bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA methylation and regulation of the human beta-globin-like genes in mouse erythroleukemia cells containing human chromosome 11.
Authors: Ley T J , Chiang Y L , Haidaris D , Anagnou N P , Wilson V L , Anderson W F
Published: May 2006

The human beta-globin gene is expressed--but the human fetal (gamma) and embryonic (epsilon) globin genes are not--in an induced mouse erythroleukemia cell line (M11-X) that contains most of human chromosome 11. A 24-hr exposure of M11-X cells to 5-azacytidine before induction causes "global" DNA hypomethylation but selective activation of the human gamma-globin genes. Genomic DNA is remethylated 2-3 days after exposure to 5-azacytidine, but sequences near the human and mouse globin genes remain hypomethylated, suggesting that the remethylation process is inhibited in these regions.
Heat-labile alkaline phosphatase from Antarctic bacteria: Rapid 5′ end-labeling of nucleic acids
Authors: Kobori Hiromi , Sullivan Cornelius W. , Shizuya Hiroaki
Published: May 2006

A heat-labile alkaline phosphatase has been purified to near homogeneity from HK47, a bacterial strain isolated from Antarctic seawater. The active form of the enzyme has a molecular weight of 68,000 and is uniquely monomeric. The optimal temperature for the enzymatic activity is 25°C. Complete and irreversible thermal inactivation of the enzyme occurs in 10 min at 55°C. By using this heat-labile enzyme for dephosphorylation followed by a 10-min heat treatment, rapid end-labeling of nucleic acids by T4 polynucleotide kinase has been achieved.
Regulation of T-cell function by antibodies: enhancement of the response of human T-cell clones to hepatitis B surface antigen by antigen-specific monoclonal antibodies.
Authors: Celis E , Zurawski V R , Chang T W
Published: May 2006

Eight mouse monoclonal antibodies specific for hepatitis B surface antigen (HBsAg) were examined for their effects on the antigen-induced proliferative response and lymphokine production of human HBsAg-specific T-cell clones in vitro. While all specifically enhanced the T-cell proliferative response, antibodies of the IgG class were generally more effective than those of the IgM class. Both the divalent F(ab')2 and the monovalent Fab fragments of an IgG monoclonal antibody had no effects, indicating that the Fc portion of the antibody molecules was required. Since antigen-presenting cells bear surface receptors for the Fc of IgGs and fewer or none for that of IgMs, the above results also suggest that antibodies enhance the capture of antigens by antigen-presenting cells as a result of the binding of antigen-antibody complexes to the Fc receptors on these cells. In addition to potentiating the proliferation of the T-cell clones, antibodies also increased the antigen-induced production of interferon-gamma by these cells. The present in vitro studies suggest that antibodies may regulate immune responses and do so by enhancing antigen presentation and thus augmenting antigen-induced activation and clonal expansion of T cells.
In vivo nuclear magnetic resonance chemical shift imaging by selective irradiation.
Authors: Bottomley P A , Foster T H , Leue W M
Published: May 2006

NMR images of preselected chemically shifted species can be obtained by selective irradiation of the remainder of the NMR chemical shift spectrum prior to application of a conventional NMR imaging sequence. The chemical-selective irradiation consists of narrow-bandwidth pi/2 or saturation radio-frequency pulses applied in the absence of imaging gradients. The technique permits substantial reductions in scan and reconstruction times over standard three- and four-dimensional Fourier transform chemical-shift-imaging methods, when images of few spectral peaks are desired. It is also suitable for the elimination of chemical shift artifacts in conventional high-field NMR imaging. In vivo applications of the technique to the head and limbs in a 1.5-T magnetic field yield 1H H2O and -CH2-images, with little detectable -CH2- in muscle and brain.
Inhibition of human natural killer cell activity by (14R,15S)-14,15-dihydroxy-5Z,8Z,10E,12E- icosatetraenoic acid.
Authors: Ramstedt U , Serhan C N , Lundberg U , Wigzell H , Samuelsson B
Published: May 2006

