bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of antibodies to human T-lymphotropic virus type III by using a synthetic peptide of 21 amino acid residues corresponding to a highly antigenic segment of gp41 envelope protein.
Authors: Wang J J , Steel S , Wisniewolski R , Wang C Y
Published: May 2006

A peptide of 21 amino acids with the sequence Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp- Gly-Cys - Ser encoded by a segment in the env gene of human T-lymphotropic virus type III (HTLV-III), corresponding to amino acids 586-606 of the precursor envelope glycoprotein, has been synthesized by the Merrifield solid-phase method. Combined serological and chemical analyses of this peptide and related peptides have revealed the importance of certain amino acid residues in the antigenic determinant of the relevant peptide. Enzyme immunoassay (EIA) employing this peptide as an antigen adsorbent was shown to reproducibly detect antibodies in sera of patients with HTLV-III infection. This assay provided positive results with all sera that were reactive with gp41 envelope protein of HTLV-III in electrophoretic immunoblot analysis. Thus far, no false-positive sera have been encountered in control populations. Our EIA with this peptide as the coating antigen is shown to have advantages over that with the whole HTLV-III virus as an immunoadsorbent.
Entropy, matter, and cosmology
Authors: Prigogine I. , Géhéniau J.
Published: May 2006

The role of irreversible processes corresponding to creation of matter in general relativity is investigated. The use of Landau-Lifshitz pseudotensors together with conformal (Minkowski) coordinates suggests that this creation took place in the early universe at the stage of the variation of the conformal factor. The entropy production in this creation process is calculated. It is shown that these dissipative processes lead to the possibility of cosmological models that start from empty conditions and gradually build up matter and entropy. Gravitational entropy takes a simple meaning as associated to the entropy that is necessary to produce matter. This leads to an extension of the third law of thermodynamics, as now the zero point of entropy becomes the space-time structure out of which matter is generated. The theory can be put into a convenient form using a supplementary “C” field in Einstein's field equations. The role of the C field is to express the coupling between gravitation and matter leading to irreversible entropy production.
A DNA binding protein specific for an origin of replication of herpes simplex virus type 1.
Authors: Elias P , O'Donnell M E , Mocarski E S , Lehman I R
Published: May 2006

We have identified a protein that binds specifically to an origin of replication (oris) of the herpes simplex virus type 1 genome. The oris binding protein, detectable only in nuclear extracts of infected cells, shows the same time course of appearance as the herpesvirus-induced DNA polymerase and the DNA binding protein ICP8. The partially purified oris binding protein generates a DNase I "footprint" that spans 18- of the 90-base-pair minimal oris sequence. The oris binding protein may, therefore, be analogous to other origin-specific binding proteins that are required for the initiation of viral and chromosomal DNA replication.
Interaction of a common factor with conserved promoter and enhancer sequences in histone H2B, immunoglobulin, and U2 small nuclear RNA (snRNA) genes.
Authors: Sive H L , Roeder R G
Published: May 2006

We have examined the interaction of factors in HeLa cell nuclear extracts with a human histone H2B gene (H2B) promoter. Protein-DNA mobility-shift and DNase I protection assays detected a factor(s) binding to a 15-base-pair consensus element that is essential for efficient H2B transcription in vitro. Part of this consensus sequence is the octanucleotide ATTTGCAT, which is apparently a functional component of several non-histone genes. A subset of these genes, including a human U2 small nuclear RNA (snRNA) gene promoter, a mouse immunoglobulin heavy chain enhancer, and a mouse light chain promoter, were shown to interact with the H2B consensus sequence-binding factor(s). These results suggest that a common factor or closely related factors may contribute to the regulation of these and other genes that share the octanucleotide sequence.
Inhibition of oligosaccharide processing and membrane morphogenesis in retinal rod photoreceptor cells.
Authors: Fliesler S J , Rayborn M E , Hollyfield J G
Published: May 2006

Castanospermine (Cas), an inhibitor of alpha-glucosidase I, blocks "trimming" of the N-linked oligosaccharide Glc3Man9GlcNAc2, thus preventing normal glycoprotein maturation. With use of a dual-label protocol, Xenopus retinas incubated in the presence of Cas exhibited at least a 2.3-fold increase in the incorporation of [3H]mannose into total retina Cl3CCOOH-precipitable material, whereas incorporation of [14C]leucine was not significantly affected, relative to controls. Analysis of NaDodSO4/PAGE fluorograms of solubilized retinas and rod outer segment (ROS) membranes indicated a relatively selective effect of Cas on opsin (the rod visual pigment apoglycoprotein). The apparent molecular mass of opsin was increased by approximately 2500 in the presence of Cas; the incorporation of [3H]mannose into opsin was enhanced about 2.3-fold without a significant effect on [14C]leucine incorporation, relative to controls. Electron microscopic autoradiography of retinas incubated for 4 hr with [3H]mannose showed that the number of newly formed ROS discs in Cas-treated retinas was not significantly different from controls, but the silver grain density over those discs was about 2.6-fold greater than in controls. The morphology of the newly formed discs was comparable under both conditions. Thus, opsin bearing abnormally large oligosaccharides can be accommodated in the process of disc morphogenesis. These results suggest that the structural requirements for opsin's oligosaccharides, with regard to their potential role as determinants of disc morphogenesis, are not stringent. Furthermore, post-translational processing of N-linked oligosaccharides is not essential for the normal intracellular routing and cell surface expression of membrane glycoproteins.
Intracellular potassium depletion in IM-9 lymphocytes suppresses the slowly dissociating component of human growth hormone binding and the down-regulation of its receptors but does not affect insulin receptors.
Authors: Ilondo M M , Courtoy P J , Geiger D , Carpentier J L , Rousseau G G , De Meyts P
Published: May 2006

