bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations induced by methylene blue plus light in single-stranded M13mp2.
Authors: McBride T J , Schneider J E , Floyd R A , Loeb L A
Published: May 2006

Reactive oxygen species are generated by a variety of cellular processes. These endogenously generated, reactive intermediates produce a multiplicity of DNA alterations and mutations and have been implicated in the pathogenesis of several human diseases. We report here that treatment of single-stranded M13mp2 bacteriophage DNA with methylene blue and white light generates increased levels of 8-hydroxydeoxyguanosine and that mutagenesis is both highly specific and dependent on the SOS response. Lesions produced block the progression of DNA synthesis one base preceding template guanines. In SOS-induced Escherichia coli, 97% of all methylene blue-induced mutations in the lacZ alpha gene of M13mp2 DNA are single-base substitutions opposite template guanines. The most frequent mutations are G----C transversions. The G----T transversions expected from the presence of 8-hydroxydeoxyguanosine in the template strand occur, but at a lower frequency. Sequence data together with SOS dependency and the presence of replication blockage demonstrate that while 8-hydroxydeoxyguanosine may serve as an important marker to monitor oxygen-induced DNA damage in humans, it does not account for either the observed blockage to replication or the mutagenesis by methylene blue plus light in SOS-induced E. coli. Instead, an as yet unidentified lesion generated by active oxygen species is a more potent mutagenic event.
Secretion of a soluble, chimeric gamma delta T-cell receptor-immunoglobulin heterodimer.
Authors: Eilat D , Kikuchi G E , Coligan J E , Shevach E M
Published: May 2006

Soluble derivatives of T-cell antigen receptors (TCRs) should prove invaluable for studying the interaction of these receptors with antigens and major histocompatibility complex molecules, for structural studies, and for the identification of unknown ligands. We have engineered chimeric proteins, containing the extracellular domains of the mouse V gamma 1.1-C gamma 4 and V delta 6.2-C delta (V, variable; C, constant) TCR chains fused to the hinge region, CH2 (H, heavy), and CH3 domains of human IgG1 heavy chain, and expressed them by transient transfection in COS cells. We show here that TCR gamma-IgH and TCR delta-IgH chimeric chains are produced intracellularly in significant amounts, that the two chains can assemble correctly to form disulfide-linked, glycosylated heterodimers, and that a selective mechanism allows secretion of correctly paired receptor chains into the medium. Identity of the chimeric secreted TCR gamma delta-IgH heterodimer was confirmed by immunoblot analysis using V gamma 1-specific anti-peptide antiserum and immunoprecipitation analysis using the monoclonal antibody UC7, which is shown to be specific for the TCR delta chain. In addition, the soluble TCR gamma delta-IgH heterodimer can be immunoprecipitated with the anti-clonotypic monoclonal antibody F10/56, which suggests that the fusion protein likely has a structural conformation similar to that of the native TCR. The COS cell expression system may prove useful for the production of additional TCR-IgH fusion proteins.
Aminoguanidine inhibits oxidative modification of low density lipoprotein protein and the subsequent increase in uptake by macrophage scavenger receptors.
Authors: Picard S , Parthasarathy S , Fruebis J , Witztum J L
Published: May 2006

Aminoguanidine decreases the formation of advanced glycosylation end products that occurs during chronic hyperglycemia. Presumably this occurs because early glycosylation products preferentially bind to aminoguanidine rather than to lysine groups of adjacent proteins. Because oxidative modification of low density lipoprotein (LDL) also involves derivatization of lysine residues of apolipoprotein (apo) B by reactive aldehydes formed during the decomposition of oxidized fatty acids, we postulated that aminoguanidine might also inhibit the oxidatively induced modification of LDL protein. To test this hypothesis we oxidized LDL by incubation with Cu2+ or with endothelial cells in the absence or presence of aminoguanidine. Aminoguanidine prevented apo B lysine modification, as measured by fluorescence spectroscopy, and inhibited in a dose-dependent manner the oxidatively induced increase in subsequent macrophage uptake. At concentrations that inhibited apo B modification (5-10 mM), aminoguanidine increased the lag time in diene conjugation but did not affect the plateau value reached. These data indicate that aminoguanidine inhibits oxidative modification of LDL protein in large part by binding reactive aldehydes formed during lipid peroxidation and preventing their subsequent conjugation to apo B. Thus, aminoguanidine (and related compounds) may be of dual benefit in inhibiting atherosclerosis, both by inhibiting formation of advanced glycosylation end products and by inhibiting the modification of LDL apo B that makes it a ligand for scavenger receptors.
A high-molecular-weight squid neurofilament protein contains a lamin-like rod domain and a tail domain with Lys-Ser-Pro repeats.
Authors: Way J , Hellmich M R , Jaffe H , Szaro B , Pant H C , Gainer H , Battey J
Published: May 2006

