylation reactions, but was caused by a simultaneous release of water‐soluble compounds, which, according to their chromatographic properties, were assumed to contain inositol polyphosphates. Although the [32P]‐labeled lipids, as well as the inositol polyphosphates, were not identified unequivocally by chemical analysis, these findings point to an auxin‐mediated control of a phosphoinositidase C‐like reaction similar to the hormone‐stimulated phosphoinositide response in animals. Exogenously applied inositol (1,4,5)trisphosphate [(1,4,5)IP3] was found to release 45Ca2+ from preloaded membrane vesicles of carrot cells. Both the detection of the auxin‐stimulated phosphoinositide response and the (1,4,5)IP3‐mediated Ca2+ release on isolated cell membranes offer new experimental approaches for the identification of the putative auxin receptor and its signal transduction pathway.
Purification and partial characterization of a hepatocyte antiproliferative glycopeptide
Authors: Auger Geneviève , Blanot Didier , Van Heijenoort Jean , Nadal Claude , Gournay Marie‐Françoise , Winchenne Jean‐Jacques , Boffa Georges A. , Lambin Patrick , Maes Pierrette , Tartar André 
Published: December 2004

AbstractA low molecular weight compound, which inhibits the G1‐S transition in rat hepatocytes, was obtained by tryptic hydrolysis of human α2‐macroglobulin followed by ultrafiltration at pH 10. It was purified by high‐performance liquid chromatography on μBondapak C18 and μBondapak NH2 with a practically quantitative yield; from 5.1 g of α2‐macroglobulin, 2.8 μg of purified compound were recovered. Inactivation by specific enzymes and chemical analyses showed that the inhibitor is a sialylated glycopeptide whose peptide moiety contains a pyroglutamyl residue. Its molecular mass, estimated by gel permeation chromatography, would be in the interval 3,500–4,600. However, amino acid analyses indicated that it is not yet pure. All these data suggest that α2‐macroglobulin could be the carrier of the precursor form of the glycopeptide.
Thermal properties of young red blood cells are indicative of an age‐dependent regulation of membrane‐skeleton interaction
Authors: Grimaldi Paola , Minetti Maurizio , Pugliese Pellegrina , Isacchi Giancarlo 
Published: December 2004

AbstractThe effects of red blood cell (RBC) age on membrane thermal properties have been investigated by using a 16‐nitroxide stearic acid spin probe. We detected in unfractionated and most dense cells (2% fraction of circulating cells) a thermal transition at 40°C that in young cells (1% fraction) was lowered at 33–35°C. Spectrin seems to be directly involved in the transition detected in both young and unfractionated cells, as showed by the disappearance of the breaks after low salt extraction of spectrin. A further indication for a role of spectrin in this transition comes from its characteristic thermal unfolding above 40°C.However, young cells did not show changes either in the thermal unfolding of spectrin or in the distribution of spectrin dimer, tetramer, and high oligometric forms. These data rule out that spectrin of young RBC is modified in its thermal properties and indicate that young cells may have a different spectrin‐membrane interaction.Treatment of unfractionated ghosts with an antibody specific for a fragment of the 10K domain of protein 4.1, which is fully competent for the spectrin‐actin binding, produced an evident lowering of the transition temperature. The same antibody did not affect the thermal transition of young ghosts. Our results suggest that spectrin‐membrane interactions may be regulated during RBC lifespan.
Can glutathione‐S‐transferases function as intracellular heme carriers?
Authors: Kirschner‐Zilber Ilana , Laufer Hava , Shaklai Nurith 
Published: December 2004

AbstractThe possibility that glutathione‐S‐transferases can serve as heme carriers in cells was studied via the following two characteristics: the ability to bind hemin reversibly and the coordination between heme and glutahione‐S‐transferases level in the cell. Two erythroleukemic cell lines that can be induced to synthesize hemoglobin were studied, K‐562 and Friend murine erythroleukemia cells. It was found that hemin‐associated glutathione‐S‐transferase tends to lose its native structure as expressed by partial irreversible inhibition of glutathione conjugation activity. In K‐562 cells, a small increase in heme synthesis was induced, but under no condition could glutathione‐S‐transferase be elevated. In addition, introduction of high hemin from without caused large hemoglobin production but did not inducechanges in the glutathione‐S‐transferase content. Dimethyl sulfoxide‐induced Friend murine eryth‐roleukemia cells synthesized a large amount of endogenous hemin that had to be transported from the mitochondria for hemoglobin synthesis. Although a concomitant increase in glutathione‐S‐transferase level (20–40%) was observed, it was only short‐lived, unlike hemin, which continued to increase. These data indicate a lack of correlation between glutathione‐S‐transferase and hemin or hemoglobin levels. Finally, dimethyl sulfoxide‐induced cells were treated with succinyl acetone to inhibit heme synthesis. These cells showed the same increased levels and time‐dependent pattern of gluathione‐S‐transferase as untreated cells. A similar phenomenon was observed when different substrates were used to measure the activities of glutathione‐S‐transferases. These results raise doubts about the possibility of glutathione‐S‐transferases functioning as heme carriers in cells.
Developmental changes in expression of a tumor‐associated protein in the rat fetus
Authors: Runge Steven W. , Larroya Saroj , Schumm Dorothy E. , Webb Thomas E. 
Published: December 2004

