bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the Na+, K(+)-ATPase in Narcine brasiliensis.
Authors: Blum H , Nioka S , Johnson R G
Published: May 2006

The in vivo activation and turnover rates of the sodium pump (Na+, K(+)-ATPase) were investigated in the electrocytes of the electric organ of the elasmobranch Narcine brasiliensis. The Narcine electric organ appears to be an excellent model for the study of sodium pump activation in an excitable tissue. The sodium transmembrane gradient and high-energy phosphagens were concurrently measured by 23Na and 31P NMR spectroscopy. The resting electric organ, which depends primarily on anaerobic metabolism, displays a high concentration of phosphocreatine (PCr). It has an intracellular sodium concentration ([Na+]i) of 20 +/- 10 milliequivalents/liter as estimated by NMR. Electrical stimulation of the nerves innervating the electric organ results in an increase in [Na+]i in the electrolyte and rapid depletion of PCr. Ouabain causes an 85% decrease in utilization of high-energy phosphagens, indicating that rapid PCr turnover in this tissue is mainly due to Na+, K(+)-ATPase activity. From these data we can determine that the rate of sodium pump turnover increases by greater than 3 orders of magnitude within several hundred milliseconds. In excised unstimulated electric organ slices, changes in [Na+]i equivalent to those occurring with stimulation, but induced by hyperosmolar conditions, do not result in increased PCr hydrolysis. We conclude that cholinergic stimulation of the electric organ causes a rapid and extremely large increase in sodium pump turnover, which is regulated predominantly by factors other than [Na+]i.
Use of nonradioactive 2-deoxyglucose to study compartmentation of brain glucose metabolism and rapid regional changes in rate.
Authors: McDougal D B , Ferrendelli J A , Yip V , Pusateri M E , Carter J G , Chi M M , Norris B , Manchester J , Lowry O H
Published: May 2006

A method is presented for measuring rapid changes in the rate of glucose phosphorylation in mouse brain with nonradioactive 2-deoxyglucose (DG). After times as short as 1 min after DG injection, the mouse is frozen rapidly, and selected brain regions are analyzed enzymatically for DG, 2-deoxyglucose 6-phosphate (DG6P), and glucose. The rate of glucose phosphorylation can be directly calculated from the rate of change in DG6P, the average levels of DG and glucose, and a constant derived from direct comparison of the rate of changes in glucose and DG6P after decapitation. Experiments with large brain samples provided evidence for a 2% per min loss of DG6P and at least two compartments differing in their rates of glucose metabolism, one rapidly entered by DG with glucose phosphorylation almost double that of average brain and another more slowly entered with a much lower phosphorylation rate. The method is illustrated by changes in phosphorylation within 2 min after injection of a convulsant or an anesthetic and over a 48-min time course with and without anesthesia. The sensitivity of the analytical methods can be amplified as much as desired by enzymatic cycling. Consequently, the method is applicable to very small brain samples. Examples are given for regions with volumes of 5 x 10(-4) microliters, but studies with samples as small as single large cell bodies are feasible.
Identification of five putative yeast RNA helicase genes.
Authors: Chang T H , Arenas J , Abelson J
Published: May 2006

The RNA helicase gene family encodes a group of eight homologous proteins that share regions of sequence similarity. This group of evolutionarily conserved proteins presumably all utilize ATP (or some other nucleoside triphosphate) as an energy source for unwinding double-stranded RNA. Members of this family have been implicated in a variety of physiological functions in organisms ranging from Escherichia coli to human, such as translation initiation, mitochondrial mRNA splicing, ribosomal assembly, and germinal line cell differentiation. We have applied polymerase chain reaction technology to search for additional members of the RNA helicase family in the yeast Saccharomyces cerevisiae. Using degenerate oligonucleotide primers designed to amplify DNA fragments flanked by the highly conserved motifs V L D E A D and Y I H R I G, we have detected five putative RNA helicase genes. Northern and Southern blot analyses demonstrated that these genes are single copy and expressed in yeast. Several members of the RNA helicase family share sequence identity ranging from 49.2% to 67.2%, suggesting that they are functionally related. The discovery of such a multitude of putative RNA helicase genes in yeast suggests that RNA helicase activities are involved in a variety of fundamentally important biological processes.
Hemimethylation of DNA prevents chromatin expression.
Authors: Deobagkar D D , Liebler M , Graessmann M , Graessmann A
Published: May 2006

The activity of hemimethylated herpes simplex virus thymidine kinase DNA and chromatin was analyzed by microinjection and thymidine incorporation into the DNA of thymidine kinase-negative Rat2 cells. Hemimethylated DNA was obtained by in vitro replication of single-stranded M13 DNA constructs and of chromatin produced by in vitro reconstitution of the DNA with purified chicken histone octamers. We found that methylation of either the coding or the noncoding DNA strand was sufficient to block expression of the hemimethylated chromatin. In contrast, the hemimethylated DNA was as active as the unmethylated control DNA after microinjection until chromatin formation occurred in the recipient cells. Microinjection of chromatin hemimethylated by bacterial Hae III methyltransferase excluded the possibility that inactivation was caused by symmetrical methylation of the injected molecules.
lac repressor: crystallization of intact tetramer and its complexes with inducer and operator DNA.
Authors: Pace H C , Lu P , Lewis M
Published: May 2006

