bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of chimeric human transferrin genes in vitro
Authors: Fischbach K. , Lu Y. , Tiffany‐Castiglioni E. , Minter A. , Bowman B. H. , Adrian G. S. 
Published: January 2005

AbstractTransferrin (TF), a major plasma protein, binds and transports ferric iron. Evidence exists for unique roles for TF in brain in oligodendrocyte differentiation, myelination and neuronal development. In this study, 5' flanking regions of the TF gene important in regulating gene expression were identified by transfected cell studies and a comparison of 5' flanking sequences of the human TF and TF receptor genes. Human glioma cell lines HTB‐16 and HTB‐17 were shown to synthesize TF identical in size and immunological reaction to TF synthesized by liver. The expression of a series of human chimeric TF genes in glioma cells was compared with hepatoma and HeLa cells. A difference in transient expression was observed in hepatoma and glioma cells transfected with TF chimeric genes containing 3.9 kb of the 5' region; hepatoma cells demonstrated significantly more expression than did glioma cells, suggesting that a DNA region present in the 3.9‐kb construct is important either in liver‐specific expression or in repression of brain expression, or in both. Smaller constructs containing ≤ 0.622 kb of the 5' regulatory region of the TF gene failed to demonstrate cell‐specific expression; they were expressed in HeLa cells, a line that does not synthesize TF. High levels of expression of 0.15‐kb TF constructs were also observed in hepatoma and glioma cell lines, but not in transgenic mice. Possible explanations of differences observed in expression of shorter TF constructs in vitro and in vivo are discussed.
Monoclonal antibodies to the cell surface and a soluble form of the human nerve growth factor receptor
Authors: Clagett‐Dame M. , Chung C. , Chao M. V. , DiStefano P. S. 
Published: January 2005

AbstractMonoclonal antibodies (designated IIIG5, VIID1, VIIIC8, and XIF1) have been produced that bind to the human nerve growth factor receptor (NGF‐R) as well as to a soluble, truncated form of the receptor (NGF‐Rt). The antibodies were generated against partially purified NGF‐Rt from the conditioned medium of E9b cells, a transfected mouse fibroblast cell line (Ltk−) that expresses large numbers of the low affinity form of the human NGF‐R on its cell surface (Chao MV, Bothwell MA, Ross AH, Koprowski H, Lanahan A A, Buck CR, Sehgal A [1986]: Science 232:518–521). Hybridomas were screened by radiometric immunosorbent assay (RISA) and by immunoprecipitation of solubilized cell surface receptor covalently cross‐linked to [125‐I]‐NGF. Four positive lines were cloned by limiting dilution and were found to secrete monoclonal antibodies of the IgGl, k subclass. All monoclonal antibodies bound to both NGF‐R and NGF‐Rt. Two monoclonal antibodies (VIIDl, XIF1) immunoblotted the NGF‐R from E9b cell preparations resolved on non‐reducing sodium dodecyl sulfate (SDS)‐polyacrylamide gels. The antibodies immunoprecipitated NGF‐R from both E9b cells and from SH‐SY5Y human neuroblastoma cells. The monoclonal antibodies bound to monkey (rhesis and cynomolgus) NGF‐Rt, but did not cross‐react with NGF‐R from chick or rat. Results of antibody competition studies demonstrated that three antibodies bound to a similar or overlapping epitope on the NGF‐Rt and one monoclonal antibody (IIIG5) recognized a distinct receptor epitope. Antibodies that bound to different sites on the receptor were used to develop a sensitive 2‐site RISA. The 2‐site RISA can be used to rapidly quantitate NGF‐R and NGF‐Rt in large numbers of biological samples in the absence of added [125‐I]‐labeled NGF.
Differential effect of lithium on fos protooncogene expression mediated by receptor and postreceptor activators of protein kinase C and cyclic adenosine monophosphate: Model for its antimanic action
Authors: Divish M. M. , Sheftel G. , Boyle A. , Kalasapudi V. D. , Papolos D. F. , Lachman Herbert M. 
Published: January 2005