We have investigated whether the slowly dissociating component of insulin and human growth hormone (hGH) binding and the homologous down-regulation of their receptors in IM-9 cultured human lymphocytes are due to distinct conformations of the receptor or, rather, to a redistribution within the cell. To do so, we used intracellular K+ depletion, which has been shown to inhibit reversibly coated-pit formation and ligand internalization in some cell lines. IM-9 cells incubated in K+-free buffer after a hypotonic shock rapidly lost their K+, which was stabilized at +/- 50% of control by incubation in K+-free binding assay buffer. In K+-depleted cells, the hGH dissociation kinetics became monoexponential and, in contrast with control cells, compatible with the equilibrium constant derived from saturation and association data using a simple model. The loss of hGH receptors during competition studies was abolished. The down-regulation by unlabeled hGH was decreased by 80%. In contrast, insulin receptor kinetics remained unchanged (non-first-order) in the K+-depleted cells; the negative cooperativity and the down-regulation (60%) were identical to those of control cells. Quantitative electron microscopic autoradiography showed a decrease of +/- 50% in the fraction of 125I-labeled hGH internalized. The number of visible coated pits in the membrane was reduced by 80%. Thus, in IM-9 cells, association with coated pits and endocytosis appear to play a major role in the kinetics of hGH binding and in the down-regulation of its receptors, but not in insulin-receptor binding kinetics and down-regulation.
Transfection of mouse erythroleukemia cells with myc sequences changes the rate of induced commitment to differentiate.
Authors: Lachman H M , Cheng G H , Skoultchi A I
Published: May 2006

We have examined the role of the c-myc protooncogene in chemically induced differentiation of mouse erythroleukemia (MEL) cells by transfecting the cells with recombinant plasmids in which c-myc coding sequences were cloned downstream from the mouse metallothionein I promoter in sense and antisense orientations. We previously showed that treatment of MEL cells with inducers of differentiation leads to a rapid (less than 2 hr) decrease in the level of c-myc mRNA. c-myc mRNA is then transiently restored to pretreatment levels approximately 12-18 hr later. These events occur prior to the detection of cells that are irreversibly committed to erythroid differentiation. MEL cell transfectants containing the plasmid with myc in the sense orientation express a chimeric MT-myc mRNA, which also decreases shortly after addition of inducer. However, these clones reexpress myc RNA more rapidly than the parental line and they also differentiate more rapidly. On the other hand, transfectants containing the plasmid with myc in the antisense orientation exhibited a delay in the reexpression of c-myc mRNA and were found to differentiate more slowly than parental cells. Thus, we find a correlation between the time at which myc RNA is reexpressed following inducer treatment and the rate of entry of cells into the terminal differentiation program.
Characterization of mutations induced by 2-(N-acetoxy-N-acetyl)aminofluorene in the dihydrofolate reductase gene of cultured hamster cells.
Authors: Carothers A M , Urlaub G , Steigerwalt R W , Chasin L A , Grunberger D
Published: May 2006

To determine the types of alterations in gene structure that are induced by the carcinogen 2-(N-acetoxy-N-acetyl)aminofluorene, we used this compound to generate mutations at the dihydrofolate reductase (DHFR) locus (DHFR) in Chinese hamster ovary cells. Twenty-nine independent enzyme-deficient mutants were isolated. A profile of the 26-kilobase (kb)-long gene was obtained by Southern blot analysis of the mutant and parental DNAs digested with BstEII/Kpn I. Hybridization to a mixed probe of 10 DHFR genomic and cDNA fragments revealed 12 bands that scan 34 kb. Twenty-one DHFR- clones (72%) contained small mutations (changes less than 100 base pairs in size). Large or small deletions involving various parts of the gene occurred in eight of the mutants (28%). A large deletion (greater than 35 kb) with 5' and 3' breakpoints mapping to approximately the same location was noted in four mutants. One mutant has undergone a deletion of 550-900 bp that eliminated the first coding exon. Concomitantly, a chromosomal event (either translocation, insertion, or inversion) has separated the 5' flank from the body of the gene. In another mutant, four deletions have occurred at the DHFR 5' end and internally. Restriction fragment length polymorphism analysis of the mutant DNAs with exon-specific probes localized three mutations. One mutant has lost a Taq I (TCGA) site, and another has lost a Sac I (GAGCTC) site. In a third, a GC----TA transversion has created a BstEII (GGTNACC) site. Finally, we used HPLC to determine the ratio of acetylated (12%) to deacetylated (88%) 2-aminofluorene adducts formed in the parental cells. A correlation between the mutational specificities and the conformational changes induced by the two types of DNA adducts is discussed.
On the origin and spread of beta-thalassemia: recurrent observation of four mutations in different ethnic groups.
Authors: Wong C , Antonarakis S E , Goff S C , Orkin S H , Boehm C D , Kazazian H H
Published: May 2006