The intact lac repressor tetramer, which regulates expression of the lac operon in Escherichia coli, has been crystallized in the native form, with an inducer, and in a ternary complex with operator DNA and an anti-inducer. The crystals without DNA diffract to better than 3.5 A. They belong to the monoclinic space group C2 and have cell dimensions a = 164.7 A, b = 75.6 A, and c = 161.2 A, with alpha = gamma = 90 degrees and beta = 125.5 degrees. Cocrystals have been obtained with a number of different lac operator-related DNA fragments. The complex with a blunt-ended 16-base-pair strand yielded tetragonal bipyramids that diffract to 6.5 A. These protein-DNA cocrystals crack upon exposure to the gratuitous inducer isopropyl beta-D-thiogalactoside, suggesting a conformational change in the repressor-operator complex.
An adenovirus E4 gene product trans-activates E2 transcription and stimulates stable E2F binding through a direct association with E2F.
Authors: Neill S D , Hemstrom C , Virtanen A , Nevins J R
Published: May 2006

The adenovirus E4 gene encodes a trans-activating function that can stimulate the E2 promoter. E2 promoter sequences required for E4 trans-activation are identical to those required for E1A trans-activation, and these principally are the E2 promoter binding factor (E2F) binding sites. Furthermore, full activation of E2F DNA binding activity requires both E1A and E4 action. Analysis of a series of mutant E4 viruses identifies open reading frame (orf) 6/7 of the E4 transcription unit as that required for activation of E2F binding activity. In addition, the assay of various E4 cDNAs demonstrates that the E4 orf 6/7 also is responsible for the trans-activation of E2 transcription. Translation of the E4 orf 6/7 mRNA, but not a control mRNA, in a reticulocyte extract generates an activity that can stimulate cooperative binding of E2F in vitro, consistent with recent in vivo assays that demonstrate a role for the E4 gene in E2F stable complex formation. This stimulation is due to a direct interaction of the E4 protein with E2F since an antibody that recognizes the E4 orf 6/7 polypeptide detects this E4 protein in the E2F-DNA complex. We conclude that the E4 orf 6/7 product interacts with the E2F factor altering binding to allow formation of a stable complex that results in a stimulation of transcription.
Mutations that increase the activity of a transcriptional activator in yeast and mammalian cells.
Authors: Gill G , Sadowski I , Ptashne M
Published: May 2006

Activating region I of GAL4 protein is a stretch of amino acids, positioned adjacent to the DNA-binding region, that activates transcription in yeast and, as we show here, in mammalian cells. Here we describe mutations located throughout a 65-amino acid region that increase the activation function of region I. Most of these mutations replace positively charged amino acids in the region with neutral ones, although we also describe substitutions at one position that do not alter the charge of the region. Mutations of region I that alter the activation function in yeast have similar effects on activation when assayed in mammalian cells. When individual mutations that raise the acidity of the activating region are recombined, the activities of the mutant proteins increase with increasing negative charge in both yeast and mammalian cells. These results extend and modify the correlation between acidity and activation and suggest that the requirements for a strong activating region are conserved in yeast and mammals.
Defined neurofilament, tau, and beta-amyloid precursor protein epitopes distinguish Alzheimer from non-Alzheimer senile plaques.
Authors: Arai H , Lee V M , Otvos L , Greenberg B D , Lowery D E , Sharma S K , Schmidt M L , Trojanowski J Q
Published: May 2006

Eight antisera and one monoclonal antibody to synthetic peptides that corresponded to domains extending over the entire length of the beta-amyloid precursor protein (beta-APP), and an antiserum to the full-length 695-amino acid form of the beta-APP, were raised to probe the composition of the core and corona of senile plaques (SPs). We localized distinct beta-APP domains, including the beta-amyloid protein or A4 region, within the SPs of 13 end-stage Alzheimer disease (AD) and 13 age-matched control samples of hippocampus and entorhinal cortex. The composition of SPs also was probed with antibodies to defined epitopes in tau (tau) as well as the large and mid-size neurofilament (NF) proteins. The most important observations were that beta-APP domains outside the A4 region were largely restricted to SP coronas in the AD samples, together with tau and NF determinants, whereas the same epitopes were absent from A4-positive blood vessels and exceptionally rare in non-AD SPs. Indeed, samples from a subset of the non-AD cases contained a considerable number of A4-positive SPs totally devoid of any of the other beta-APP, tau, and NF epitopes. These observations suggest that the deposition of the A4 protein in AD SPs results from the local processing of beta-APPs in association with tau and NF protein fragments. It is unclear whether this association is fortuitous or linked by common mechanisms. However, differences between the complement of beta-APP, tau, and NF protein epitopes in AD versus non-AD brains implicate a defect involving one or more steps in the posttranslational modification, degradation, or elimination of these proteins in AD brains, and this may account for the massive numbers of SPs that characterize AD.
Functional analysis of the human tissue factor promoter and induction by serum.
Authors: Mackman N , Fowler B J , Edgington T S , Morrissey J H
Published: May 2006