AbstractLithium salts are the most effective agents used in treating manic‐depressive illness. It has been suggested that lithium's therapeutic efficacy could be due to an inhibitory effect on either inositol phospholipid (IP) and/or cyclic nucleotide metabolism. We have investigated the effect of lithium on these two signal transduction pathways in PC12 pheochromocytoma cells by studying a common effector target, expression of the fos protooncogene. We find that lithium, at therapeutic doses, has an augmenting effect on phosphatidylinositol (PI)‐mediated fos expression induced by activating a muscarinic cholinergic pathway, whereas it has no effect, at tenfold the therapeutic dose, on fos expression induced by receptor or postreceptor activators of cyclic adenosine monophosphate (cAMP). The lithium augmenting effect is also observed when the cells are treated with phorbol esters, which directly activate protein kinase C (PKC), suggesting that the level of lithium's interaction with the IP pathway is at the postreceptor level. We also show that phorbol esters induce extensive down regulation of subsequent cholinergic and phorbol ester responsiveness as well as heterologous down regulation of cAMP responses. Treatment of down‐regulated cells with lithium leads to an enhanced responsiveness when cells are rechallenged with agonists that activate PKC but not by agonists that stimulate cAMP. We also show that carbamazepine, another antimanic agent, has an inhibitory effect on cAMP‐mediated fos but no effect on the IP pathway. The opposite effects of lithium and carbamazepine on two critical transducing systems suggest a model for the antimanic action of these agents.
Serial passage of embryonic human astrocytes in serum‐free, hormone‐supplemented medium
Authors: Loo D. T. , Sakai Y. , Rawson C. L. , Barnes D. W. 
Published: January 2005

AbstractWe applied serum‐free cell culture methods that allow extended proliferation of mouse astrocyte precursor cells to the multipassagc culture of embryonic human brain cells. Cells were cultured in nutrient medium supplemented with insulin, transferrin, epidermal growth factor, fibreblast growth factor, heparin, high‐density lipoprotein, and fibronectin. Cultures were maintained for a maximum of 70 population doublings before proliferation ceased. The cells synthesized glial fibrillary acidic protein, an astrocyte marker, and expression of this protein was increased by incubation of the cells with transforming growth factor beta or serum. These results identify extracellular factors important for proliferation and differentiation of embryonic human astrocytes and provide a controlled system for multipassage culture.
Nerve growth factor‐induced increase in calcium uptake by PC12 cells
Authors: Nikodijevic B. , Guroff G. 
Published: January 2005

AbstractTreatment of PC 12 cells with nerve growth factor (NGF) produces a rapid and transient increase in calcium uptake into the cells. The increased uptake is maximal after 5 minutes of NGF treatment, but after 15 minutes of NGF treatment, no such increase can be observed. The effect of NGF is partially inhibited by blockers of L‐type calcium channels. K‐252a, an alkaloid‐like kinase inhibitor that usually is found to inhibit the actions of NGF on PC 12 cells, produces an increase in calcium uptake similar to, but smaller than, that seen with NGF. NGF had no effect on calcium release under these conditions.
K+ channel properties in cultured mouse Schwann cells: Dependence on extracellular K+
Authors: Verkhratsky A. , Hoppe D. , Kettenmann H. 
Published: January 2005

AbstractIn cultured Schwann cells, single‐channel and whole‐cell K+ currents can be activated by depolarizing the membrane to values more negative than −50 mV. In elevated extracellular K+ concentration ([K+]o), however, single‐channel activity and whole‐cell currents could be recorded at more negative potentials. Thus, the threshold of current activation was shifted to more negative potentials. This shift in the activation threshold was only observed with normal (50‐60 mM) intracellular [K+] levels; it was not appparent when [K+]j was elevated to 145 mM. The control of [K+ ]0 on the gating properties of K+ channels may serve to en lance the capability of the Schwann cell to take up [K+ ]o and thus may serve for [K+] homeostasis in the peripheral nerve.
Multidrug transport in human glioblastoma cells is inhibited by transforming growth factors type β1, β2, and β1.2
Authors: Schluesener H. J. 
Published: January 2005