AbstractMonoclonal antibodies specific for a rat tumor‐associated protein cross‐react with a similar protein present in the cytosol of the rat fetus. The oncofetal protein exists as two species of approximate molecular weight 50 and 55 kDa which promote the transport of RNA from isolated nuclei. During rat fetal development, the protein first increases in concentration from approximately 12 to 16 days gestation and then drops to non‐detectable levels perinatally.
Modulation of transforming growth factor alpha‐dependent expression of epidermal growth factor receptor gene by transforming growth factor beta, triiodothyronine, and retinoic acid
Authors: Fernandez‐Pol J. A. , Klos D. J. , Hamilton P. D. 
Published: December 2004

AbstractWe have investigated the actions of transforming growth factor (TGF) type α on epidermal growth factor (EGF) receptor mRNA expression in MDA‐468 human mammary carcinoma cells in serum‐free media. We found that exposure of MDA‐468 cells to TGFα results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGFβ1 enhanced the accumulation of EGF receptor mRNA induced by TGFα in a time and dose‐dependent manner. We also found that triiodothyronine at physiological concentrations exerts synergistic control on the action of TGFα alone, or in association with TGFβ1, on EGF receptor mRNA expression. Similarly, retinoic acid treatment also enhanced in a time‐ and dose‐dependent manner the TGFα‐dependent response of EGF receptor mRNA and acted synergistically with TGFβ1. The results described here suggest that optimum regulation of EGF receptor gene expression by TGFα is a complex process involving synergistic interactions with heterologous growth factors and hormones.
ras‐Transformation of MDCK cells alters responses to phorbol ester without altering responses to bradykinin
Authors: R. Slivka Sandra , Godson Catherine , A. Insel Paul 
Published: December 2004

AbstractThe results of studies to evaluate the hypothesis that the 21 kDa GTP‐binding protein derived from the ras oncogene is involved in regulation and coupling of hormone receptors to phospholipase activity have thus far been inconsistent. We therefore examined the effect of H‐ras transformation on basal, tumor‐promoting phorbol ester (TPA)‐stimulated, and bradykinin‐mediated phospholipid hydrolysis in Madin Darby canine kidney cells (MDCK) by comparing H‐ras‐transformed MDCK cells (MDCK‐RAS) to two non‐transformed strains of MDCK cells (MDCK‐DI and MDCK‐ATCC). In unstimulated MDCK‐RAS, diacylglycerol (DAG), inositol phosphate accumulation, and choline phosphate release were increased while arachidonic acid and arachidonic acid metabolite (AA) release was not increased, suggesting that ras transformation increased phospholipase C activity. Protein kinase C (PK‐C) activity was decreased, and specific binding of (3H) phorbol ester was reduced in MDCK‐RAS relative to the non‐transformed MDCK cells suggesting that elevated DAG may activate and thereby down‐regulate PK‐C. Consistent with this finding in MDCK‐RAS, TPA‐stimulated AA release and subsequent prostaglandin E2 production were decreased, while TPA‐stimulated choline phosphate release was increased. Bradykinin receptor‐stimulated phospho‐lipid hydrolysis in MDCK‐RAS was similar to that of non‐transformed cells, suggesting that the ras‐derived protein does not directly couple bradykinin receptors to phospholipases in MDCK cells. However, the ability of TPA‐treatment to inhibit bradykinin‐stimulated phosphoinositide hydrolysis and enhance bradykinin‐stimulated AA release was attenuated in MDCK‐RAS. Additionally, in MDCK‐RAS the conversion of arachidonic acid to prostaglandin E, was substantially reduced. We conclude that rus transformation of MDCK cells increases DAG levels, thereby activating and, in turn, down‐regulating PK‐C and certain responses to TPA. Since activation of PK‐C may result in a variety of effects on signal transduction pathways, we propose that increased DAG and altered PK‐C levels associated with rus transformation may account for the inconsistent effects previously observed in studies evaluating the effect of rus transformation on phospholipases and other signal transduction systems.
Modulation of HPV18 and BPV1 transcription in human keratinocytes by simian virus 40 large T antigen and adenovirus type 5 E1A antigen
Authors: Bernard Bruno A. , Bailly Catherine , Lenoir Marie‐Cecile , Darmon Michel Y. 
Published: December 2004