The interactions between products of the 15-lipoxygenase cascade and human natural killer (NK) cell activity have been studied. Addition of human leukocyte-derived (14R,15S)-14,15-dihydroxy-5Z,8Z,10E,12E-ic osatetraenoic acid (14,15-DiHETE) to the NK cytotoxicity assay against K562 target cells resulted in inhibition of NK cell activity, whereas addition of other 15-lipoxygenase-associated metabolites [i.e., (15S)-15-hydroperoxy-5Z,8Z,11Z,13E-icosatetra eno ic acid, (15S)-15-hydroxy-5Z,8Z,11Z,13E-icosatetraenoic acid, and (8R,15S)- and (8S,15S)-8,15-dihydroxy-5Z,9E,-11E,13E-icosat etr aenoic acid isomers] resulted in little or no inhibition of NK function. Dose-response studies indicate that leukocyte-derived 14,15-DiHETE and 14,15-DiHETE methyl ester, at micromolar concentrations, inhibit NK function even in the presence of 2.5% fetal calf serum. Synthetic 14,15-DiHETE prepared by total organic synthesis displayed similar biological activities over identical dose ranges. These icosanoids do not inhibit NK target cell binding and they exert only a variable effect in either antibody-dependent cytotoxicity or cytotoxic T-lymphocyte assays. These results demonstrate that the 14,15-DiHETE inhibits NK cell function in vitro. Moreover, they suggest that activation of the 15-lipoxygenase cascade and formation of 14,15-DiHETE in vivo may provide a mode of immune regulation.
Distribution of reversing factor in reticulocyte lysates during active protein synthesis and on inhibition by heme deprivation or double-stranded RNA.
Authors: Thomas N S , Matts R L , Petryshyn R , London I M
Published: May 2006

We have recently shown a direct correlation between protein synthetic activity and the function of reversing factor (RF) as a catalyst of GDP-GTP exchange in whole reticulocyte lysates under normal conditions and on inhibition of protein synthesis by heme deficiency, double-stranded RNA, or oxidized glutathione. In this paper we report that RF is detectable as a nonribosomal complex with eukaryotic initiation factor 2 phosphorylated in its alpha subunit [eIF-2(alpha P)] in whole lysates inhibited by heme deprivation or by double-stranded RNA. The complex contains no unphosphorylated eIF-2 alpha, and the GDP present is freely dissociable. All nonribosomal eIF-2(alpha P) is complexed with RF in fully inhibited lysates; we have not detected free eIF-2(alpha P). RF in this [RF X eIF-2(alpha P)] complex is unavailable to catalyze the release of GDP from eIF-2-GDP. Dephosphorylation of eIF-2(alpha P) present in nonribosomal fractions releases active RF, which is able to carry out its normal guanine nucleotide exchange function.
Creatine kinase protein sequence encoded by a cDNA made from Torpedo californica electric organ mRNA.
Authors: West B L , Babbitt P C , Mendez B , Baxter J D
Published: May 2006

Creatine kinase (ATP creatine N-phosphotransferase, EC 2.7.3.2) is important in the maintenance of ATP levels in high energy-requiring tissues such as muscle and brain. A complete understanding of its function requires knowledge of its amino acid sequence. To obtain cDNA clones encoding creatine kinase sequences, a cDNA bank was constructed using mRNA from the electric organ of Torpedo californica and was screened by comparing differential colony hybridization of electric organ and liver-derived 32P-labeled cDNAs. Cloned DNAs have been isolated that can arrest the abundant synthesis of Mr 40,000-43,000 material seen after in vitro translation of electric organ mRNA. One of the clones, CK52g8, was sequenced by the dideoxy M13 method and was found to encode a Mr 42,941 protein, which is 68% homologous to a known partial sequence of rabbit muscle creatine kinase and which has a composition similar to creatine kinases from chicken and rabbit tissues. By contrast, no significant homology was found with the known sequences of kinases that use other substrates. RNA blot hybridization analysis indicated that CK52g8 is complementary to a 1600-base-pair mRNA. Primer extension analysis indicated that CK52g8 is only 5 nucleotides short of a full-length cDNA, implying that it encodes a complete protein sequence. The availability of this complete sequence should be useful in further studies of creatine kinase structure and function using techniques such as site-specific mutagenesis.
A membrane protein encoded by Epstein-Barr virus in latent growth-transforming infection.
Authors: Hennessy K , Fennewald S , Hummel M , Cole T , Kieff E
Published: May 2006