Previous studies have shown that two low molecular-weight neurofilament (NF) proteins (NF-60 and NF-70) from the squid Loligo pealei are translated from mRNAs that are splice variants of a single squid NF gene. In this study, we report the isolation and characterization of cDNA clones encoding a high-molecular-weight squid NF protein (NF-220), the mRNA of which derives from the same squid NF gene. All three proteins are identical in their amino-terminal and lamin-like rod domains but differ in their carboxyl-terminal tail regions. In contrast to the short tail domains of NF-60 and NF-70, the NF-220 protein has a longer tail domain containing an acidic cluster of amino acids immediately followed by repeated copies of the sequence motif Lys-Ser-Pro. The Lys-Ser-Pro domain is similar to that of mammalian medium NF (NF-M) and high NF (NF-H) proteins, where the serines are highly phosphorylated. Except for these Lys-Ser-Pro motifs, there is surprisingly little structural similarity between the squid NF-220 protein and mammalian NF-M and NF-H proteins. Furthermore, the location of introns in squid NF-220 protein shows that it is more closely related to nuclear lamins and type III intermediate-filament proteins than to vertebrate NF proteins.
Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA.
Authors: Michaels M L , Cruz C , Grollman A P , Miller J H
Published: May 2006

It has been previously shown both in vivo and in vitro that DNA synthesis past an oxidatively damaged form of guanine, 7,8-dihydro-8-oxoguanine (8-oxoG), can result in the misincorporation of adenine (A) opposite the 8-oxodG. In this study we show that MutY glycosylase is active on a site-specific, oxidatively damaged A/8-oxoG mispair and that it removes the undamaged adenine from this mispair. Strains that lack active MutY protein have elevated rates of G.C----T.A transversions. We find that the mutator phenotype of a mutY strain can be fully complemented by overexpressing MutM protein (Fpg protein) from a plasmid clone. The MutM protein removes 8-oxoG lesions from DNA. In addition, we have isolated a strain with a chromosomal mutation that suppresses the mutY phenotype and found that this suppressor also overexpresses MutM. Finally, a mutY mutM double mutant has a 25- to 75-fold higher mutation rate than either mutator alone. The data strongly suggest that MutY is part of an intricate repair system directed against 8-oxoG lesions in nucleic acids and that the primary function of MutY in vivo is the removal of adenines that are misincorporated opposite 8-oxoG lesions during DNA synthesis.
Potentiation of DNA adduct formation in HL-60 cells by combinations of benzene metabolites.
Authors: Lévay G , Bodell W J
Published: May 2006

Using P1 nuclease enhanced 32P postlabeling, we investigated DNA adduct formation in HL-60 promyelocytic leukemia cells treated with the benzene metabolites hydroquinone, catechol, and 1,2,4-benzenetriol. Comparison of the slopes of the dose-response curves showed that hydroquinone was 7-9 times more effective than 1,2,4,-benzenetriol and catechol at inducing DNA adducts. Comparison of hydroquinone with catechol showed a good correlation between adduct formation and cytotoxicity. Similar comparisons of hydroquinone and 1,2,4,-benzenetriol suggest that cellular processes in addition to DNA adduct formation contributed to cytotoxicity. In cells treated with the combination of hydroquinone and either catechol or 1,2,4,-benzenetriol, DNA adduct formation was 3-6 times greater than the sum of adduct formation produced by single-agent treatments. Treatment with hydroquinone and 1,2,4,-benzenetriol produced DNA adducts not detected after treatment with either metabolite alone. The synergistic interaction of benzene metabolites in the production of DNA adducts may play an important role in the genotoxic effects of benzene in vivo.
Central role for differential gene expression in mammalian hibernation.
Authors: Srere H K , Wang L C , Martin S L
Published: May 2006

Mammalian hibernators experience dramatic reductions in body temperature, metabolic rate, respiratory rate, and heart rate during hibernation. These changes are precisely controlled and reversible with only internally driven mechanisms, suggesting specific biochemical regulation. We present a model that integrates our observations of differential liver gene expression during preparation for, and maintenance of, the hibernating state, with the known phylogenetic interspersion of hibernating species in several major mammalian lineages. This model predicts a major role for the differential expression of existing mammalian genes in the biochemical regulation of hibernation.
Elimination of smooth muscle cells in experimental restenosis: targeting of fibroblast growth factor receptors.
Authors: Casscells W , Lappi D A , Olwin B B , Wai C , Siegman M , Speir E H , Sasse J , Baird A
Published: May 2006

Factors in plasma and platelets do not fully account for the proliferation of smooth muscle cells in vascular injury, implying that additional factors are involved. Recently, we and others have observed that vascular injury regulates basic fibroblast growth factor, suggesting a further role for this pleiotropic factor. We report here that injury of rat arteries leads to an increase in fibroblast growth factor receptors in vascular smooth muscle cells. This up-regulation makes smooth muscle cells susceptible, in vitro and in vivo, to the lethal effects of a conjugate of basic fibroblast growth factor with the ribosome inactivator saporin. Saporin alone has no effect, whereas the conjugate kills proliferating, but not quiescent, smooth muscle cells in vitro. In vivo, one to three doses inhibit neointimal proliferation but have no apparent effect on the uninjured artery. Thus, the up-regulation of fibroblast growth factor receptors in vascular injury suggests new therapeutic possibilities for such refractory conditions as restenosis following balloon angioplasty.
The 3' untranslated region of localized maternal messages contains a conserved motif involved in mRNA localization.
Authors: Gottlieb E
Published: May 2006