Seven beta-thalassemia genes were characterized after they were identified as candidates for previously undescribed mutations based upon the close association of DNA polymorphism haplotypes in the beta-globin gene cluster with specific ethnic mutations. The molecular defect in four of these genes was identical, a frameshift deletion of four nucleotides (-CTTT) within codons 41 and 42. This gene represents a common Southeast Asian mutation shared by a Laotian beta-thalassemia gene, [framework 1 (FR1)], a Vietnamese (FR1), and two Chinese patients (FR3 Asian and FR1). The deletion has been observed previously in Chinese (FR1) and Asian Indians (FR2) and is an example of independent origins of the same molecular defect, possible interallelic gene conversion (as it is seen on two different beta-globin gene frameworks in Chinese), and mutant gene migration in the Asian countries. A second example of mutant gene migration was identified in an Iranian patient with a nucleotide insertion (G) between codons 8 and 9, the same mutation previously found in an Asian Indian in the same chromosomal background. The last two genes examined represent further strong evidence for independent origins of mutation. A C-to-T substitution at position -88 in an Asian Indian has been identified previously in an American Black on a different beta-globin gene framework, and a G-to-A transition at nucleotide 1 of intervening sequence 2 found in an American Black has been observed previously on a different chromosome background in Mediterraneans. This study suggests that there are not many common beta-thalassemia mutations remaining to be discovered. It also suggests that certain sequences in the beta-globin gene are relatively mutation sensitive.
cDNA clones encoding murine IgE-binding factors represent multiple structural variants of intracisternal A-particle genes.
Authors: Kuff E L , Mietz J A , Trounstine M L , Moore K W , Martens C L
Published: May 2006

Previously [Moore, K. W., Jardieu, P., Mietz, J. A., Trounstine, M. L., Kuff, E. L., Ishizaka, K. & Martens, C. L. (1986) J. Immunol. 136, 4283-4290], we examined a T-hybridoma-derived cDNA clone, 8.3, that encodes a biologically active murine IgE-binding factor (IgE-BF), and we showed that it was a variant member of the endogenous retroviral gene family related to mouse intracisternal A particles (IAPs). We have now characterized four more IgE-BF cDNA clones by heteroduplex and restriction enzyme analysis and found that they all represent different structural variants of the full-size IAP genomic element. In clones 8.3 and 10.2, which have been fully sequenced, the open reading frames span deletions 3.4 and 1.9 kilobases (kb) long, respectively, and specify different gag-pol fusion polypeptides. Clone 9.5 contains a 2.1-kb deletion entirely within the pol region. Two other clones (4.2 and 11.7) contain no internal deletion and may represent truncated cDNA copies of full-size (7.2 kb) IAP gene transcripts. Structural variants very similar to clone 10.2 are common in the mouse genome, and clone 9.5 is also probably not a unique gene form. The sequences of clones 8.3 and 10.2 are different in detail, but each is closely homologous to a randomly cloned mouse genomic IAP element throughout the gag-related portions of their open reading frames. Antibodies against two oligopeptides specified by the sequence of clone 8.3 immunoprecipitated IAP-related proteins from mouse neuroblastoma and myeloma cells, confirming that the IgE-BF produced by this clone shares sequence with expressed IAP elements in different cell types. Thus, information related to the IgE-BF is an integral part of the murine IAP retrotransposon gag gene.
Nicotinic acetylcholine receptor contains multiple binding sites: evidence from binding of alpha-dendrotoxin.
Authors: Conti-Tronconi B M , Raftery M A
Published: May 2006

We have studied the stoichiometry of the binding of the long alpha-neurotoxins from the venom of Dendroaspis viridis (alpha-dendrotoxin) and Naja naja siamensis (alpha-cobratoxin) to the membrane-bound acetylcholine receptor (AcChoR) from Torpedo californica electric organ. The number of toxin molecules bound to one AcChoR molecule was determined by simultaneous-quantitative gas-phase microsequencing of all the amino acid sequences present in AcChoR-alpha-neurotoxin complexes. This method permits the use of homogeneous (nonradiolabeled) preparations of native toxins to obtain molar ratios of neurotoxin-receptor complexes. The stoichiometry obtained for alpha-cobratoxin was 2.1 +/- 0.2 (n = 4), in agreement with the accepted view that alpha-cobratoxin, like alpha-bungarotoxin, binds to the two alpha subunits, which are constituent polypeptides of the AcChoR molecule. alpha-Dendrotoxin gave a stoichiometry of 4.1 +/- 0.5 (n = 12); therefore, the AcChoR molecule contains four binding sites for this alpha-neurotoxin, two of which are recognized by alpha-cobratoxin. In support of this contention we have also found that when the AcChoR is saturated with alpha-bungarotoxin, addition of alpha-dendrotoxin markedly accelerates the dissociation of the bound alpha-bungarotoxin, demonstrating that the occupancy of the additional two sites by the latter toxin influences and decreases the affinity of the former toxin for its two binding sites. The fact that the AcChoR molecule is a pseudosymmetric complex of five highly homologous peptides suggests the possibility that as many as five binding sites for cholinergic ligand could be present, one on each subunit.
Synthesis of coenzyme A ester of retinoic acid: intermediate in vitamin A metabolism.
Authors: Kutner A , Renstrom B , Schnoes H K , DeLuca H F
Published: May 2006

Coenzyme A esters of all-trans- and 13-cis-retinoic acid were synthesized for use in studying vitamin A metabolism. The esters were obtained by two different synthetic methods starting from retinoic acids, which were converted to activated succinimidyl esters or anhydrides. These in turn were coupled with coenzyme A to form their respective thioesters. The retinoyl coenzyme A esters were purified by reverse-phase high performance liquid chromatography.
Translocation of the precursor of 5-
enol
pyruvylshikimate-3-phosphate synthase into chloroplasts of higher plants
in vitro
Authors: Della-Cioppa Guy , Bauer S. Christopher , Klein Barbara K. , Shah Dilip M. , Fraley Robert T. , Kishore Ganesh M.
Published: May 2006