The nucleotide sequence of an Epstein-Barr virus gene expressed in latently infected growth-transformed cells is known to include a long open reading frame containing a 33-base-pair repeat element. A bacterial fusion protein constructed from a portion of the reading frame and Escherichia coli beta-galactosidase was used to produce sera in rabbits against the previously unidentified gene product. The viral protein detected with these sera in latently infected cells varies in size with the number of copies of the DNA repeat element. Translation of the RNA in vitro yields a protein of similar size. As expected from its primary sequence, the protein is a membrane protein. Immunofluorescence studies with the rabbit antisera suggest that the protein is in the plasma membrane. Thus, this protein could be the lymphocyte-determined membrane antigen (LYDMA) responsible for the generation of T-cell immunity to latently infected cells.
Inducible expression of a cloned heat shock fusion gene in sea urchin embryos.
Authors: McMahon A P , Novak T J , Britten R J , Davidson E H
Published: May 2006

A fusion gene construct, in which the coding sequence for bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA: chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) was placed under the control of the regulatory region of the Drosophila gene encoding the 70-kilodalton heat shock protein [Di Nocera, P.P. & Dawid, I.B. (1983) Proc. Natl. Acad. Sci. USA 80, 7095-7098], was microinjected into the cytoplasm of unfertilized sea urchin eggs. Pluteus-stage embryos developing from the injected eggs were exposed to high temperature conditions that we found would elicit an endogenous sea urchin heat shock response. These embryos express the gene for CAT and, after heat treatment, display 8-10 times more CAT enzyme activity than do extracts from control embryos cultured at normal temperatures. The injected DNA is present in high molecular weight concatenates and, during development, is amplified about 100-fold. Amplified sequences are responsible for all or most of the induced CAT enzyme activity.
Selective killing of leukemic cells by merocyanine 540-mediated photosensitization.
Authors: Sieber F , Spivak J L , Sutcliffe A M
Published: May 2006

In vitro incubation of leukemic bone marrow with the lipophilic fluorescent dye merocyanine 540 and simultaneous exposure to light reduced the concentration of L1210 leukemia cells by about 5 orders of magnitude but spared enough normal pluripotent hematopoietic stem cells to allow hematopoietic reconstitution of lethally irradiated syngeneic mice. This simple and rapid procedure may find an application in the purging of tumor cells from autologous bone marrow grafts.
Nucleotide sequence of the gene encoding the fusion (F) glycoprotein of human respiratory syncytial virus.
Authors: Collins P L , Huang Y T , Wertz G W
Published: May 2006

The nucleotide sequence of the mRNA encoding the F protein of respiratory syncytial (RS) virus (strain A2) was determined from cDNA clones that contain the complete mRNA sequence. The mRNA is 1899 nucleotides long exclusive of polyadenylylate. The single major open reading frame encodes a protein of 574 amino acids, with a calculated molecular weight of 63,453. Major structural features predicted from the amino acid sequence include an NH2-terminal signal sequence (residues 1-22), hydrophobic transmembrane anchor sequence (residues 525-550), five potential acceptor sites for asparagine-linked carbohydrate, and a potential site (residues 131-136) for the proteolytic cleavage that generates the disulfide-linked F1 and F2 subunits, which, by analogy to other paramyxoviruses, constitute the biologically active form of the F protein. The sequence also contains an internal hydrophobic domain (residues 137-154) that, as a consequence of the activating proteolytic cleavage described above, would become the NH2 terminus of the larger, F1 subunit. The amino acid sequence of the hydrophobic terminus of the F1 subunit is known to be highly conserved among several paramyxoviruses but is markedly dissimilar for RS virus. The F2 subunit is relatively hydrophilic and contains four of the five potential carbohydrate acceptor sites. The subunit order is NH2-F2-F1-COOH. The nucleotide sequences at the 5' and 3' mRNA termini are conserved among the eight RS viral mRNAs sequenced to date. The conserved sequences are: 5' G-G-G-G-C-A-A-A-U ... A-G-U-AU-A-(N)0-2-AU-U-poly(A). These are candidates to be signals for viral transcription. The nucleotide and amino acid sequences described further define the relationship between RS virus and other paramyxoviruses.
A separate editing exonuclease for DNA replication: the epsilon subunit of Escherichia coli DNA polymerase III holoenzyme.
Authors: Scheuermann R H , Echols H
Published: May 2006