Messenger RNA (mRNA) localization is emerging as a means of regulating gene expression. This process is operational in fly and frog development, where a subset of maternally inherited RNAs are asymmetrically distributed and thought to impart axial polarity to the embryo. Since most maternal mRNAs are uniformly distributed, an apparatus must exist to recognize and specifically transport these rare localized species. Here I report the identification of a nine-nucleotide motif, YUGUUYCUG, common to the 3' untranslated regions of four sequenced messages of this class: Drosophila bicoid and nanos mRNAs and Xenopus An2 and Vg1 mRNAs. To test the role of this nonamer sequence in the localization process, a Drosophila transient assay has been established. The assay reveals that bicoid mRNA specifically lacking this nonamer is partially mislocalized. In contrast, nonamer deletion is inconsequential to message stability. The existence of specific and general mRNA localization signals is proposed and it is suggested that this conserved motif belongs to the latter category.
Cloning, expression, and localization of a rat brain high-affinity glycine transporter.
Authors: Guastella J , Brecha N , Weigmann C , Lester H A , Davidson N
Published: May 2006

A cDNA clone encoding a glycine transporter has been isolated from rat brain by a combined PCR and plaque-hybridization strategy. mRNA synthesized from this clone (designated GLYT1) directs the expression of sodium- and chloride-dependent, high-affinity uptake of [3H]glycine by Xenopus oocytes. [3H]Glycine transport mediated by clone GLYT1 is blocked by sarcosine but is not blocked by methyl-aminoisobutyric acid or L-alanine, a substrate specificity similar to that described for a previously identified glycine-uptake system called system Gly. In situ hybridization reveals that GLYT1 is prominently expressed in the cervical spinal cord and brainstem, two regions of the central nervous system where glycine is a putative neurotransmitter. GLYT1 is also strongly expressed in the cerebellum and olfactory bulb and is expressed at lower levels in other brain regions. The open reading frame of the GLYT1 cDNA predicts a protein containing 633 amino acids with a molecular mass of approximately 70 kDA. The primary structure and hydropathicity profile of GLYT1 protein reveal that this protein is a member of the sodium- and chloride-dependent superfamily of transporters that utilize neurotransmitters and related substances as substrates.
Dynamic NMR spectral analysis and protein folding: identification of a highly populated folding intermediate of rat intestinal fatty acid-binding protein by 19F NMR.
Authors: Ropson I J , Frieden C
Published: May 2006

The folding of intestinal fatty-acid binding protein has been monitored by 19F NMR after incorporation of 6-fluorotryptophan into the protein. The two resonances resulting from the two tryptophans of this protein showed different dependencies on denaturant concentration. One of the resonances was in slow chemical exchange between two resonance frequencies, native and completely unfolded. The changes for this resonance occurred over a denaturant concentration range identical to that monitored by circular dichroism or fluorescence during unfolding. The other resonance continued to show changes at concentrations of denaturant well above that needed to complete the unfolding transition as monitored by optical techniques. Site directed mutagenesis showed that tryptophan-82 was the residue responsible for the unexpected behavior. We conclude, based on complete line-shape analysis, that there are significant concentrations of one or more intermediates in equilibrium with the native and unfolded forms. The structure of the intermediate(s) is more similar to the completely unfolded form of the protein than to the native structure, since little if any secondary structure is present. Further, these structure(s) persist at high denaturant concentrations and may represent local initiating sites in the folding of this beta-sheet protein.
The glial voltage-gated sodium channel: cell- and tissue-specific mRNA expression.
Authors: Gautron S , Dos Santos G , Pinto-Henrique D , Koulakoff A , Gros F , Berwald-Netter Y
Published: May 2006

Previous electrophysiological and pharmacological studies on central and peripheral glia revealed the presence of voltage-gated Na channels with properties that are similar but not identical to those of neuronal Na channels. Here we report the isolation and characterization of a cDNA encoding the C-terminal portion of a putative glial Na-channel (Na-G) alpha subunit. The amino acid sequence deduced from this cDNA indicates that the Na-G represents a separate molecular class within the mammalian Na-channel multigene family. By Northern blot, RNase protection, and in situ hybridization assays, we demonstrate that, in addition to brain astroglia, the Na-G mRNA is expressed in cultures of Schwann cells derived from dorsal root ganglia or from sciatic nerve. In vivo, the Na-G mRNA is detected not only in brain, dorsal root ganglia, and sciatic nerve, but also in tissues outside the nervous system including cardiac and skeletal muscle and lung. Its level varies according to the tissue and is developmentally regulated. The sequence and expression data concur in designating Na-G as an distinct type of Na channel, presumably with low sensitivity to tetrodotoxin.
Role of loop-helix interactions in stabilizing four-helix bundle proteins.
Authors: Chou K C , Maggiora G M , Scheraga H A
Published: May 2006