Tissue factor (TF) is the primary initiator of the coagulation protease cascades. This cell surface glycoprotein is the receptor and essential cofactor for the serine protease factor VIIa. TF is constitutively expressed in some extravascular cell types and is transiently induced in monocytes, endothelial cells, and fibroblasts. Inducible expression is implicated in cellular immune responses, inflammation, and intravascular coagulation. Transcriptional regulation of the TF promoter was analyzed in COS-7 cells under conditions of (i) high-level expression and (ii) serum induction. The region comprising nucleotides -209 to +121 (relative to the transcription start site) supports high-level transcriptional activity and can be divided into two distinct regions: a region (-111 to +121) that exhibited low promoter activity and a region (-209 to -112) that enhanced transcriptional activity to a high level. The role of further upstream sequences is still to be established, although two consensus binding sites for the transcriptional activator protein AP-1 did enhance low-level promoter activity. In serum-starved COS-7 cells TF expression was transiently increased 20-fold by serum. All transcriptionally active constructs were responsive to serum, indicating the presence of at least one serum response element, whose function was retained in the immediate 5' aspect of the gene, at -111 to +14. Based on this functional map, we propose that the elaborate pattern of TF expression by cells results from a relatively complex promoter.
Tissue and strain-specific patterns of endogenous proviral hypomethylation analyzed by two-dimensional gel electrophoresis.
Authors: Mietz J A , Kuff E L
Published: May 2006

Proviral sequences related to the intracisternal A particle (IAP) are amplified and dispersed in the mouse genome. Their expression is associated with hypomethylation at CpG sites in the 5' long terminal repeat. We have used two-dimensional agarose gel electrophoresis to examine patterns of IAP hypomethylation in mouse DNA. The method is sensitive to both the methylation status of a conserved Hae II site in the 5' long terminal repeat and the location of the closest BamHI site in the flanking DNA upstream of each hypomethylated long terminal repeat. The method also defects restriction fragments derived from IAP elements that are themselves methylated but have an unmethylated Hae II site in their 5' adjacent DNA. DNAs from each of four inbred mouse strains (BALB/c, C3H/He, C57BL/6, and DBA/2) gave distinctive two-dimensional patterns of BamHI/Hae II restriction fragments detected by hybridization with an IAP probe. This constitutive pattern was largely conserved among several tissues of each strain, but some tissue-specific variations were observed. The site-specific hypomethylations reflected in the two-dimensional patterns were heritable properties, since DNA from progeny of an interstrain cross contained both parental sets of fragments. IAP elements may be useful endogenous reporters of genomic methylation patterns.
Engrailed, a homeodomain protein, can repress in vitro transcription by competition with the TATA box-binding protein transcription factor IID.
Authors: Ohkuma Y , Horikoshi M , Roeder R G , Desplan C
Published: May 2006

Engrailed (En) is a homeodomain protein that binds to a consensus sequence (NP) and plays an important role during Drosophila development. Purified En, which is produced in Escherichia coli, binds not only to this consensus sequence but also to the TATA box of the Drosophila Hsp70 promoter and of other eukaryotic promoters. Interestingly, En represses transcription of these promoters in an in vitro-reconstituted mammalian transcription system and footprint analyses show that En competes with the TATA box-binding protein transcription factor IID for binding to the TATA box. In contrast, a stable template-committed complex formed by preincubation of transcription factor IID with the promoter is not disrupted by addition of En, and in this case transcription is not repressed. These in vitro studies suggest a transcriptional repression mechanism, involving competition between En and transcription factor IID for TATA box binding, that may be involved in En-mediated repression in vivo.
The regulatory subunit of Escherichia coli aspartate carbamoyltransferase may influence homotropic cooperativity and heterotropic interactions by a direct interaction with the loop containing residues 230-245 of the catalytic chain.
Authors: Newton C J , Kantrowitz E R
Published: May 2006

A recent x-ray structure of aspartate carbamoyltransferase (carbamoyl-phosphate: L-aspartate carbamoyl-transferase, EC 2.1.3.2) with phosphonoacetamide bound [Gouaux, J. E. & Lipscomb, W. N. (1990) Biochemistry 29, 389-402] shows an interaction between Asp-236 of the catalytic chain and Lys-143 of the regulatory chain. Asp-236 is part of the loop containing residues 230-245 (240s) of the catalytic chain that undergoes a significant conformational change between the tight and the relaxed states of the enzyme. Furthermore, side-chain interactions between the 240s loop and other portions of the enzyme have been shown to be important for the low activity and low affinity of the tight state and the high activity and high affinity of the relaxed state. To determine whether the intersubunit link between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain is important for either homotropic cooperativity and/or the heterotropic interactions in aspartate carbamoyltransferase, site-specific mutagenesis was used to replace Asp-236 with alanine. The mutant enzyme exhibits full activity and a loss of both homotropic cooperativity and heterotropic interactions. Furthermore, the aspartate concentration at half the maximal observed specific activity is reduced by approximately 8-fold. The mutant enzyme exhibits normal thermal stability but drastically altered reactivity toward p-hydroxymercuribenzoate. The catalytic subunit of the mutant and wild-type enzymes have very similar properties. These results, in conjunction with previous experiments, suggest that the intersubunit link involving Asp-236 is involved in the stabilization of the 240s loop in its tight-state position and that the regulatory subunits exert their effect on the catalytic subunits by influencing the position of the 240s loop.
Characterization of cDNA encoding a human sperm membrane protein related to A4 amyloid protein.
Authors: Yan Y C , Bai Y , Wang L F , Miao S Y , Koide S S
Published: May 2006

A rat testis lambda gt11 cDNA library was screened with a monoclonal antibody raised against a human sperm membrane protein designated YWK-II. A clone was found with a cDNA insert composed of 1837 base pairs that contained an open reading frame coding for 191 amino acid residues. The deduced polypeptide contained a segment with high homology to the transmembrane-cytoplasmic domains of the A4 amyloid protein found in brain plaques of Alzheimer disease patients. A sequence of basic amino acid residues, Arg-Lys-Arg, was found instead of Lys-Lys-Lys at the probable membrane-cytoplasmic junction that may be a unique property of sperm membrane proteins.
Expression of recombinant glutathione S-transferase pi, Ya, or Yb1 confers resistance to alkylating agents.
Authors: Puchalski R B , Fahl W E
Published: May 2006