DNA polymerase III (polIII) holoenzyme of Escherichia coli has 3'----5' exonuclease ("editing") activity in addition to its polymerase activity, a property shared by other prokaryotic DNA polymerases. The polymerization activity is carried by the large alpha subunit, the product of the dnaE gene. Mutations affecting the fidelity of DNA replication in vivo and the activity of 3'----5' exonuclease assayed in vitro are found in the dnaQ gene, which specifies the epsilon subunit. To determine whether epsilon carries the 3'----5' exonuclease activity, we have used an overproduction protocol to purify epsilon separately from the other subunits of polIII holoenzyme. We find that epsilon has 3'----5' exonuclease activity indistinguishable from that of polIII core, the subassembly of polIII holoenzyme consisting of the alpha, epsilon, and theta subunits. We conclude that the editing and polymerization activities of polIII holoenzyme reside on distinct subunits, in contrast to DNA polymerase I of E. coli and DNA polymerase of phage T4. This functional separation may provide for regulation of exonucleolytic editing independently of polymerization, allowing cellular control of replication fidelity.
Identification of a recent recombination event within the human beta-globin gene cluster.
Authors: Gerhard D S , Kidd K K , Kidd J R , Egeland J A , Housman D E
Published: May 2006

In a detailed study of inheritance of DNA sequence polymorphism in a large reference pedigree, an individual was identified with an apparent genetic recombination event within the human beta-globin gene cluster. Analysis of the haplotypes of relevant individuals within this pedigree suggested that the meiotic crossing-over event is likely to have occurred within a 19.8-kilobase-pair region of the beta-globin gene cluster. Analysis of other DNA markers closely linked to the beta-globin gene cluster--segment 12 of chromosome 11 (D11S12) and loci for insulin, the cellular oncogene c-Ha-ras, and preproparathyroid hormone--confirmed that a crossover event must have occurred within the region of chromosome 11 between D11S12 and the beta-globin gene cluster. It is suggested that the event observed has occurred within a DNA region compatible with recombinational "hot spots" suggested by population studies.
Promoter-regulatory region of the major immediate early gene of human cytomegalovirus.
Authors: Thomsen D R , Stenberg R M , Goins W F , Stinski M F
Published: May 2006

The DNA templates containing immediate early (IE) genes of human cytomegalovirus (CMV) were transcribed in vitro by using a HeLa cell extract. When IE region 1, 2, and 3 were used, transcription was detected qualitatively only from IE region 1. Transcription was detected with DNA representing IE region 2 when the IE region 1 promoter was not present. DNA sequence analysis of the upstream regulatory region of IE region 1 detected two distinct repeats of 19 and 18 nucleotides, both being repeated four times. A putative cruciform structure could form through the surrounding sequences with each 18-nucleotide repeat being located in the unpaired region. The potential secondary structure and the repeat sequences in the regulatory region of IE region 1 are presumably related to the high level of transcription of this IE gene.
A unique sequence related to the ecotropic murine leukemia virus is associated with the Fv-4 resistance gene.
Authors: Kozak C A , Gromet N J , Ikeda H , Buckler C E
Published: May 2006

Several strains of laboratory and wild-derived mice from Japan carry the dominant allele at the Fv-4 locus (Fv-4r) that is responsible for resistance to infection by exogenous ecotropic murine leukemia virus. We have used blot hybridization with a probe specific for the ecotropic viral envelope to show that a unique envelope-reactive sequence is present in the Japanese mouse Mus musculus molossinus and in four independently derived partially congeneic strains carrying Fv-4r. Analysis of 31 backcross mice shows complete concordance between inheritance of this fragment and resistance to Friend virus complex-induced erythroblastosis. Inheritance of this sequence also suppresses spontaneous expression of the endogenous ecotropic viruses carried by M. m. molossinus. Restriction enzyme analysis shows that the Fv-4r-associated sequence is different from the full-length ecotropic proviruses of laboratory mice. Infectious virus cannot be induced from mice carrying only the Fv-4r-associated sequence. Examination of other wild-derived mice resistant to Friend virus complex shows that Mus cervicolor cervicolor also contains the Fv-4r sequence. Our data indicate that a unique or incomplete provirus containing ecotropic envelope-related sequences is responsible for Fv-4-mediated resistance to both exogenous and endogenous ecotropic virus in various Asian mice.
Detection of a secreted form of the murine H-2 class I antigen with an antibody against its predicted carboxyl terminus.
Authors: Maloy W L , Coligan J E , Barra Y , Jay G
Published: May 2006