5-
enol
Pyruvylshikimate-3-phosphate synthase (EPS
P
synthase; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC 2.5.1.19) is a chloroplast-localized enzyme of the shikimate pathway in plants. This enzyme is the target for the nonselective herbicide glyphosate (
N
-phosphonomethylglycine). We have previously isolated a full-length cDNA clone of EPS
P
synthase from
Petunia hybrida
. DNA sequence analysis suggested that the enzyme is synthesized as a cytosolic precursor (pre-EPS
P
synthase) with an amino-terminal transit peptide. Based on the known amino terminus of the mature enzyme, and the 5′ open reading frame of the cDNA, the transit peptide of pre-EPS
P
synthase would be maximally 72 amino acids long. To confirm this prediction and to assay directly for translocation of pre-EPS
P
synthase into chloroplasts
in vitro
, we cloned the full-length cDNA into an SP6 transcription system to produce large amounts of mRNA for
in vitro
translation. The translation products, when analyzed by NaDodSO
4
/PAGE autoradiography, indicate a relative molecular mass for pre-EPS
P
synthase of ≈55 kDa. Uptake studies with intact chloroplasts,
in vitro
, indicate that pre-EPS
P
synthase was rapidly taken up into chloroplasts and proteolytically cleaved to the mature ≈48-kDa enzyme. The transit peptide was shown to be essential for import of the precursor enzyme into the chloroplast. To our knowledge, post-translational import into chloroplasts of a precursor enzyme involved in amino acid biosynthesis has not been reported previously. Furthermore, enzymatic analysis of translation products indicates that pre-EPS
P
synthase is catalytically active and has a similar sensitivity to the herbicide glyphosate as the mature enzyme. To our knowledge, pre-EPS
P
synthase represents the only example of a catalytically competent chloroplast-precursor enzyme.
Homologous ribosomal proteins in bacteria, yeast, and humans.
Authors: Chen I T , Dixit A , Rhoads D D , Roufa D J
Published: May 2006

We describe sequences of two human ribosomal proteins, S14 and S17, and messenger RNAs that encode them. cDNAs were used as molecular hybridization probes to recognize complementary genes in rodent, Drosophila, and yeast chromosomal DNAs. Human ribosomal protein sequences are compared to analogous Chinese hamster, yeast, and bacterial genes. Our observations suggest that some ribosomal protein genes have been conserved stringently in the several phylogenetic lines examined. These genes apparently were established early in evolution and encode products that are fundamental to the translational apparatus. Other ribosomal protein genes examined, although similar enough to heterologous DNA sequences to indicate their structural relationships, appear to have diverged substantially during evolution, probably reflecting adaptations to different genetic environments.
Significant potential secondary structures in the Epstein-Barr virus genome.
Authors: Karlin S
Published: May 2006

This paper identifies all statistically significant dyad symmetry combinations in the Epstein-Barr virus genome. The distribution of long dyad symmetry pairings emphasizes two regions, the 5' third of the 3.1-kilobase-pair (kbp) repeat and the oriP region, the latter essential for Epstein-Barr virus replication during latency. A 600-base-pair (bp) stretch in the 3.1-kbp repeat can establish an extended hairpin loop of stem length in excess of 208 bp of predominantly G + C stacking. Moreover, the 3.1-kbp repeat has the potential to form a wide variety of secondary structures based on juxtapositions of sizable palindromes, close dyad symmetry pairings, and direct repeats. The 3.1-kbp repeat presents several features that portend it as an important control region. The oriP region contains an abundance of statistically significant dyad symmetry combinations that strongly correlate with the "21 X 30 bp" tandem repeat units and four truncated copies of this repeat unit 1 kbp downstream. Each of the units centers on the same approximately 30-bp palindrome. Contrasts in the content and the secondary structure formations associated with the 3.1-kbp repeat units versus those of the oriP region are discussed in relation to viral or cellular function.
Anti-idiotypic antibodies that inhibit immediate-type skin reactions in unsensitized monkeys on challenge with staphylococcal enterotoxin.
Authors: Bamberger U , Scheuber P H , Sailer-Kramer B , Bartsch K , Hartmann A , Beck G , Hammer D K
Published: May 2006

The staphylococcal enterotoxin B (SEB)-induced immediate-type skin reaction in unsensitized monkeys was used as a nonimmunological mast cell stimulus to examine whether the toxin exerts its effect via specific receptors on the target cell membrane. Anti-idiotypic antibodies (anti-Id) were raised in BALB/c mice against monoclonal anti-SEB antibodies (anti-SEB) and purified by idiotype affinity chromatography. The anti-Id nature of the antibody was demonstrated by its ability to inhibit the binding of 125I-labeled anti-SEB to the ligand in a concentration-dependent manner. Moreover, binding of anti-SEB to anti-Id was antagonized by the SEB ligand in a competitive way. These antibodies completely abolished skin reactions in unsensitized monkeys on challenge with SEB and impeded those provoked by staphylococcal enterotoxins A and C1 but did not have the biological activity of the toxin. These data are compatible with the view that receptors for staphylococcal enterotoxins may exist on the membrane of mast cells in the skin of unsensitized monkeys. The data suggest an experimental approach for producing anti-cell receptor antibodies that are of potential value to influence the course of staphylococcal enterotoxin-mediated effects.
Crystal structure of a prokaryotic ribosomal protein.
Authors: Wilson K S , Appelt K , Badger J , Tanaka I , White S W
Published: May 2006