Increased levels of glutathione S-transferase (GST; RX:glutathione R-transferase; EC 2.5.1.18) mRNA, protein, and activity in tumor biopsy samples and in drug-resistant cultured cells are associated with resistance to anticancer drugs. We report that each of three full-length cloned GST cDNAs, that for pi (acidic), Ya (basic), and Yb1 (neutral), can confer drug resistance when expressed in cultured mammalian cells. In one approach, stably transfected mouse C3H/10T1/2 cells that express GST pi, Ya, or Yb1 were cloned and analyzed for drug resistance in colony-forming assays. Transiently transfected COS cells that were sorted on a fluorescence-activated cell sorter were used in the second approach to avoid interclonal variation in factors other than the recombinant GST and to show that reversion of transient GST expression correlated with loss of drug resistance. A sorting technique, developed to separate the 20% of the electroporated COS cell population that transiently expressed GST pi, Ya, or Yb1 from the nonexpressing population, was based on a GST-catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane. GST Ya conferred the greatest increase in resistance to chlorambucil and melphalan (1.3- to 2.9-fold), Yb1 conferred the greatest increase in resistance to cisplatin (1.5-fold), and pi conferred the greatest increase in resistance to a racemic mixture of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene and 7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene and doxorubicin (1.5- and 1.3-fold) relative to controls. These resistance values to alkylating agents are commensurate with values observed clinically. Cytotoxicity curves representing recombinant GST+ populations were significantly different from their controls with P values ranging from 0.005 to 0.0001. No resistance to vinblastine was detected. Conferred drug resistance was proportional to the magnitude of GST Ya expression, and reversion of transient expression in GST Ya+ COS cell clones to a GST Ya- phenotype was associated with total loss of drug resistance.
Cloned manganese superoxide dismutase reduces oxidative stress in Escherichia coli and Anacystis nidulans.
Authors: Gruber M Y , Glick B R , Thompson J E
Published: May 2006

The Mn superoxide dismutase gene of Escherichia coli was subcloned into the E. coli-Anacystis nidulans shuttle vector pSG111 to make the plasmid pMYG1. Transformation of E. coli HB101 with pMYG1 resulted in a 6-fold increase in superoxide dismutase activity. There was also induction of Mn superoxide dismutase in the transformants upon exposure to paraquat, as evidenced by dramatically increased levels of the Mn superoxide dismutase polypeptide in cytoplasmic extracts and a 16-fold further increase in superoxide dismutase activity. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth. Anacystis nidulans, a cyanobacterium that has no detectable Mn superoxide dismutase and is, consequently, very sensitive to oxidative stress, was also transformed with pMYG1. The transformants had detectable levels of Mn superoxide dismutase protein and showed resistance to paraquat-mediated inhibition of growth and photobleaching of pigments. Paraquat is known to promote formation of the superoxide radical anion, O2-., and thus the data have been interpreted as indicating that the cloned Mn superoxide dismutase provides protection in both E. coli and A. nidulans against damage attributable to O2-..
Evidence that the synthesis of glucosylphosphodolichol in yeast involves a 35-kDa membrane protein.
Authors: Palamarczyk G , Drake R , Haley B , Lennarz W J
Published: May 2006

In an effort to identify the polypeptide chain of glucosylphosphodolichol synthase (EC 2.4.1.117), yeast microsomal membranes were allowed to react with 5-azido[beta-32P]UDPGlc, a photoactive analogue of UDPGlc, which is a substrate for this enzyme. Upon photolysis the 32P-labeled probe was shown to link covalently to a 35-kDa protein present in microsomal membranes prepared from several wild-type yeast strains. Binding was either reduced or absent in the microsomal membranes from two yeast mutants (alg5 and dpg1) that are known to be defective in the synthesis of glucosylphosphodolichol. The microsomes isolated from a heterozygous diploid strain alg5::dpg1 generated from these two mutants exhibited partial restoration of both the ability to photolabel the 35-kDa protein and the ability to catalyze the synthesis of glucosylphosphodolichol. Microsomal membranes from a mutant strain that synthesized glucosylphosphodolichol but lacked the ability to transfer the glucosyl residue to the growing lipid-linked oligosaccharide (alg6) exhibited labeling with 5-azido[beta-32P]UDPGlc comparable to that found in microsomes from the wild-type strain. In all cases photoinsertion of the probe into the 35-kDa protein correlated with the level of synthase assayed in the microsomal membranes. These results strongly support the conclusion that the 35-kDa protein labeled in these experiments is a component of glucosylphosphodolichol synthase.
Cloning of a cDNA for a major human protein-tyrosine-phosphatase.
Authors: Chernoff J , Schievella A R , Jost C A , Erikson R L , Neel B G
Published: May 2006