One of the critical issues regarding proteins with a four-helix bundle motif is which interactions play the major role in stabilizing this type of folded structure: the interaction among the four alpha-helices or the interaction between the loop and helix segments. To answer this question, an energetic analysis has been carried out for three proteins with a four-helix bundle--namely, methemerythrin, cytochrome b-562, and cytochrome c'. The structures on which the analysis has been made were derived from their respective crystallographic coordinates. All three proteins have long helices (16-26 residues) and most of their loops are short (3-5 residues). However, it was found in all three proteins that loop-helix interactions were stronger than helix-helix interactions. Moreover, not only the nonbonded component but also the electrostatic component of the interaction energy were dominated by loop-helix interactions rather than by interhelix interactions, although the latter involve favorable helix-dipole interactions due to the antiparallel arrangement of neighboring helices. The results of the energetic analysis indicate that the loop segments, whether they are in a theoretical model or in real proteins, play a significant role in stabilizing proteins with four-helix bundles.
Toward rules relating zinc finger protein sequences and DNA binding site preferences.
Authors: Desjarlais J R , Berg J M
Published: May 2006

Zinc finger proteins of the Cys2-His2 type consist of tandem arrays of domains, where each domain appears to contact three adjacent base pairs of DNA through three key residues. We have designed and prepared a series of variants of the central zinc finger within the DNA binding domain of Sp1 by using information from an analysis of a large data base of zinc finger protein sequences. Through systematic variations at two of the three contact positions (underlined), relatively specific recognition of sequences of the form 5'-GGGGN(G or T)GGG-3' has been achieved. These results provide the basis for rules that may develop into a code that will allow the design of zinc finger proteins with preselected DNA site specificity.
Somatotopic organization in rat striatum: evidence for a combinational map.
Authors: Brown L L
Published: May 2006

2-Deoxy-D-[14C]glucose autoradiography was used in awake rats to map neural activity in the sensorimotor sector of striatum. Stimulation of hindlimb, trunk, or forelimb activated primary sensory cortex in a localized columnar pattern, indicating activation of somatosensory receptors and a discrete cortical functional unit. In sensorimotor striatum, an image analysis detection technique revealed regions of maximal activity, or features, that formed a patchy pattern of activation reminiscent of the known anatomic patterns of cortico-striate terminals. Ipsilateral as well as contralateral activation was observed. The activated areas revealed a body map in striatum that was organized in a manner consistent with cortical topography (dorsoventrally: hindlimb, trunk, forelimb) at most anteroposterior levels, similar to that found in other species. However, at other levels, a different organization (e.g., trunk, hindlimb, forelimb) was observed. Furthermore, the arrangements of body region and side were also unique at different anteroposterior levels. Thus, functional activity showed multiple, different juxtapositions of body elements--i.e., a combinational map. The data suggest that striatum may provide an anatomic substrate for different combinations of inputs necessary to select and integrate movement.
Wild-type p53 is a cell cycle checkpoint determinant following irradiation.
Authors: Kuerbitz S J , Plunkett B S , Walsh W V , Kastan M B
Published: May 2006

Cell cycle checkpoints appear to contribute to an increase in cell survival and a decrease in abnormal heritable genetic changes following exposure to DNA damaging agents. Though several radiation-sensitive yeast mutants have been identified, little is known about the genes that control these responses in mammalian cells. Recent studies from our laboratory have demonstrated a close correlation between expression of wild-type p53 genes in human hematopoietic cells and their ability to arrest in G1 phase after certain types of DNA damage. In the present study, this correlation was first generalized to nonhematopoietic mammalian cells as well. A cause and effect relationship between expression of wild-type p53 and the G1 arrest that occurs after gamma irradiation was then established by demonstrating (i) acquisition of the G1 arrest after gamma irradiation following transfection of wild-type p53 genes into cells lacking endogenous p53 genes and (ii) loss of the G1 arrest after irradiation following transfection of mutant p53 genes into cells with wild-type endogenous p53 genes. A defined role for p53 (the most commonly mutated gene in human cancers) in a physiologic pathway has, to our knowledge, not been reported previously. Furthermore, these experiments illustrate one way in which a mutant p53 gene product can function in a "dominant negative" manner. Participation of p53 in this pathway suggests a mechanism for the contribution of abnormalities in p53 to tumorigenesis and genetic instability and provides a useful model for studies of the molecular mechanisms of p53 involvement in controlling the cell cycle.
Distal transcript of the dystrophin gene initiated from an alternative first exon and encoding a 75-kDa protein widely distributed in nonmuscle tissues.
Authors: Hugnot J P , Gilgenkrantz H , Vincent N , Chafey P , Morris G E , Monaco A P , Berwald-Netter Y , Koulakoff A , Kaplan J C , Kahn A
Published: May 2006