AbstractThe transforming growth factors type β1, β2 and β1.2 suppress multidrug transport in human pat‐1 glioblastoma cells and even in cells that strongly over‐express mdr genes and are resistant to inhibition of multidrug transport by chemosensitizers. Thus, inhibition of multidrug transport by cytokines might be a new approach to increase cellular accumulation of chemotherapeutic agents in multidrug resistant glial tumor cells. Interestingly, a member of the more distantly related decapentaplegic subgroup of transforming growth factors, the bone morphogenetic protein BMP 2, did not inhibit multidrug transport.
Effect of nerve growth factor on C‐1300 murine neuroblastoma tumor growth and catecholamine content in neonatally sympathectomized mice
Authors: Fink D. W. , Mirkin B. L. 
Published: January 2005

AbstractThe in situ C‐1300 murine neuroblastoma (MNB) tumor model was used to investigate the influence of exogenously administered nerve growth factor (NGF) on tumor growth and tissue catecholamine concentra tion in mice Sympathectomized with 6‐hydroxy‐dopamine (6‐OHDA) on postnatal days 4–10. Mice were implanted with 1 × 106 disaggregated MNB cells 3 days after termination of 6‐OHDA administra tion. NGF (12‐15 μg/mouse/day) treatment was ini tiated at the time of MNB cell implantation and con tinued until sacrifice of the animal. The time interval between tumor cell implantation and detection of pal pable tumor (tumor onset time), transverse tumor diameter, tumor weight, tumor weight to body weight ratio, and tumor catecholamine concentration were determined. Neonatal sympathectomy caused a de crease in myocardial norepinephrine concentration of 88% compared with vehicle‐treated animals as well as a significant reduction in total body and organ weight Average body, brain, heart, and spleen weights were decreased 31% 16%, 25%, and 42%, respectively, below control values. The daily injection of NGF, from the time of MNB tumor implantation to sacrifice, did not prevent these effects of chemical sympathectomy from being expressed. Tumor onset time following implantation of MNB cells was signif icantly increased in neonatally Sympathectomized mice and was not altered by treatment with NGF. In contrast, the decrease in MNB tumor growth rate observed in Sympathectomized mice was reversed by administration of NGF. Mean tumor weight and mean tumor to body weight ratio were 89% and 115% of comparable control values, respectively, in Sympathectomized mice receiving exogenous NGF. The norepinephrine (NE) and dopamine (DA) con centrations of tumors removed 8 days after tumor onset were increased 2.8‐ and 3. 6‐fold, respectively, in neonatally Sympathectomized mice. Administration of NGF prevented the increase in tumor NE and DA concentration associated with chemical sympa thectomy. Cell surface receptors for NGF were present on cultured C‐1300 MNB cells. These data suggested the presence of two types of binding sites with KD values of 45.9 pM (high affinity) and 2.4 nM (low affinity). The inhibition of MNB tumor growth and elevation in tumor catecholamine content pro duced by neonatal sympathectomy may be Immorally mediated since they were selectively reversed by the administration of NGF.
Expression of the β‐nerve growth factor gene in male sex organs of the mouse, rat, and guinea pig
Authors: MacGrogan D. , Desprès G. , Romand R. , Dicou E. 
Published: January 2005

AbstractSteady‐state nerve growth factor (NGF) mRNA levels were estimated in male sex organs of the mouse, rat, and guinea pig by RNA blot hybridization analysis. The abundance of NGFmRNAs was in the order vas deferens < epididymis ≥ seminal vesicles > testis. NGF mRNA levels in these organs were compared with those estimated for other rat peripheral tissues and were found to correlate with the density of their sympathetic innervation, with the exception of guinea pig prostate. Castration had no significant effect on NGF mRNA levels in the guinea pig prostate, suggesting that NGF synthesis in this tissue is not under direct androgen control. NGF‐like and proNGF‐like immunoreactivities were localized by immunohistochemical techniques in the secretory cells of the glandular epithelium of the guinea pig prostate and in germ cells in the seminiferous tubules of the mouse testis.
Quantitation of tyrosine hydroxylase and neuropeptide Y immunoreactivity in single rat sympathetic neurons: Effects of preganglionic nerve activity
Authors: Gurusinghe C. J. , Harris P. J. , Abbott D. F. , Messina A. , Bell Christopher 
Published: January 2005