The structure of ribosomal protein L30 from Bacillus stearothermophilus has been solved to a resolution of 2.5 A. The molecule is somewhat elongated and contains two helices and a three-stranded, anti-parallel beta-pleated sheet. The protein fold, in which helices pack on the same side of the sheet, generates a simple helix-sheet, two-layered motif. It is possible to distinguish three hydrophobic patches on the molecular surface, and one end has six isolated arginine and lysine residues. It is proposed that these reflect sites of protein-protein and protein-RNA interaction, respectively. The protein fold is very similar to that of the only other known ribosomal protein structure, L7/L12 from Escherichia coli, and, based on this similarity, an attempt is made to align the amino acid sequences of the two proteins.
Islet-activating protein inhibits leukotriene D4- and leukotriene C4- but not bradykinin- or calcium ionophore-induced prostacyclin synthesis in bovine endothelial cells.
Authors: Clark M A , Conway T M , Bennett C F , Crooke S T , Stadel J M
Published: May 2006

Incubation of the bovine endothelial cell line, CPAE, with leukotriene D4, leukotriene C4, bradykinin, or the calcium ionophore A23187 results in the release of arachidonic acid metabolites including 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. Pretreatment of these cells with the pertussis toxin islet-activating protein (IAP) results in a dose-dependent inhibition of the release of arachidonic acid metabolites and prostacyclin in response to leukotriene D4 and leukotriene C4. In contrast, similar responses evoked by bradykinin or ionophore were not significantly altered by the IAP pretreatment of the cells. IAP in the presence of [32P]NAD specifically [32P]ADP-ribosylates a 41-kDa protein in membranes prepared from CPAE cells. Pretreatment of the intact cells with IAP resulted in a dose-dependent inhibition of subsequent 32P labeling of the toxin substrate in the membranes and correlates with the uncoupling of the leukotriene responses. These results suggest that the 41-kDa IAP substrate, presumably a guanine nucleotide regulatory protein, mediates the response of CPAE cells to leukotriene D4 and leukotriene C4, but not to bradykinin or the calcium ionophore.
A temperature-dependent pBR322 copy number mutant resulting from a Tn5 position effect.
Authors: Lupski J R , Projan S J , Ozaki L S , Godson G N
Published: May 2006

In the process of randomly mutagenizing a recombinant pBR322 clone with transposon Tn5, a high copy number plasmid mutant, pLO88, has been isolated. The copy number phenotype of pLO88 is observed only at elevated temperatures, greater than or equal to 37 degrees C, and is due to the precise position of a Tn5 insertion. Nucleotide sequence of the Tn5-pBR322 junction reveals that Tn5-88 has inserted into an open reading frame that codes for a 63 amino acid protein previously shown to negatively regulate pBR322 plasmid copy number. By deleting portions of the Tn5 it is shown that the copy number phenotype is due not only to the insertion of Tn5 in pBR322 but also to the requirement that some Tn5 sequences remain intact. It appears that an outwardly directed Tn5 promoter initiates the synthesis of a transcript (RNA X) that interferes with the normal repressor RNA (RNA I)-primer RNA (RNA II) interaction at elevated temperatures.
Expression of T-cell-activating protein in peripheral lymphocyte subsets.
Authors: Yeh E T , Reiser H , Benacerraf B , Rock K L
Published: May 2006

T-cell-activating protein (TAP) is an allelic 12-kDa membrane protein that participates in T-cell activation. Soluble anti-TAP monoclonal antibodies can trigger antigen-specific, major histocompatibility complex-restricted T-cell hybridomas to produce interleukin 2 and are mitogenic for normal T cells and thymocytes. TAP is expressed on 10% of thymocytes, which are mainly cortisone-resistant and mature. In the periphery, TAP is expressed on 70% of resting T cells but not on resting B cells. In this report, we analyze in detail the nature of TAP expression on peripheral lymphocyte subsets by immunofluorescence techniques. We show that all inducer (L3T4+) T cells are TAP+. In contrast, only 50% of Lyt-2+ T cells express detectable TAP. Functional studies demonstrated that at least part of the heterogeneity of TAP expression is present in the Lyt-2+ cytolytic T-cell (CTL) subset. Unstimulated CTL precursors are TAP- but are induced to express TAP in the effector state. Furthermore, this reflects actual synthesis of TAP, as TAP is detectable on activated Lyt-2+ CTLs passaged in vitro under conditions where passive acquisition can be ruled out. To extend this observation, we have studied the expression of TAP on activated T and B cells. Upon activation, all T and B cells became TAP+. Furthermore, the TAP molecules on B and T cells are indistinguishable by NaDodSO4/polyacrylamide gel electrophoresis. This suggests that TAP expression defines further heterogeneity of lymphocytes, with activation being one parameter influencing its expression.
Isolation and characterization of a primitive interleukin-1-like protein from an invertebrate, Asterias forbesi.
Authors: Beck G , Habicht G S
Published: May 2006