We have isolated a cDNA clone encoding the major protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) of human placenta. Degenerate oligonucleotides, based on the amino acid sequence of the protein, were used to amplify an internal fragment of the gene from human placental cDNA by the polymerase chain reaction. This fragment was then used to probe a human placental cDNA library. A 3.3-kilobase (kb) insert was isolated and sequenced. The insert has a single extended open reading frame that predicts a 435 amino acid protein of Mr approximately 50,000. From the amino terminus to residue 321, the deduced amino acid sequence is identical to that previously determined by peptide sequencing [Charbonneau, H., Tonks, N. K., Kumar, S., Diltz, C. D., Harrylock, M., Cool, D. E., Krebs, E. G., Fischer, E. H. & Walsh, K. A. (1989) Proc. Natl. Acad. Sci. USA 86, 5252-5256]; however, the sequence predicts that the protein contains an additional 114 amino acids not present in the reported peptide sequence. In vitro translation of the 3.3-kb insert produces a protein of Mr 56,000, in general agreement with the predicted size. The phosphatase gene appears to be present as a single copy in human genomic DNA and is transcribed into a 3.5-kb message in a variety of tissues.
Rapid cloning of HLA-A,B cDNA by using the polymerase chain reaction: frequency and nature of errors produced in amplification.
Authors: Ennis P D , Zemmour J , Salter R D , Parham P
Published: May 2006

A method for cloning full-length HLA-A,B cDNA (1.1 kilobases) by using the polymerase chain reaction (PCR) is described. Six HLA-A,B alleles (HLA-A2, -A25, -B7, -B37, -B51, and -B57) were cloned, and their structures were determined. Multiple PCR clones for each allele were sequenced to obtain both an accurate consensus sequence and an "authentic" clone having that sequence. Sequences from 50 clones encoding five different alleles permit assessment of the frequency and nature of PCR-produced errors. These include recombinations, deletions, and insertions in addition to point substitutions. Authentic clones were obtained at a frequency of between 30% and 70%, and analysis of three or four clones generally should be sufficient for characterization of an allele.
Fetal IgG specificities against Trypanosoma cruzi antigens in infected newborns.
Authors: Reyes M B , Lorca M , Muñoz P , Frasch A C
Published: May 2006

A panel of Trypanosoma cruzi antigens produced by recombinant DNA techniques was used to analyze the IgM and IgG specificities present in sera from 22 mothers with chronic Chagas disease and their newborn infants. Ten of the newborns were congenitally infected and the other 12 children were healthy. While in most cases IgG specificities in the newborns mirrored those of their mothers, congenitally infected newborns had, in addition, IgG specificities that were undetectable in their mothers. The new IgG specificities observed most frequently were against a shed acute-phase antigen (SAPA), and less frequently, against other nine different parasite antigens. Thus, SAPA is able to identify new fetal IgGs because antibodies against this antigen are generated during the acute phase of the infection and not in their chronically infected mothers. Sera from congenital cases also had IgMs against several parasite antigens, but again, SAPA was the most frequently detected. Neither IgMs nor new IgG specificities were detected in healthy children born to mothers with Chagas disease. We conclude that individual antigens can be used to detect new IgG specificities present in the cord blood from infected newborns. Furthermore, detection of IgMs and new fetal IgGs with recombinant antigens may be used to sort out congenitally infected infants from uninfected ones, a method that might be applied to other infectious diseases.
Conversion of a helix-turn-helix motif sequence-specific DNA binding protein into a site-specific DNA cleavage agent.
Authors: Ebright R H , Ebright Y W , Pendergrast P S , Gunasekera A
Published: May 2006

Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. & Buc, H. (1984) Science 224, 831-838; and Pabo, C. & Sauer, R. (1984) Annu. Rev. Biochem. 53, 293-321]. In this work, CAP has been converted into a site-specific DNA cleavage agent by incorporation of the chelator 1,10-phenanthroline at amino acid 10 of the helix-turn-helix motif. [(N-Acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP binds to a 22-base-pair DNA recognition site with Kobs = 1 x 10(8) M-1. In the presence of Cu(II) and reducing agent, [(N-acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP cleaves DNA at four adjacent nucleotides on each DNA strand within the DNA recognition site. The DNA cleavage reaction has been demonstrated using 40-base-pair and 7164-base-pair DNA substrates. The DNA cleavage reaction is not inhibited by dam methylation of the DNA substrate. Such semisynthetic site-specific DNA cleavage agents have potential applications in chromosome mapping, cloning, and sequencing.
Involvement of GTP-binding proteins in actin polymerization in human neutrophils.
Authors: Bengtsson T , Särndahl E , Stendahl O , Andersson T
Published: May 2006

The motility of human neutrophils, which is of vital importance for the role of these cells in host defense, is based on rapid and dynamic changes of the filamentous actin F-actin) network. Consequently, to understand how neutrophils move and ingest particles, we need to know how polymerization and depolymerization of actin are regulated. Previous studies by several investigators have, based on indirect evidence obtained with pertussis toxin, suggested a role for GTP-binding protein(s) (G protein) in chemotaxis-induced, but not phagocytosis-induced, reorganization of the F-actin network. The aim of the present investigation was to study the effects of directly activated G proteins (i.e., without prior ligand-receptor complex formation) on the F-actin content in human neutrophils. AlF4- induced a pronounced and sustained increase in F-actin in intact neutrophils. This effect coincided with an increase in cytosolic free Ca2+, indicating that phospholipase C and the subsequent transduction mechanism were also activated. Inhibition of phospholipase C activity by extensive depression of the cytosolic free Ca2+ level (less than 20 nM) only marginally affected the AlF4(-)-induced rise in F-actin content. The major part of the AlF4(-)-induced rise in F-actin content was also resistant to pertussis toxin, suggesting that pertussis toxin-insensitive G proteins in neutrophils are also able to trigger actin polymerization. The specificity of AlF4- in activating G proteins was also tested in permeabilized cells. In this case the effect was more rapid and could be totally abolished by guanosine 5'-[beta-thio]diphosphate. In analogy, in permeabilized cells guanosine 5'-[gamma-thio]triphosphate mimicked the effect of AlF4- on actin polymerization, and the effect induced by this nonhydrolyzable GTP analogue could also be totally abolished by guanosine 5'-[beta-thio]diphosphate. In summary, the present data support our previous hypothesis that G proteins are intimately linked to actin polymerization in human neutrophils.
Use of a tuberculin purified protein derivative--Asn-Ala-Asn-Pro conjugate in bacillus Calmette-Guérin primed mice overcomes H-2 restriction of the antibody response and avoids the need for adjuvants.
Authors: Lussow A R , Del Giudice G , Rénia L , Mazier D , Verhave J P , Verdini A S , Pessi A , Louis J A , Lambert P H
Published: May 2006