AbstractUsing computerised densitometry to measure immunocytochemical reaction product in a model system, we established conditions that produced a linear relationship between the logarithm of antigen concentration and the measured intensity of staining. We then applied the densitometric technique to assess the changes in tyrosine hydroxylase (TH) and neuropep‐tide tyrosine (NPY) within sympathetic neurons of rat superior cervical ganglion following chronic decentralization and following reserpine treatment. One week after surgical or pharmacological decentralization, there was appreciable reduction of neuronal levels of both TH and NPY. However, there remained considerable variation in the immunoreactivities of individual cells. Three days of treatment with reserpine elevated TH levels but substantially reduced NPY. Both these effects were prevented by prior decentralization of the ganglia. No differences were seen between normotensive and the Otago strain of genetically hypertensive rats, either in basal TH or NPY immunoreactivities or in responses to the maneouvres performed. Comparison of our findings with previous biochemical data indicate that densitometric immunocytochemistry provides an accurate index of neuronally localised antigen concentrations but also allows analysis of interneuronal differences that are not otherwise apparent.
Synthesis, purification, and characterization of human ciliary neuronotrophic factor from E. coli
Authors: Negro A. , Corona G. , Bigon E. , Martini I. , Grandi C. , Skaper S. D. , Callegaro L. 
Published: January 2005

AbstractThe cDNA for human ciliary neuronotrophic factor (CNTF) has been cloned into an expression vector under the control of the T7 promoter. The BL21 strain of E. coli was transformed with this vector. Human CNTF accounted for about 30% of the total bacterial protein after induction with isopropyl‐B‐D‐thiogalactopyranoside. This human CNTF was purified to homogeneity from inclusion bodies by a combination of ion exchange chromatography and reverse‐phase high performance liquid chromatography. The amino‐terminal amino acid sequence of the purified protein was identical to the deduced amino acid sequence; however, the methionyl residue has been removed. On SDS‐PAGE gels, human CNTF displayed a molecular weight of about 24 kDa, in accord with its deduced molecular mass; a pI of 5.8 indicates the acidic nature of the molecule. A proposed structure for human CNTF includes major alpha helical regions. The ED50 of purified human CNTF was approximately 30 pM, using cultured embryonic day 10 chicken dorsal root ganglion neurons; no activity was observed with neurons from embryonic day 8 ganglia. Polyclonal antibodies prepared against both a synthetic peptide of CNTF and the entire human CNTF protein recognized a single 24 kDa band on Western blots, corresponding to human CNTF. However, only the antibodies against intact CNTF blocked its biological activity. This represents the first molecular expresson and purification of human CNTF.
Identification of a cDNA clone specific for the oligodendrocyte‐derived repulsive extracellular matrix molecule J1‐160/180
Authors: Fuss B. , Pott U. , Fischer P. , Schwab M. E. , Schachner M. 
Published: January 2005

AbstractA cDNA clone specific for the oligodendrocyte‐de‐rived extracellular matrix glycoproteins Jl‐160/180 was obtained from a àZAPII expression library using polyclonal antibodies generated against mouse JI‐160. The library was constructed from poly(A) + ‐RNA isolated from O1 antigen‐positive rat oligodendrocytes. The cDNA clone expressed a fusion protein that was recognized by the J1‐160/180‐specific monoclonal antibodies 596, 619, and 620, and, weakly, 597. The fusion protein was not recognized by polyclonal antibodies to mouse J1/tenascin. The cDNA clone with an insert of approximately 5.6 kb in size contained the nucleotide sequence coding for the amino acid sequence of the N‐terminus of a tryptic peptide derived from mouse J1‐160. The developmental and tissue distribution of the mRNA recognized by the cDNA clone is in agreement with the described expression of the J1‐160/180 proteins.
Age‐related acute depletion of cerebral glutathione by peroxidative stress
Authors: Benzi G. , Curti D. , Marzatico F. , Pastoris O. 
Published: January 2005

AbstractCyclohexene‐1‐one and cycloheptene‐1‐one cause a severe age‐related depletion of reduced glutathione (GSH) in the forebrain of 5‐ or 15‐ or 25‐month‐old rats. Chronic pretreatment with phosphatidylcholine partially inhibits the GSH depletion in old forebrains by the prooxidants tested, suggesting that in aged animals alterations in mitochondrial inner membrane phospholipid composition and/or cytochrome oxidase activity might play a role in oxygen free‐radical production.
A molecular dissection of the carboxyterminal tails of the major neurofilament subunits NF‐M and NF‐H
Authors: Harris J. , Ayyub C. , Shaw G. 
Published: January 2005