A transcript generated by the distal part of the Duchenne Muscular Dystrophy (DMD) gene was initially detected in cells where the full size 14-kilobase (kb) messenger RNA is not found at a significant level. This transcript, approximately 4.5 kb long, corresponds to the cysteine-rich and carboxyl-terminal domains of dystrophin. It begins with a novel 80- to 100-nucleotide exon containing an ATG start site for a new coding sequence of 17 nucleotides in-frame with the consecutive dystrophin cDNA sequence from exon 63. This result suggests the existence of a third promoter that would be localized about 8 kilobases upstream from exon 63 of the DMD gene. The distal transcript is widely distributed but is absent in adult skeletal and myometrial muscle. It is much more abundant in fetal tissues. With an antibody directed against the dystrophin carboxyl terminus, the protein corresponding to this transcript was detected as a 70- to 75-kDa entity on Western blots. It was found in all tissues analyzed except in skeletal muscle. It was not found in lymphoblastoid cells from a Duchenne patient with a complete deletion of the dystrophin gene. The role and subcellular localization of this protein is not known. It may explain extramuscular symptoms exhibited by some Duchenne patients.
Statistical analysis of sparse infection data and its implications for retroviral treatment trials in primates.
Authors: Spouge J L
Published: May 2006

Reports on retroviral primate trials rarely publish any statistical analysis. Present statistical methodology lacks appropriate tests for these trials and effectively discourages quantitative assessment. This paper describes the theory behind VACMAN, a user-friendly computer program that calculates statistics for in vitro and in vivo infectivity data. VACMAN's analysis applies to many retroviral trials using i.v. challenges and is valid whenever the viral dose-response curve has a particular shape. Statistics from actual i.v. retroviral trials illustrate some unappreciated principles of effective animal use: dilutions other than 1:10 can improve titration accuracy; infecting titration animals at the lowest doses possible can lower challenge doses; and finally, challenging test animals in small trials with more virus than controls safeguards against false successes, "reuses" animals, and strengthens experimental conclusions. The theory presented also explains the important concept of viral saturation, a phenomenon that may cause in vitro and in vivo titrations to agree for some retroviral strains and disagree for others.
Regional mapping of prion proteins in brain.
Authors: Taraboulos A , Jendroska K , Serban D , Yang S L , DeArmond S J , Prusiner S B
Published: May 2006

Scrapie is characterized by the accumulation of a protease-resistant isoform of the prion protein PrPSc. Limited proteolysis and chaotropes were used to map the distribution of PrPSc in cryostat sections of scrapie-infected brain blotted onto nitrocellulose membranes, designated histoblots. Proteolysis was omitted in order to map the cellular isoform of the prion protein (PrPC) in uninfected brains. Compared with immunohistochemistry, histoblots increased the sensitivity for PrPSc detection and showed different patterns of PrPSc accumulation. In Syrian hamsters with Sc237 scrapie, the most intense PrPSc signals occurred in sites with relatively little PrPC, suggesting that aberrant localization of prion protein may be an important feature in the pathogenesis of prion diseases. Immunostaining of PrPSc in white-matter tracts suggested that prions spread along neuroanatomical pathways. PrPSc immunostaining in histoblots was quantitated by densitometry, permitting assessment of the extent of PrPSc accumulation within specific structures. Histoblots were also useful in localizing PrPCJD and beta/A4-amyloid peptide in the brains of patients with Creutzfeldt-Jakob disease and Alzheimer disease, respectively.
Selective expression of interleukin 10, interferon gamma, and granulocyte-macrophage colony-stimulating factor in ovarian cancer biopsies.
Authors: Pisa P , Halapi E , Pisa E K , Gerdin E , Hising C , Bucht A , Gerdin B , Kiessling R
Published: May 2006

The variable clinical response seen with most cancer immunotherapy suggests that there is a large interindividual variation in immunologic response to tumors. One of the key functional parameters of an immune response is the local production of cytokines. As a method to survey the immune status of tumor-infiltrating cells, we have investigated the constitutive expression of cytokine mRNA in biopsies from epithelial ovarian carcinomas by using a PCR-assisted mRNA amplification assay. Using a set of cytokine-specific primers for 10 different cytokines, we have found selective expression of interleukin 10 (IL-10), granulocyte-macrophage colony-stimulating factor, and interferon gamma mRNA in ovarian tumor tissue as compared to normal ovaries and ovarian tumor cell lines. Such differences could not be explained by the extent of T-cell infiltration, since comparing samples with the same intensity of T-cell receptor (TCR) constant region alpha-chain product from the tumor and normal biopsies demonstrated different cytokine patterns. No IL-2 gene expression was detected in the tumor biopsies. IL-2 mRNA, however, became expressed after stimulation of the tumor-derived cells via the CD3 molecule but not after growth in recombinant IL-2 alone. Using the same methodology, we also analyzed the TCR variable region beta-chain gene repertoire. No restriction or biased expression of these genes was observed.
pH transients evoked by excitatory synaptic transmission are increased by inhibition of extracellular carbonic anhydrase.
Authors: Chen J C , Chesler M
Published: May 2006

Excitatory synaptic transmission has been associated with a rapid alkalinization of the brain extracellular space. These pH shifts are markedly increased by acetazolamide, an inhibitor of carbonic anhydrase. Although this effect can be readily explained by inhibition of extracellular carbonic anhydrase, this enzyme has been considered strictly intracellular in the central nervous system. To determine whether these alkaline shifts are regulated by extracellular carbonic anhydrase, we studied the effects of a membrane impermeant, dextran-bound inhibitor of this enzyme. Extracellular alkaline transients, measured with pH-sensitive microelectrodes, were generated in the CA1 region of rat hippocampal slices by repetitive electrical stimulation of Schaeffer collateral fibers or by local ejection of glutamate. More direct alkalinizations were elicited by focal ejection of NaOH in the vicinity of a pH microelectrode. These pH transients were reversibly enhanced by addition of the dextran-bound inhibitor. We conclude that there is significant carbonic anhydrase activity in the extracellular space of the brain. We postulate that this enzyme functions in the regulation and modulation of extracellular pH transients associated with neuronal activity.
Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function.
Authors: Tiruppathi C , Malik A B , Del Vecchio P J , Keese C R , Giaever I
Published: May 2006