Because of its immunodominancy, and because it is conserved in different geographical isolates of Plasmodium falciparum, the repetitive sequence of the circumsporozoite protein, (Asn-Ala-Asn-Pro)n [(NANP)n], has been envisaged for the development of an anti-falciparum malaria subunit vaccine. However, the murine immune response to (NANP)n peptides, either carrier-free or coupled to carrier proteins, was shown to be inducible only by using strong (e.g., Freund's) adjuvants. Furthermore, response to the carrier-free peptide, administered in adjuvant, is genetically restricted to I-Ab mice. In the present paper, we report that high titers of antibodies against the NANP repetitive epitope were obtained in responder C57BL/6 (H-2b) mice when they were primed with live BCG (bacillus Calmette-Guérin Mycobacterium tuberculosis var. bovis) and immunized once with the synthetic peptide (NANP)40 coupled to tuberculin purified protein derivative (PPD) without the use of any adjuvant. This approach also led to the production of high titers of anti-NANP antibodies in ASW (H-2s), B10.RIII (H-2r), BALB/c (H-2d), C3H/He (H-2k), and DBA/1 (H-2q) nonresponder mice after two injections of the conjugate. In both cases, BCG priming was obligatory for the induction of antibodies reacting with the synthetic peptide. The levels of anti-NANP antibodies in nonresponder BALB/c mice were demonstrated to be comparable to the levels induced after PPD-(NANP)40 immunization in Freund's complete or incomplete adjuvant. The antibodies induced were also capable of recognizing P. falciparum sporozoites in immunofluorescence assays and, furthermore, these antibodies inhibited the penetration of live sporozoites into human hepatocytes in vitro. This system functioned independently of the subjects' resistance or susceptibility to BCG infection. Given the widespread natural exposure to mycobacterial antigens and the extensive use of BCG and PPD in the human population, this approach might be envisaged for vaccination with malaria peptides.
A trans-activator function is generated by integration of hepatitis B virus preS/S sequences in human hepatocellular carcinoma DNA.
Authors: Caselmann W H , Meyer M , Kekulé A S , Lauer U , Hofschneider P H , Koshy R
Published: May 2006

The X gene of wild-type hepatitis B virus or integrated DNA has recently been shown to stimulate transcription of a variety of enhancers and promoters. To further delineate the viral sequences responsible for trans-activation in hepatomas, we cloned the single hepatitis B virus insert from human hepatocellular carcinoma DNA M1. The plasmid pM1 contains 2004 base pairs of hepatitis B virus DNA subtype adr, including truncated preS/S sequences and the enhancer element. The X promoter and 422 nucleotides of the X coding region are present. The entire preC/C gene is deleted. In transient cotransfection assays using Chang liver cells (CCL 13), pM1 DNA exerts a 6- to 10-fold trans-activating effect on the expression of the pSV2CAT reporter plasmid. The trans-activation occurs by stimulation of transcription and is dependent on the simian virus 40 enhancer in the reporter plasmid. Deletion analysis of pM1 subclones reveals that the trans-activator is encoded by preS/S and not by X sequences. A frameshift mutation within the preS2 open reading frame shows that this portion is indispensable for the trans-activating function. Initiation of transcription has been mapped to the S1 promoter. A comparable trans-activating effect is also observed with cloned wild-type hepatitis B virus sequences similarly truncated. These results show that a transcriptional trans-activator function not present in the intact gene is generated by 3' truncation of integrated hepatitis B virus DNA preS/S sequences.
Cloning and expression of the delayed-rectifier IsK channel from neonatal rat heart and diethylstilbestrol-primed rat uterus.
Authors: Folander K , Smith J S , Antanavage J , Bennett C , Stein R B , Swanson R
Published: May 2006

cDNAs encoding a delayed-rectifier-type K+ channel were cloned from both neonatal rat heart and ovariectomized, diethylstilbestrol-primed rat uterus by using the polymerase chain reaction. Both clones have nucleotide sequences identical to that encoding the rat kidney IsK channel [Takumi, T., Ohkubo, H. & Nakanishi, S. (1988) Science 242, 1042-1045] and encode a putative protein of 130 amino acids. Injection of RNA transcripts of the cDNAs into Xenopus oocytes resulted in the expression of a slowly activating, voltage-dependent K+ current. An antisense oligonucleotide, derived from the sequence of the clone, specifically inhibited the expression of the slow, outward current observed in cells injected with mRNAs isolated from the parent tissues (i.e., kidney, heart, and uterus), indicating that the cloned gene underlies the major K+ current expressed from RNA isolated from these tissues.
Human trophoblast-extracellular matrix (ECM) interactions in vitro: ECM thickness modulates morphology and proteolytic activity.
Authors: Kliman H J , Feinberg R F
Published: May 2006