Analysis of H-2 class I-specific cDNA clones has suggested the synthesis by the liver of a class I molecule that is secreted rather than membrane bound. To detect this putative class I-related molecule, we have predicted a unique region of amino acid sequence located toward the carboxyl terminus of the molecule that is not expected to be shared with any of the classical H-2 class I antigens, and we have generated specific antibodies to a synthetic peptide corresponding to this sequence. Indirect immunoprecipitation with this antibody led to the identification of a Mr 40,000 polypeptide in association with beta 2-microglobulin in the serum of mice of five different H-2 haplotypes. This class I molecule is also detected in the liver together with lower molecular weight components, which are presumably underglycosylated precursors. Synthesis of this molecule is not detected in thymus, spleen, kidney, or testis. This class I serum component has no detectable reactivity with either a broad-specificity alloantiserum against H-2b or a xenoantiserum against purified H-2a class I molecules. The availability of a specific antibody against the secreted class I molecule offers a means to purify this protein for structural and functional studies.
N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen.
Authors: Dahlin D C , Miwa G T , Lu A Y , Nelson S D
Published: May 2006

N-acetyl-p-benzoquinone imine (NAPQI) has been proposed as the toxic metabolite of acetaminophen for the past 10 years, although it has never been detected as an enzymatic oxidation product of acetaminophen. We report (i) direct detection of NAPQI formed as an oxidation product of acetaminophen by cytochrome P-450 and cumene hydroperoxide and (ii) indirect evidence that is compelling for NAPQI formation from acetaminophen by cytochrome P-450, NADPH, and NADPH-cytochrome P-450 reductase. Evidence is provided for the rapid reduction of NAPQI back to acetaminophen by NADPH and NADPH-cytochrome P-450 reductase such that steady-state levels of NAPQI were below our detection limits of 6.7 X 10(-8) M. In mouse liver microsomal incubations, radiolabeled analogs of both NAPQI and acetaminophen bound covalently to microsomal protein with the loss of approximately equal to 20% of the acetyl group as acetamide. The binding in each case was decreased by glutathione with concomitant formation of 3-S-glutathionylacetaminophen. The binding also was decreased by L-ascorbic acid, NADPH, and NADH with reduction of NAPQI to acetaminophen. Results of partitioning experiments indicate that NAPQI is a major metabolite of acetaminophen, a considerable fraction of which is rapidly reduced back to acetaminophen.
Mouse yolk sac and intraembryonic tissues produce factors able to elicit differentiation of erythroid burst-forming units and colony-forming units, respectively.
Authors: Labastie M C , Thiery J P , Le Douarin N M
Published: May 2006

This work was aimed at elucidating the environmental conditions that account for the production of embryonic erythrocytes in the mouse yolk sac (YS), while the adult-type hemoglobin and erythrocytes are generated in the fetal liver. Differentiation of YS hemopoietic stem cells (YS-HSC) of 9.5-day mouse embryos (prior to the colonization of the liver rudiment by HSC) was investigated in vitro. The influence of well-characterized erythroid growth factors, burst-promoting activity (BPA) and erythropoietin (EPO), which trigger the differentiation of early erythroid burst-forming units (BFU-E) and late erythroid colony-forming units (CFU-E), respectively, was tested on the YS-HSC in two different systems of culture: (i) organ culture and (ii) clonal culture in methylcellulose. When studied in organ culture, where the YS microenvironment was maintained, YS-HSC required only additional EPO to attain complete maturation into adult erythrocytes within 7 days. In contrast, YS hemopoietic single cells grown in methylcellulose, and thus released from the influence of the YS, required the presence of both BPA and EPO to generate BFU-E-derived colonies synthesizing high concentrations of hemoglobin. It is concluded that 9.5-day YS from mouse embryos is by itself able to promote the first differentiation steps of the adult lineage YS-HSC due to an intrinsic production of a BPA-like activity. In contrast, these experiments demonstrate that EPO or an EPO-like activity is not produced by YS tissues. As demonstrated earlier, if embryonic tissue is added to YS organ culture, although separated from it by a filter preventing cell contact, YS-HSC differentiate into adult erythrocytes producing adult-type hemoglobins. This shows that, in contrast to YS tissues, the early embryo produces EPO or a factor that can substitute for EPO.
Transformation of Aspergillus nidulans by using a trpC plasmid.
Authors: Yelton M M , Hamer J E , Timberlake W E
Published: May 2006