We have developed an electrical method to study endothelial cell shape changes in real time in order to examine the mechanisms of alterations in the endothelial barrier function. Endothelial shape changes were quantified by using a monolayer of endothelial cells grown on a small (10(-3) cm2) evaporated gold electrode and measuring the changes in electrical impedance. Bovine pulmonary microvessel endothelial cells and bovine pulmonary artery endothelial cells were used to study the effects of alpha-thrombin on cell-shape dynamics by the impedance measurement. alpha-Thrombin produced a dose-dependent decrease in impedance that occurred within 0.5 min in both cell types, indicative of retraction of endothelial cells and widening of interendothelial junctions because of "rounding up" of the cells. The alpha-thrombin-induced decrease in impedance persisted for approximately 2 hr, after which the value recovered to basal levels. Pretreatment of endothelial cells with the protein kinase C inhibitor, calphostin C, or with 8-bromoadenosine 3',5'-cyclic monophosphate prevented the decreased impedance, suggesting that the endothelial cell change is modulated by activation of second-messenger pathways. The alpha-thrombin-induced decrease in impedance was in agreement with the previously observed increases in transendothelial albumin permeability and evidence of formation of intercellular gaps after alpha-thrombin challenge. The impedance measurement may be a valuable in vitro method for the assessment of mechanisms of decreased endothelial barrier function occurring with inflammatory mediators. Since the rapidly occurring changes in endothelial cell shape in response to mediators such as thrombin are mediated activation of second-messenger pathways, the ability to monitor endothelial cell dynamics in real time may provide insights into the signal-transduction events mediating the increased endothelial permeability.
Equilibrium and kinetic constants for the thiol-disulfide interchange reaction between glutathione and dithiothreitol.
Authors: Rothwarf D M , Scheraga H A
Published: May 2006

The equilibrium and rate constants for the reaction between oxidized and reduced glutathione and oxidized and reduced dithiothreitol have been determined at several pH values and temperatures. The measurements involve approach to equilibrium from both directions, quenching of the reaction by lowering the pH or by addition of methyl methanethiosulfonate, separation of reactants and products by reverse-phase HPLC, and determination of their concentrations. Analysis of reaction mixtures was carried out at various times to assure that equilibrium had been reached and to determine kinetic constants prior to the attainment of equilibrium.
A region of herpes simplex virus VP16 can substitute for a transforming domain of Epstein-Barr virus nuclear protein 2.
Authors: Cohen J I
Published: May 2006

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is essential for EBV-induced B-cell transformation in vitro. EBNA-2 contains a 14-amino acid domain that directly activates transcription and is required for transformation. To determine whether another transcriptional activator can substitute for this function, a chimeric virus was constructed that contained a portion of the transcriptional activation domain from the herpes simplex virus VP16 protein inserted in place of the 14-amino acid domain of EBNA-2. The chimeric virus was able to transform B cells efficiently and transactivate expression of EBV and B-cell genes. Randomization of the 14-amino acid sequence in the domain markedly reduced its transcriptional activating activity and the transforming efficiency of the recombinant EBV. Mutation of a tryptophan within the 14-amino acid domain of EBNA-2 completely abolished transcriptional activation and B-cell transformation. These experiments indicate that EBNA-2 and VP16 activate transcription by similar mechanisms and that transcriptional activation is required for EBV-induced B-cell transformation.
Binding of the Drosophila transformer and transformer-2 proteins to the regulatory elements of doublesex primary transcript for sex-specific RNA processing.
Authors: Inoue K , Hoshijima K , Higuchi I , Sakamoto H , Shimura Y
Published: May 2006

Sex-specific alternative processing of double-sex (dsx) precursor messenger RNA (pre-mRNA) is one of the key steps that regulates somatic sexual differentiation in Drosophila melanogaster. By transfection analyses using dsx minigene constructs, we identified six copies of the 13-nucleotide sequences TC(T/A)(T/A)C(A/G)ATCAACA in the female-specific fourth exon that act as the cis elements for the female-specific splicing of dsx pre-mRNA. UV-crosslinking experiments revealed that both female-specific transformer (tra) and transformer-2 (tra-2) products bind to the 13-nucleotide sequences of dsx pre-mRNA. These results strongly suggest that the female-specific splicing of dsx pre-mRNA is activated by binding of these proteins to the 13-nucleotide sequences.
Simian virus 40 large tumor antigen alone or two cooperating oncogenes convert REF52 cells to a state permissive for gene amplification.
Authors: Perry M E , Commane M , Stark G R
Published: May 2006