Trophoblast invasion of the uterine extracellular matrix, a critical process for human implantation and uteroplacental vascular development, is a striking example of controlled invasiveness. To examine cellular behavior relevant to this process, human trophoblasts were cultured on (i) Millicell filters prelayered with Matrigel and (ii) coverslips precoated with a gentle slope of Matrigel (Matribeach). Histologic sections of the Millicell system demonstrated significant invasion. However, on Matribeach the cells exhibited markedly different characteristics depending on the thickness of the Matrigel. On zone 1 (1-4 microns thick), flat aggregates and syncytia were seen. In contrast, cells on zone 2 (4-14 microns) formed rounded aggregates with intercellular processes. In this zone, prominent degradation of pericellular Matrigel proteins was assessed by both light microscopy and scanning electron microscopy. Treatment with 8-bromo-cAMP inhibited this proteolytic process. On zone 3 (14-60 microns), unicellular trophoblasts or small aggregates caused minimal matrix degradation. JEG-3 human choriocarcinoma cells exhibited similar morphologic and degradative properties on Matribeach, but zone 2 proteolysis was not affected by 8-bromo-cAMP. Our results suggest that extracellular matrix thickness has profound effects on cellular morphology and proteolytic activity. Furthermore, while both normal and malignant human trophoblasts can degrade extracellular matrix proteins, only normal trophoblast extracellular matrix degradation is inhibited by 8-bromo-cAMP.
Somatic diversification of chicken immunoglobulin light chains by point mutations.
Authors: Parvari R , Ziv E , Lantner F , Heller D , Schechter I
Published: May 2006

The light-chain locus of chicken has 1 functional V lambda 1 gene, 1 J gene, and 25 pseudo-V lambda-genes (where V = variable and J = joining). A major problem is which somatic mechanisms expand this extremely limited germ-line information to generate many different antibodies. Weill's group [Reynaud, C. A., Anquez, V., Grimal, H. & Weill, J. C. (1987) Cell 48, 379-388] has shown that the pseudo-V lambda-genes diversify the rearranged V lambda 1 by gene conversion. Here we demonstrate that chicken light chains are further diversified by somatic point mutations and by V lambda 1-J flexible joining. Somatic point mutations were identified in the J and 3' noncoding DNA of rearranged light-chain genes of chicken. These regions were analyzed because point mutations in V lambda 1 are obscured by gene conversion; the J and 3' noncoding DNA are presented in one copy per haploid genome and are not subject to gene conversion. In rodents point mutations occur as frequently in the V-J coding regions as in the adjacent flanking DNA. Therefore, we conclude that somatic point mutations diversify the V lambda 1 of chicken. The frequency (0-1%) and distribution of the mutations (decreasing in number with increased distance from the V lambda 1 segment) in chicken were as observed in rodents. Sequence variability at the V lambda 1-J junctions could be attributed to imprecise joining of the V lambda 1 and J genes. The modification by gene conversion of rearranged V lambda 1 genes in the bursa was similar in chicken aged 3 months (9.5%) or 3 weeks (9.1%)--i.e., gene conversion that generates the preimmune repertoire in the bursa seems to level off around 3 weeks of age. This preimmune repertoire can be further diversified by somatic point mutations that presumably lead to the formation of antibodies with increased affinity. A segment with structural features of a matrix association region [(A + T)-rich and four topoisomerase II binding sites] was identified in the middle of the J-C lambda intron (where C = constant).
Molecular cloning of the CD3 eta subunit identifies a CD3 zeta-related product in thymus-derived cells.
Authors: Jin Y J , Clayton L K , Howard F D , Koyasu S , Sieh M , Steinbrich R , Tarr G E , Reinherz E L
Published: May 2006

The CD3 eta subunit of the T-cell antigen receptor forms a heterodimeric structure with the CD3 zeta subunit in thymus-derived lymphoid cells and is apparently involved in signal transduction through the receptor. Here we report the primary structure of murine CD3 eta as deduced from protein microsequencing and cDNA cloning. The mature protein is divided into three domains: a 9-amino acid extracellular segment, a 21-amino acid transmembrane segment including a negatively charged residue characteristic of CD3 subunits, and a 155-amino acid cytoplasmic tail. The NH2-terminal sequences of CD3 eta and CD3 zeta are identical through amino acid 122 of each mature protein but then diverge in the remainder of their respective COOH-terminal regions, consistent with alternatively spliced products of a common gene. The cytoplasmic domain of CD3 eta is 42 amino acids larger than that of CD3 zeta but lacks one of six potential tyrosine phosphorylation sites as well as a putative nucleotide binding site previously identified in CD3 zeta. These structural features presumably account for the difference between CD3 eta and CD3 zeta function and are consistent with the notion that CD3 eta may be an important component of a T-cell receptor isoform(s) during thymic development.
Cisplatin-resistant cells express increased levels of a factor that recognizes damaged DNA.
Authors: Chu G , Chang E
Published: May 2006