We constructed a chimeric plasmid carrying a complete copy of the trifunctional trpC gene from the Ascomycete fungus Aspergillus nidulans. This plasmid, designated pHY201, replicates in Escherichia coli, where it confers resistance to ampicillin and chloramphenicol and complements trpC mutants lacking phosphoribosylanthranilate isomerase activity. We used pHY201 to transform an A. nidulans trpC- strain to trpC+ at frequencies of greater than 20 stable transformants per microgram of DNA. Southern blot analysis of DNA from transformants showed that pHY201 DNA had integrated into the A. nidulans chromosomes in a majority of cases. Most of the integration events appeared to occur at the site of the trpC- allele of the recipient strain. In several instances, we succeeded in recovering pHY201, or derivatives thereof, from A. nidulans transformants by restriction endonuclease digestion of chromosomal DNA, ligation, and transformation of E. coli.
Dark-adapted bacteriorhodopsin contains 13-cis, 15-syn and all-trans, 15-anti retinal Schiff bases.
Authors: Harbison G S , Smith S O , Pardoen J A , Winkel C , Lugtenburg J , Herzfeld J , Mathies R , Griffin R G
Published: May 2006

13C NMR spectra of lyophilized dark-adapted [14-13C]retinyl-labeled bacteriorhodopsin show a large anomalous upfield shift for the 13C-14 resonance assigned to the 13-cis isomer, relative to both the all-trans isomer and model compounds. We attribute this to the so-called gamma effect, which results from a steric interaction between the C-14 retinal proton and the protons on the epsilon CH2 of the lysine. As a consequence of this observation, we infer that dark-adapted bacteriorhodopsin is composed of a mixture of all-trans, 15-anti (trans or E) and 13-cis, 15-syn (cis or Z) isomers. These occur in an approximate 4:6 ratio and are commonly identified as bR568 and bR548. This conclusion is based on an examination of the isotropic and anisotropic chemical shifts and a comparison with 13C shifts of the carbons adjacent to the C = N linkage in protonated ketimines. Other possible origins for the anomalous shift are examined and shown to be insufficient to account for either the size of the shift or the nature of the shift tensor. We discuss the consequences of this finding for the structure and photochemistry of bacteriorhodopsin.
A genomic gene encoding the b5 rabbit immunoglobulin kappa constant region: implications for latent allotype phenomenon.
Authors: Emorine L , Sogn J A , Trinh D , Kindt T J , Max E E
Published: May 2006

We previously reported that domestic rabbits of five immunoglobulin kappa allotype strains (b4v, b4, b5, b6, and b9) harbor at least two DNA sequences that hybridize strongly to kappa constant region probes in Southern blots. One of these sequences ("type A") has been identified as encoding the constant region of the kappa 2 isotype, an immunoglobulin chain that most rabbits express only at low levels, if at all. We identified the second sequence--for rabbits of the b4 allotype--as encoding the nominal b4 kappa chain (or kappa 1 isotype), but for rabbits of other allotypes no definite identification for this "type B" sequence could be made. Here we suggest that the type B sequence in rabbits of the other domestic allotypes also encodes the nominal kappa 1 immunoglobulin chain. We show this directly for the b5 allotype; a type B sequence cloned from b5 DNA has been found to contain an apparently functional gene encoding the b5 constant region sequence. Indirect arguments suggest the corresponding conclusion for the b4v, b6, and b9 allotypes. We have considered the implications of these results for the phenomenon of "latent allotype" expression.
The Ro small cytoplasmic ribonucleoproteins: identification of the antigenic protein and its binding site on the Ro RNAs.
Authors: Wolin S L , Steitz J A
Published: May 2006