Gene amplification is characteristic of tumors and continuous cell lines but not of primary, normal, diploid, senescing cells. However, the rat cell line REF52, which resembles primary cells in requiring expression of cooperating oncogenes for transformation, is unusual among cell lines as it is not permissive for amplification. REF52 cells did not form colonies in N-(phosphonacetyl)-L-aspartate (PALA), a drug for which the only known mechanism of resistance is amplification of the carbamoylphosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD) gene. Colonies did form in a low concentration of methotrexate but did not contain amplified dihydrofolate reductase genes. Expression of two cooperating oncogenes in REF52 cells converted them to a state permissive for amplification. Cells expressing only the 12S E1A mRNA of adenovirus 5 did not give rise to PALA-resistant colonies, but expression of an activated ras gene together with E1A readily allowed the cells to form resistant colonies in which the CAD gene was amplified. Cells expressing E1A plus ras were fully transformed, but expression of simian virus 40 large tumor antigen alone converted REF52 cells to a state permissive for amplification without transforming them fully. The ability to manipulate gene amplification in REF52 cells by expression of oncogenes should contribute to an understanding of the nature of the permissive state.
Composition of transcription factor B-TFIID.
Authors: Timmers H T , Meyers R E , Sharp P A
Published: May 2006

Initiation of transcription by RNA polymerase II requires a TFIID factor, which can recognize the TATA element common to many promoters. Two distinct multisubunit TFIID factors can be resolved from extracts of mammalian cells, and both of them contain the well-characterized TATA-binding protein (TBP) and are capable of supporting RNA polymerase II transcription in an in vitro reaction system. The smaller complex, B-TFIID, was purified and its subunit composition was determined. B-TFIID consists of two subunits: the TBP and a TBP-associated factor (TAF) of 170 kDa. This TAF is specific for B-TFIID and appears not to be present in the D-TFIID complex. Furthermore, it was found that the highly purified B-TFIID fractions have (d)ATPase activity.
Glucose and glucosamine regulate growth factor gene expression in vascular smooth muscle cells.
Authors: McClain D A , Paterson A J , Roos M D , Wei X , Kudlow J E
Published: May 2006

We have investigated the regulation of the expression of two growth factors found in vascular smooth muscle, transforming growth factor alpha (TGF alpha) and basic fibroblast growth factor (bFGF). Cells cultured in medium containing 30 mM glucose exhibited a 2-fold increase in TGF alpha mRNA and a 3-fold increase in bFGF mRNA compared with cells grown in normal (5.5 mM) glucose. Glucosamine was more potent than glucose, leading to a 6-fold increase in TGF alpha mRNA. TGF alpha protein levels were also increased by glucosamine treatment, and the predominant species present was the membrane-bound precursor form of TGF alpha. To examine further the regulation of growth factors by sugars, cultured rat aortic smooth muscle cells were transfected with a plasmid construct consisting of a 1.2-kilobase-pair fragment of the TGF alpha promoter linked to a luciferase reporter gene. Increasing the concentration of glucose in the culture medium from 5.5 mM to 30 mM led to a rapid, 1.7-fold increase in the activity of the TGF alpha promoter. Glucosamine was much more potent than glucose in this stimulation, with 2 mM glucosamine causing a 12-fold increase in TGF alpha promoter activity. Insulin had no effect on luciferase activity in either the presence or the absence of added sugars. The glucose response element of the TGF alpha gene maps to a 130-base-pair segment that includes three potential binding sites for the transcription factor Sp1. We conclude that high glucose concentrations such as are reached in diabetes mellitus can stimulate the transcription of the genes for growth factors in vascular smooth muscle cells. This signaling pathway apparently involves the metabolism of glucose to glucosamine. This effect could be representative of nutritional regulation of a family of genes and could contribute to the toxicity of hyperglycemia and the vascular complications of diabetes.
Identification, characterization, and localization of the dopamine D3 receptor in rat brain using 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin.
Authors: Lévesque D , Diaz J , Pilon C , Martres M P , Giros B , Souil E , Schott D , Morgat J L , Schwartz J C , Sokoloff P
Published: May 2006

We have identified 7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin ([3H]7-OH-DPAT) as a selective probe for the recently cloned dopamine D3 receptor and used it to assess the presence of this receptor and establish its distribution and properties in brain. In transfected Chinese hamster ovary (CHO) cells, it binds to D3 receptors with subnanomolar affinity, whereas its affinity is approximately 100-, 1000-, and 10,000-fold lower at D2, D4, and D1 receptors, respectively. Specific [3H]7-OH-DPAT binding sites, with a Kd of 0.8 nM and a pharmacology similar to those at reference D3 receptors of CHO cells, were identified in rat brain. D3 receptors differ from D2 receptors in brain by their lower abundance (2 orders of magnitude) and distribution, restricted to a few mainly phylogenetically ancient areas--e.g., paleostriatum and archicerebellum--as evidenced by membrane binding are autoradiography studies. Native D3 receptors in brain are characterized by an unusually high nanomolar affinity for dopamine and a low modulatory influence of guanyl nucleotides on agonist binding. These various features suggest that D3 receptors are involved in a peculiar mode of neurotransmission in a restricted subpopulation of dopamine neurons.
Canonical bases in tensor products.
Authors: Lusztig G
Published: May 2006