Cancer treatment with the drug cisplatin is often thwarted by the emergence of drug-resistant cells. To study this phenomenon, we identified two independent cellular factors that recognize cisplatin-damaged DNA. One of the two factors, designated XPE binding factor, is deficient in complementation group E of xeroderma pigmentosum, an inherited disease characterized by defective repair of DNA damaged by ultraviolet radiation, cisplatin, and other agents. Human tumor cell lines selected for resistance to cisplatin showed more efficient DNA repair and increased expression of XPE binding factor. These results suggest that XPE binding factor may be responsible, at least in part, for the development of cisplatin resistance in human tumors and that the mechanism may be increased DNA repair.
High-level beta-globin expression after retroviral transfer of locus activation region-containing human beta-globin gene derivatives into murine erythroleukemia cells.
Authors: Novak U , Harris E A , Forrester W , Groudine M , Gelinas R
Published: May 2006

The locus activation region (LAR) of the human beta-globin-like gene cluster is characterized by a group of four DNase I hypersensitive sites, which arise specifically in erythroid tissues and are required for a normal pattern of beta-globin-like gene expression. The hypersensitive sites are found at positions 6.1, 10.9, 14.7, and 18 kilobase pairs (kbp) 5' of the epsilon-globin gene. Recently functional assays of the LAR that tested determinants for all four hypersensitive sites showed that expression of the human beta-globin gene was increased to normal or near-normal levels in both transgenic mice and erythroid cells. We constructed retroviral vectors with a human beta-globin gene and the determinant for a single hypersensitive site and measured beta-globin gene expression after retroviral infection of murine erythroleukemia cells. Fragments for the hypersensitive sites at -18 or -10.9 kbp increased human beta-globin RNA levels respectively to 35% or 132% of the endogenous mouse beta maj-globin RNA level. In addition, greater expression was also observed for the neomycin phosphotransferase RNA, which was transcribed from the retroviral LTR, showing that the LAR fragments activated expression from a heterologous promoter. In the context of gene-transfer experiments ultimately aimed at gene therapy, our results show that LAR determinants lead to an increased level of human beta-globin RNA expression after retroviral transfer into erythroid cells. But inclusion of LAR determinants in retroviral vectors also entails the potential risk of activating the expression of nonglobin genes in erythroid cells.
Glutamate receptors activate Ca2+ mobilization and Ca2+ influx into astrocytes.
Authors: Glaum S R , Holzwarth J A , Miller R J
Published: May 2006

We measured changes in the molar concentration of cytosolic Ca2+ ([Ca2+]i) in individual astrocytes in culture produced by the glutamate analog quisqualate (QA) and related substances by using fura-2 digital fluorescence microscopy. In cells cultured from the cortex, hippocampus, and cerebellum, the QA analog alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA; 10 microM) produced a slow increase in [Ca2+]i that was modest in amplitude (approximately 200 nM). These effects were completely abolished by 10 microM 6-nitro-7-cyano-quinoxaline-2,3-dione (CNQX). In cerebellar astrocytes, similar effects were produced by QA. However, in cortical and hippocampal astrocytes, the response to QA was much more complex. In these cells, QA produced an initial [Ca2+]i spike that was followed by a sustained influx of Ca2+ ("plateau"). In the absence of extracellular Ca2+, this plateau was abolished but the spike remained. CNQX did not block the spike and only slightly reduced the size of the plateau in some cells. Ni2+ (10 microM) but not nimodipine (10 microM) reduced the amplitude of the plateau. Pretreatment with 100 nM phorbol 12-myristate 13-acetate for 15 min abolished the spike but not the plateau portion of the QA response. Treatment with pertussis toxin at 250 ng/ml for 12-16 hr failed to alter the response. In some instances, the latency of the QA response differed considerably for individual cells in a group. It appeared that the response began in one cell and then spread to neighboring cells. Thus, QA appears to trigger a complex response in some astrocytes consisting of Ca2+ mobilization from intracellular stores and also Ca2+ influx resulting from the activation of AMPA-sensitive and -insensitive pathways.
Trans-activation of the JC virus late promoter by the tat protein of type 1 human immunodeficiency virus in glial cells.
Authors: Tada H , Rappaport J , Lashgari M , Amini S , Wong-Staal F , Khalili K
Published: May 2006

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the JC virus (JCV), a human papovavirus. PML is a relatively rare disease seen predominantly in immunocompromised individuals and is a frequent complication observed in AIDS patients. The significantly higher incidence of PML in AIDS patients than in other immunosuppressive disorders has suggested that the presence of human immunodeficiency virus type 1 (HIV-1) in the brain may directly or indirectly contribute to the pathogenesis of this disease. In the present study we have examined the expression of the JCV genome in both glial and non-glial cells in the presence of HIV-1 regulatory proteins. We find that the HIV-1-encoded trans-regulatory protein tat increases the basal activity of the JCV late promoter, JCVL, in glial cells. In a reciprocal experiment, the JCV early protein, the large tumor antigen, stimulates expression from JCVL and HIV-1 long terminal repeat promoter in both glial and non-glial cells. This trans-activation occurs at the level of RNA synthesis, as measured by the rate of transcription, stability of the message, and translation. We conclude that the presence of the HIV-1-encoded tat protein may positively affect the JCV lytic cycle in glial cells by stimulating JCV gene expression. Our results suggest a mechanism for the relatively high incidence of PML in AIDS patients than in other immunosuppressive disorders. Furthermore, our findings indicate that the HIV-1 regulatory protein tat may stimulate other viral and perhaps cellular promoters, in addition to its own.