Interleukin 1 (IL-1) is the major immunoregulatory molecule produced by macrophages and other related cells. It acts as the molecular orchestrator of nonspecific host defense mechanisms against multifarious insults. In this study we report the characterization of an IL-1-like protein from the starfish Asterias forbesi. IL-1-like activity was found in the coelomocytes of this invertebrate and was also isolated and purified from the coelomic fluid. The coelomocytes were capable of phagocytosing latex particles and were adherent to plastic tissue culture dishes. Starfish IL-1 stimulated murine thymocyte proliferation directly and to a greater degree in the presence of submitogenic concentrations of concanavalin A. Starfish IL-1 also stimulated fibroblast proliferation and fibroblast protein synthesis. It was found to have a Mr of 29,500 and pI values of 7.5, 5.9, and 4.8. Starfish IL-1 activity was inhibited by an antibody to human IL-1. These characteristics suggest starfish IL-1 to be very similar to murine and human IL-1. The similarities to vertebrate IL-1 considered along with the evolutionary position of the starfish suggest that IL-1 is an important, ancient, and functionally conserved molecule.
Ia+ murine epidermal Langerhans cells are deficient in surface expression of the class I major histocompatibility complex.
Authors: Caughman S W , Sharrow S O , Shimada S , Stephany D , Mizuochi T , Rosenberg A S , Katz S I , Singer A
Published: May 2006

Murine epidermal Langerhans cells were analyzed with fluorescence microscopy and multicolor flow cytometry for the surface expression of major histocompatibility complex (MHC) class I and class II antigens. Langerhans cells of H-2k haplotype were identified in situ or in epidermal-cell suspensions by their surface expression of the MHC class II determinants I-Ak and I-Ek. More than 90% of class II-positive Langerhans cells in epidermal-cell suspensions expressed no or barely detectable amounts of MHC class I antigens. Quantitation by flow cytometry revealed that H-2k Langerhans cells expressed only 1.6-3.3% as much H-2Kk as did class II-negative keratinocytes in the same epidermal-cell suspensions. By fluorescence microscopy, class I MHC antigens were not detectable on Langerhans cells in situ when analyzed on sheets of intact epidermis. The deficient expression of class I MHC permitted highly purified Langerhans cell populations to be isolated from epidermal cell suspensions by treatment with anti-class I MHC monoclonal antibody and complement. It is likely that the uniquely low cell-surface expression of class I MHC antigen by Langerhans cells has relevance to both immune responses in the skin as well as to mechanisms of skin allograft rejection. In addition, it is conceivable that regulation of class I MHC expression on antigen-presenting cells in general is an important but hitherto unrecognized mechanism of immune regulation.
Analgesia induced by isolated bovine chromaffin cells implanted in rat spinal cord.
Authors: Sagen J , Pappas G D , Pollard H B
Published: May 2006

Chromaffin cells synthesize and secrete several neuroactive substances, including catecholamines and opioid peptides, that, when injected into the spinal cord, induce analgesia. Moreover, the release of these substances from the cells can be stimulated by nicotine. Since chromaffin cells from one species have been shown to survive when transplanted to the central nervous system of another species, these cells are ideal candidates for transplantation to alter pain sensitivity. Bovine chromaffin cells were implanted into the subarachnoid space of the lumbar spinal region in adult rats. Pain sensitivity and response to nicotine stimulation was determined at various intervals following cell implantation. Low doses of nicotine were able to induce potent analgesia in implanted animals as early as one day following their introduction into the host spinal cord. This response could be elicited at least through the 4 months the animals were tested. The induction of analgesia by nicotine in implanted animals was dose related. This analgesia was blocked by the opiate antagonist naloxone and partially attenuated by the adrenergic antagonist phentolamine. These results suggest that the analgesia is due to the stimulated release of opioid peptides and catecholamines from the implanted bovine chromaffin cells and may provide a new therapeutic approach for the relief of pain.
Basic fibroblast growth factor supports the survival of cerebral cortical neurons in primary culture.
Authors: Morrison R S , Sharma A , de Vellis J , Bradshaw R A
Published: May 2006

Bovine basic fibroblast growth factor (bFGF) is a potent mitogen isolated from bovine pituitary glands and brain. The addition of homogeneous bFGF to primary cultures of rat cerebral cortical neurons markedly enhances cell survival and elaboration of neurites. These effects are dose-dependent, with optimal stimulation occurring at a concentration of 500 pg/ml. Maintenance of survival and neurite outgrowth require the continuous presence of bFGF. Other growth factors, such as thrombin, platelet-derived growth factor, beta nerve growth factor, and interleukin 2, have no effect on neuronal survival or process formation. Although the cellular site(s) of bFGF synthesis has not yet been established, these results suggest that bFGF may function as a neurotrophic agent in the central nervous system.
Purification of a terminal uridylyltransferase that acts as host factor in the in vitro poliovirus replicase reaction.
Authors: Andrews N C , Baltimore D
Published: May 2006

Poliovirus RNA polymerase requires a host factor to initiate RNA synthesis in vitro. The host factor was previously purified to near homogeneity from HeLa cells but was not assigned an enzymatic activity. This report describes the purification of a terminal uridylyltransferase that can act as host factor. By all criteria examined it is identical to the factor purified previously. It has the same molecular weight (68,000), chromatographic properties, and cellular localization. We present evidence that terminal uridylyltransferase can add uridine residues to the 3' poly(A) end of virion RNA and that these anneal back to the poly(A) and form a hairpin primer for polymerase.
Computer-assisted molecular modeling of tumor promoters: rationale for the activity of phorbol esters, teleocidin B, and aplysiatoxin.
Authors: Jeffrey A M , Liskamp R M
Published: May 2006