AbstractWe have initiated a multidisciplinary project that aims to dissect and ultimately define the functions of the long and unusual C‐terminal “tail” sequences of the two high molecular weight neurofilament sub‐units, NF‐M and NF‐H. A series of recombinant fusion proteins containing selected NF‐M and NF‐H tail sequences were constructed using appropriate cDNAs. These fusion proteins were used to further define the epitopes for a variety of widely used neurofilament antibodies, including NN18 and N52, which are now available commercially from several companies. We also measured the SDS‐PAGE mobility of the fusion proteins and found that, like the native neurofilament tails, the fusion proteins ran considerably slower than predicted from their molecular weight. Since all fusion proteins produced so far exhibit this characteristic we conclude that all segments of the NF‐M and NF‐H tail share this unusual property. Finally we were able to produce novel and potentially useful polyclonal and monoclonal antibodies to selected segments of NF‐M and NF‐H sequence. These antibody studies showed that the extreme C‐termini of NF‐M and NF‐H are immunologically absolutely distinct from one another and also indicate that the extreme C‐terminus of NF‐M is immunologically much more conserved than the analogous region of NF‐H. These findings are in complete agreement with our conclusions derived from amino acid sequence analysis, and further underline the possible functional importance of the extreme C‐terminus of NF‐M. We also show that the unusual immunological properties of the bovine NF‐M tail we have previously observed do not extend to the extreme C‐terminal region, which appears immunologically no different from the analogous region of other NF‐M molecules. The peculiarities of bovine NF‐M could be explained by the presence of a KSP motif that resembles the NF‐H KSP prototype.
Identification of a tektin‐like protein associated with neurofilaments in the developing chick nervous system
Authors: Edson K. J. , Linck R. W. , Letourneau P. C. 
Published: January 2005

AbstractA 160‐kD polypeptide, which is recognized by an affinity‐purified polyclonal antibody to the 55‐kD tektin‐A polypeptide from sea urchin sperm flagellar microtubules, is associated with neurofilaments in embryonic chick nerve cells. Antibodies to tektin‐A and monoclonal antibodies to the neurofilament triplet proteins colocalize to filaments in cultured nerve cells and to filaments in extracts of chick spinal cord, using indirect immunofluorescence microscopy and immunogold electron microscopy. The antigen reacting with anti‐tektin‐A in chick brain and spinal cord extracts has been identified as a 160‐kD polypeptide by SDS‐PAGE and has been shown to be distinct from the known neurofilament‐triplet proteins by two‐dimensional immunoblot analysis. These data suggest that a unique protein with limited sequence homology to tektin‐A is a component of the neuronal cytoskeleton and is incorporated into or associated with neurofilaments.
Depletion of acetylated α‐tubulin during microtubule purification from bovine brain gray and white matter regions
Authors: Kim H. 
Published: January 2005

AbstractWe have followed the fate of acetylated α‐tubulin during microtubule (MT) purification from both gray matter‐ and white matter‐enriched bovine brain regions, using quantitative immunoblot assays employing well characterized monoclonal antibodies specific for acetylated α‐tubulin and all α‐tubulins. Our results show that crude homogenates from both gray matter and white matter brain regions contain the same proportion of acetylated α‐ to total tubulin. We have found that the acetylated isoform cycles more efficiently with MTs from gray matter than with MTs from white matter. However, the resultant purified MT preparations from both gray and white matter regions are greatly depleted in the acetylated isoform compared to the tubulin in the initial homogenates because most of the acetylated α‐tubulin from both tissue sources partitions with the cold‐insoluble fraction of the initial brain homogenate. A low percentage of the acetylated α‐tubulin in brain homogenates does become incorporated into MTs initially, but this sub‐population of acetylated α‐tubulin then becomes associated with a cold‐ and calcium‐insoluble fraction of the MT preparation. These results demonstrate that the standard brain MT and tubulin preparations used by most investigators for in vitro studies are greatly depleted in acetylated α‐tubulin, and thus provide poor model systems for the analysis of the function of this tubulin isoform.