I construct a canonical basis in the tensor product of a simple integrable highest weight module with a simple integrable lowest weight module of a quantized enveloping algebra. This basis is simultaneously compatible with many submodules of the tensor product. As an application, I obtain a construction of a canonical basis of (a modified form of) the quantized enveloping algebra.
Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor.
Authors: Burbach K M , Poland A , Bradfield C A
Published: May 2006

A cDNA encoding the murine Ah receptor (Ahb-1 allele for aromatic hydrocarbon responsiveness) has been isolated and characterized. Analysis of the deduced protein sequence revealed a region with similarity to the basic region/helix-loop-helix (BR/HLH) motif found in many transcription factors that undergo dimerization for function. In addition to the BR/HLH domain, the N-terminal domain of the Ah receptor has extensive sequence similarity to the human ARNT (aryl hydrocarbon receptor nuclear translocator) protein and two regulatory proteins of Drosophila, Sim and Per. Photoaffinity labeling and peptide mapping studies indicate that the Ah receptor binds agonist at a domain that lies within this conserved N-terminal domain. The Ah receptor appears to be a ligand-activated transcription factor with a helix-loop-helix motif similar to those found in a variety of DNA-binding proteins, including Myc and MyoD.
Leukemia inhibitory factor is expressed by the preimplantation uterus and selectively blocks primitive ectoderm formation in vitro.
Authors: Shen M M , Leder P
Published: May 2006

Among its many activities, leukemia inhibitory factor (LIF) can maintain embryonic stem cell monolayers in a pluripotent undifferentiated state. Presuming that this might reflect its physiologic role during embryogenesis, we have examined LIF expression in the embryonic environment by RNase protection assays and have determined its in vitro effect on differentiating embryonic stem cell embryoid bodies. Of all adult tissues analyzed, LIF transcripts appear only in the uterus, where their level fluctuates with the estrous cycle, peaking after ovulation. LIF expression continues in the uteri of pregnant and pseudopregnant females, with a relative peak when blastocysts are normally present. As for its effects on in vitro differentiation, we have found that LIF blocks embryoid body differentiation only partially, yet in a precise manner. Using molecular markers to follow the differentiation of defined cell types, we demonstrate that LIF selectively inhibits the formation of primitive ectoderm, while permitting the differentiation of primitive endoderm. These results suggest a specific role for LIF in preimplantation mouse development.
Mechanism of C8 alkylation of guanine residues by activated arylamines: evidence for initial adduct formation at the N7 position.
Authors: Humphreys W G , Kadlubar F F , Guengerich F P
Published: May 2006

Aromatic amines are bioactivated to electrophilic compounds that react with DNA, predominantly at the C8 position of guanine bases. This site is weakly nucleophilic and it has been proposed that the C8 adduct is the final product after initial N7-adduct formation. To consider this possibility, we reacted several C8-substituted guanine derivatives with N-acetoxy-2-aminofluorene, prepared in situ from 2-acetylsalicylic acid and N-hydroxy-2-aminofluorene. With C8,N9-dimethylguanine, an adduct was isolated in good yield that was consistent, by NMR and mass spectral characterization, with a structure involving carcinogen substitution at the N7 position of guanine and linked through the 2-aminofluorenyl nitrogen--N-(C8,N9- dimethylguanin-N7-yl)-2-aminofluorene. This adduct could be easily reduced with NaBH4, consistent with the proposed N7-adduct structure. The same reaction was also carried out with C8-methylguanosine and C8-methyldeoxyguanosine and similar adducts were isolated. In contrast, C8-bromoguanosine reacted with N-acetoxy-2-aminofluorene to yield the C8-substituted arylamine adduct N-(guanosin-C8-yl)-2-aminofluorene directly. These products are uniquely consistent with a scheme in which C8-adduct formation is preceded by an initial electrophilic substitution on the N7 atom, which is postulated to be a general reaction for activated arylamines and heterocyclic amines.
Endothelin regulation of neuropeptide release from nerve endings of the posterior pituitary.
Authors: Ritz M F , Stuenkel E L , Dayanithi G , Jones R , Nordmann J J
Published: May 2006

We have investigated the role of endothelin (ET) in the stimulus-secretion coupling mechanism in the posterior pituitary. We report that isolated nerve endings contain immunoreactive endothelin, the level of which is regulated by homeostatic mechanisms involved in control of water balance. ET-1 and ET-3 potentiate vasopressin release induced by depolarization through interaction with specific receptors of the ETA subtype and this response is antagonized by sarafotoxin S6b. The second messenger for this effect, however, remains unknown since the potentiation of depolarization-induced vasopressin release occurs in the absence of an increase in cellular calcium.
Human monoclonal islet cell antibodies from a patient with insulin-dependent diabetes mellitus reveal glutamate decarboxylase as the target antigen.
Authors: Richter W , Endl J , Eiermann T H , Brandt M , Kientsch-Engel R , Thivolet C , Jungfer H , Scherbaum W A
Published: May 2006

The autoimmune phenomena associated with destruction of the beta cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.