In the two-stage model of skin carcinogenesis, it is believed that initiators bind to DNA and that tumor promoters such as phorbol 12-tetradecanoate 13-acetate (TPA) bind noncovalently to membrane-associated high-affinity receptors, probably protein kinase C. Two other types of potent tumor-promoting substances, aplysiatoxin and teleocidin, appear to act also by binding to and activating protein kinase C, even though their chemical structures are quite different. Therefore, we have undertaken computer modeling of the special relationship of various functional groups in these three chemical classes of tumor promoters in an attempt to explain how these diverse structures bind to the same receptor molecule. We propose a stereochemical model in which the oxygens in TPA at C-3, C-4, C-9, and C-20 (O-3, O-4, O-9, and O-20) correspond to the O-11, N-13, N-1, and O-24 positions in teleocidin and the O-27, O-3, O-11, and O-30 oxygens in aplysiatoxin, respectively. In this model all distances with respect to overlap of the corresponding atoms are less than 1 A. In addition, all three types of molecules have their hydrophobic moieties oriented in a similar position. This model is further discussed with respect to other compounds showing various degrees of activity as tumor promoters, including mezerein, ingenol, and 4 alpha-TPA. The model explains how chemically diverse structures can have similar biological activity as tumor promoters and provides a basis for designing both agonists and antagonists of tumor promoters.
Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver catalase.
Authors: Furuta S , Hayashi H , Hijikata M , Miyazawa S , Osumi T , Hashimoto T
Published: May 2006

We have isolated five cDNA clones for rat liver catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6). These clones overlapped with each other and covered the entire length of the mRNA, which had been estimated to be 2.4 kilobases long by blot hybridization analysis of electrophoretically fractionated RNA. Nucleotide sequencing was carried out on these five clones and the composite nucleotide sequence of catalase cDNA was determined. The 5' noncoding region contained 83 bases and was followed by 1581 bases of an open reading frame that encoded 527 amino acids. The 3' noncoding region was 831 bases long and contained long repeats of the unit AC. The amino acid sequence deduced from the nucleotide sequence of the cDNAs showed about 90% homology with the reported primary structure of bovine liver catalase. The molecular weight of rat liver catalase was calculated to be 59,758 from the predicted amino acid sequence. The amino acid residues in contact with the heme group are completely identical for bovine liver and rat liver catalases. The amino acid sequence at the COOH terminus was confirmed by the results of carboxypeptidase P treatment of the protein purified from rat liver in the presence of leupeptin. Rat liver catalase has no cleavable signal peptide for translocation of the enzyme into peroxisomes.
Liquid crystalline phases in concentrated aqueous solutions of Na+ DNA.
Authors: Rill R L
Published: May 2006

Concentrated aqueous saline solutions of short (146-base-pair) DNA fragments suddenly become turbid and iridescent when the DNA concentration is slightly increased or the temperature is decreased. Microscopic examination through crossed polarizing filters shows that turbidity and iridescence is due to formation of a liquid crystalline DNA phase similar to cholesteric liquid crystals formed by other semirigid, but nonelectrolyte, chiral polymers. Several distinct textures of the liquid crystalline phase or phases are observed depending on DNA concentration, temperature, and method of sample preparation. Textures observed include spherulites with Maltese crosses, striated and highly colored ribbons, whorls of periodic interference fringes, and colored flakes. The liquid crystalline DNA phase coexists in metastable equilibrium with the isotropic phase over a relatively narrow temperature/concentration range--approximately 175-250 mg/ml and 25-62 degrees C (limit of measurements). At higher concentrations and temperatures above approximately equal to 25 degrees C, the solutions appear fully liquid crystalline. When concentrated solutions are cooled below room temperature, crystals form due to precipitation of supporting electrolyte. A partial phase diagram is reported for the isotropic----liquid crystal----crystal transitions of solutions of DNA in buffered saline (2 M Na+). The general features of this phase diagram and the critical DNA volume fraction for formation of the anisotropic phase are consistent with the observed and theoretically predicted phase behavior of rodlike or semirigid nonelectrolyte polymers.
Solubilization and separation of the human erythrocyte D-glucose transporter covalently and noncovalently photoaffinity-labeled with [3H]cytochalasin B.
Authors: Kurokawa T , Tillotson L G , Chen C C , Isselbacher K J
Published: May 2006

The D-glucose transporter in the human erythrocyte membranes was photoaffinity-labeled with [3H]cytochalasin B and solubilized with n-octyl beta-D-glucopyranoside (octyl glucoside). [3H]Cytochalasin B-bound proteins were further isolated by using Sephadex G-50 chromatography. The amount of [3H]cytochalasin B associated with the membrane proteins was approximately 10% of the total radioactivity in the octyl glucoside extract. The solubilized photoaffinity-labeled D-glucose transporter was isolated and found to consist of two major peaks by DEAE-Sephacel chromatography. The radioactivity of peak II was considerably greater than that of peak I. The incorporation of [3H]cytochalasin B into both peaks was blocked by the presence of D-glucose during photolysis. With preparative NaDod-SO4/polyacrylamide gel electrophoresis, the radioactivity of peak I could be released, but that of peak II remained with the D-glucose transporter. These results indicate that [3H]cytochalasin B was covalently bound to the D-glucose transporter only in peak II and that peak II could be generated by the photoaffinity labeling of peak I. However, the D-glucose transport activity was associated only with peak I. These findings suggest that the anionic domain of the D-glucose transporter becomes exposed because of conformational changes of the protein as a result of covalent binding with [3H]cytochalasin B by photoaffinity labeling.
Design of a photoreactive analogue of the Escherichia coli heat-stable enterotoxin STIb: use in identifying its receptor on rat brush border membranes.
Authors: Gariépy J , Schoolnik G K
Published: May 2006