AbstractTranscription of early open reading frames initiated from the long control region (LCR) of HPV18 and BPV1 is known to be modulated by homologous and heterologous papillomavirus E2 gene products. Using CAT constructs transfected into normal human keratinocytes, we show that SV40 large T antigen activates transcription from the LCR of both viruses, whereas Ad5‐Ela antigen represses transcription from the HPV18‐LCR but activates transcription from BPVI‐LCR. Experiments using constructs containing subfragments of the HPV18‐LCR cloned in enhancer configuration ahead of the SV40 early promoter or the HSVI‐Tk promoter suggest that the effect of Ad5‐Ela antigen on HPV18 transcription is probably due to a repression of the enhancer function of the LCR. The mechanism of transcription stimulation by SV40 large T antigen is less clear. The 230 bp Rsal‐Rsal central domain of the HPV18‐LCR seems involved both in transcriptional stimulation by SV40 large T antigen and transcriptional inhibition by adenovirus Ela antigen.
Co‐transformation of Lec1 CHO cells with N‐acetylgucosaminyltransferase 1 activity and a selectable marker
Authors: Ripka James , Pierce Michael , Fregien Nevis 
Published: December 2004

AbstractIn animal cells, the enzyme α( 1,3)‐mannoside‐β( 1,2)‐N‐acetylglucosaminyltransferase I (GlcNAc‐TI, EC.2.4.1.101) catalyzes the addition of N‐acetylglucosamine to the ASN‐linked Man GlcNAc oligosaccharide. The Chinese hamster ovary (CHO) mutant cell line Lecl is deficient in this enzyme activity and, therefore, accumulates mannose‐terminating cell surface ASN‐linked oligosaccharides. Consequently, Lecl cells are sensitive to the cytotoxic effects of the mannose‐binding lectin Concanavalin A (Con A). Lecl cells were co‐transformed with human DNA from A431 cells and eukaryotic expression plasmids containing the bacterial neo gene by calcium phosphate/DNA‐mediated transformation. Co‐transformants were selected for resistance to Con A and G‐418. DNA from a primary co‐transformant was purified and used to transform Lecl cells, resulting in secondary co‐transformants. Both primary and secondary co‐transformants exhibited in vitro GlcNAc‐TI‐specific enzyme activity, DNA gel blot analysis indicated that secondary co‐transformants contained both human and neo sequences.
Role of glycosylation in the transport of recombinant glycoproteins through the secretory pathway of lepidopteran insect cells
Authors: Jarvis Donald L. , Oker‐Blom Christian , Summers Max D. 
Published: December 2004

AbstractCell lines established from the Lepidopteran insect Spodoptera frugiperda (e.g., Sf9) are used routinely as hosts for the expression of foreign proteins by baculovirus vectors. Previously, we showed that human tissue plasminogen activator (t‐PA) was expressed, N‐glycosylated, and secreted by Sf9 cells infected with a recombinant baculovirus (Jarvis DL, Summers MD: Mol Cell Biol 9:214–223, 1989). We also showed that t‐PA secretion was blocked by tunicamycin.(TM), an inhibitor of N‐glycosylation, but not by castanospermine (CS) or N‐methyldeoxynojirimycin, inhibitors of the initial steps in N‐linked oligosaccharide processing. This suggestedthat the addition, but not the processing, of N‐linked oligosaccharides is required for the secretion of recombinant t‐PA from baculovirus‐infected Sf9 cells. In this study, we present a more generalized evaluation of the role of N‐glycosylation in the transport of recombinant glycoproteins through the Sf9 cell secretory pathway. Several different secretory or membrane‐bound glycoproteins were expressed in control, TM‐treated, or CS‐treated Sf9 cells, and their appearance in the medium or on the cell surface was measured. The results showed that TM blocked the transport of some, but not all, of these proteins, whereas CS did not block the transport of any. This suggests that N‐glycosylation is sometimes required for the transport of recombinant glycoproteins through the Sf9 secretory pathway, while processing of the oligosaccharides is not. At least two other proteins, p80 and p31, consistently coimmunoprecipitated with the nonglycosylated precursors of recombinant glycoproteins expressed in TM‐treated Sf9 cells. Neither was antigenically related to any of the recombinant proteins. Relatively larger amounts of p80 and p31 were coprecipitated when transport was completely blocked by TM compared to when transport was only reduced or was unaffected. These results suggest that p80 and p31 block the transport of some nonglycosylated glycoprotein precursors in TM‐treated Sf9 cells by binding to them and producing transport‐incompetent heterooligomeric com‐plexes. If this speculation is correct, then p80 and p31 are functionally analogous to the mammalian immunoglobulin heavy chain binding/glucose‐regulated 78 kilodalton protein (BiP/GRP78).