The Escherichia coli heat-stable enterotoxin, STIb was prepared by solid-phase peptide synthesis and purified to homogeneity by high-pressure liquid chromatography. This analogue was iodinated and shown to bind specifically to rat intestinal membranes. The radiolabeled peptide was derivatized at the amino terminus with the photoreactive heterobifunctional crosslinking agent N-hydroxysuccinimidyl p-benzoylbenzoate. This photoreactive probe also exhibited binding specificity. It was mixed with rat intestinal brush border membranes and photolyzed in the presence or absence of excess unlabeled STIb. Polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate and 2-mercaptoethanol indicated that the peptide probe was crosslinked specifically to two molecular species of 57 and 75 kDa. One or both of these molecules appear to constitute the enterotoxin receptor or to be in close proximity to it.
Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction.
Authors: Dodson M , Echols H , Wickner S , Alfano C , Mensa-Wilmot K , Gomes B , LeBowitz J , Roberts J D , McMacken R
Published: May 2006

The O protein of bacteriophage lambda localizes the initiation of DNA replication to a unique site on the lambda genome, ori lambda. By means of electron microscopy, we infer that the binding of O to ori lambda initiates a series of protein addition and transfer reactions that culminate in localized unwinding of the origin DNA, generating a prepriming structure for the initiation of DNA replication. We can define three stages of this prepriming reaction, the first two of which we have characterized previously. First, dimeric O protein binds to multiple DNA binding sites and self-associates to form a nucleoprotein structure, the O-some. Second, lambda P and host DnaB proteins interact with the O-some to generate a larger complex that includes additional DNA from an A + T-rich region adjacent to the O binding sites. Third, the addition of the DnaJ, DnaK, and Ssb proteins and ATP results in an origin-specific unwinding reaction, probably catalyzed by the helicase activity of DnaB. The unwinding reaction is unidirectional, proceeding "rightward" from the origin. The minimal DNA sequence competent for unwinding consists of two O binding sites and the adjacent A + T-rich region to the right of the binding sites. We conclude that the lambda O protein localizes and initiates a six-protein sequential reaction responsible for but preceding the precise initiation of DNA replication. Specialized nucleoprotein structures similar to the O-some may be a general feature of DNA transactions requiring extraordinary precision in localization and control.
Human delta-aminolevulinate dehydratase: nucleotide sequence of a full-length cDNA clone.
Authors: Wetmur J G , Bishop D F , Cantelmo C , Desnick R J
Published: May 2006

Two cDNAs encoding human delta-aminolevulinate dehydratase (ALA-D; porphobilinogen synthase; EC 4.2.1.24), the second enzyme in the heme biosynthetic pathway, were identified, recloned into bacteriophage M13, and sequenced by primer extension. The first clone with an 827-base-pair (bp) pEX-ALA-D cDNA insert, shown to contain DNA sequences that were colinear with four bovine ALA-D peptide sequences, was used to screen a pKT218 human liver library. A second clone containing a 1200-bp insert was identified that contained an open reading frame of 990 bp as well as 5' (66 bp)- and 3' (94 bp)-untranslated regions, the latter terminating in poly(dA). The predicted N-terminal amino acid sequence was colinear with the first 13 residues of microsequenced ALA-D purified from human erythrocytes. The ATG initiation codon was preceded by ACGCC, a functional initiation sequence, while an upstream (position -32), in-phase AACTG ATG sequence was entirely nonhomologous with the initiation consensus sequence and, therefore, presumed to be nonfunctional. The unusual polyadenylylation signal, AGTAAA, has been reported only in the human HRAS1 gene. The nucleotide sequences of the two cDNA clones differed at position 730 or 733 and encoded two differently charged amino acids. This nucleotide difference may be the basis for the polymorphic charge isozymes of human ALA-D. The sequence encoding this zinc metalloenzyme contained a cysteine- and histidine-rich binding site for zinc and an unusual region of charge complementarity surrounding the active lysine residue in the catalytic site.
Engineering of the mRNA-interfering complementary RNA immune system against viral infection.
Authors: Hirashima A , Sawaki S , Inokuchi Y , Inouye M
Published: May 2006

Creation of an artificial mRNA-interfering complementary RNA (micRNA) immune system, utilizing anti-sense RNAs to inhibit viral gene expression, has been shown to be an effective way to prevent viral infection. In the RNA coliphage SP, the gene for the maturation protein was found to be the best target for this type of immune system; mRNA-interfering complementary RNAs specific to the genes for coat protein and replicase were less effective in preventing infection. The greatest inhibitory effect was observed with a 240-base sequence encompassing the 24-base noncoding region of the maturation gene plus the 216-base coding sequence. Significantly, even a 19-base sequence covering only the Shine-Dalgarno sequence (ribosome-binding region) without the coding region exerted a strong inhibitory effect on phage proliferation. In contrast to the highly specific action against phage SP exhibited by the longer mRNA-interfering complementary RNA, the specificity with the shorter mRNA-interfering complementary RNA was broadened to phages Q beta and GA as well as SP, all of which are classified in the different groups of RNA coliphages. Therefore, this type of anti-viral reagent may be designed to have a particular breadth of specificity, thus increasing its value in various research and possibly clinical applications.