Patients with systemic lupus erythematosus and other rheumatic diseases often possess autoantibodies directed against discrete classes of small ribonucleoprotein particles (RNPs). The class of particles recognized by anti-Ro antibodies contains from two to four small cytoplasmic RNAs, depending on the mammalian species examined. We find that an antigenic polypeptide of 60 kDa is the major protein residing in Ro RNPs from human HeLa cells. To determine what common feature of Ro RNA sequence or structure is recognized by the Ro protein, we carried out ribonuclease protection experiments on isolated Ro RNPs from HeLa cells. For each of the three human Ro RNAs whose sequence is known, the most highly protected portion found in immunoprecipitates corresponded to the lower section of a stem formed by base-pairing the 5' and 3' ends of the RNA. Within this protected helix is a highly conserved region composed of seven identical base pairs with a single bulged cytidine. We discuss possible functions for the Ro RNPs.
Determination of retinal Schiff base configuration in bacteriorhodopsin
Authors: Smith Steven O. , Myers Anne B. , Pardoen Johannes A. , Winkel Chris , Mulder Patrick P. J. , Lugtenburg Johan , Mathies Richard
Published: May 2006

Resonance Raman spectra of the BR
568
, BR
548
, K
625
, and L
550
intermediates of the bacteriorhodopsin photocycle have been obtained in
1
H
2
O and
2
H
2
O by using native purple membrane as well as purple membrane regenerated with 14,15-
13
C
2
and 12,14-
2
H
2
isotopic derivatives of retinal. These derivatives were selected to determine the contribution of the C
14
—C
15
stretch to the normal modes in the 1100- to 1400-cm
-1
fingerprint region and to characterize the coupling of the C
14
—C
15
stretch with the NH rock. Normal mode calculations demonstrate that when the retinal Schiff base is in the C[unk]N
cis
configuration the C
14
—C
15
stretch and the NH rock are strongly coupled, resulting in a large (≈50-cm
-1
) upshift of the C
14
—C
15
stretch upon deuteration of the Schiff base nitrogen. In the C[unk]N
trans
geometry these vibrations are weakly coupled and only a slight (<5-cm
-1
) upshift of the C
14
—C
15
stretch is predicted upon
N
-deuteration. In BR
568
, the insensitivity of the 1201-cm
-1
C
14
—C
15
stretch to
N
-deuteration demonstrates that its retinal C[unk]N configuration is
trans
. The C
14
—C
15
stretch in BR
548
, however, shifts up from 1167 cm
-1
in
1
H
2
O to 1208 cm
-1
in
2
H
2
O, indicating that BR
548
contains a C[unk]N
cis
chromophore. Thus, the conversion of BR
568
to BR
548
(dark adaptation) involves isomerization about the C[unk]N bond in addition to isomerization about the C
13
[unk]C
14
bond. The insensitivity of the native, [14,15-
13
C
2
]-, and [12,14-
2
H
2
]K
625
and L
550
spectra to
N
-deuteration argues that these intermediates have a C[unk]N
trans
configuration. Thus, the primary photochemical step in bacteriorhodopsin (BR
568
→ K
625
) involves isomerization about the C
13
[unk]C
14
bond alone. The significance of these results for the mechanism of proton-pumping by bacteriorhodopsin is discussed.
Normal liver chromatin contains a firmly bound and larger protein related to the principal cytosolic target polypeptide of a hepatic carcinogen.
Authors: Vinores S A , Churey J J , Haller J M , Schnabel S J , Custer R P , Sorof S
Published: May 2006

A 14,000-dalton polypeptide was previously reported to be the principal protein target of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) in liver cytosol at the start of hepatocarcinogenesis in rats. The 14,000-dalton polypeptide was purified to homogeneity according to gel electrophoreses in both NaDodSO4-containing medium and acetic acid/urea and also by immunogenicity. An immunologically related form of the cytosolic target polypeptide has now been found to be present in the nuclei of normal rat liver as a 17,500-dalton polypeptide that is firmly and ionically bound to chromatin. Serial salt extractions of isolated liver nuclei or chromatin at 0.15 and 0.35 ionic strengths fail to dissolve the bound polypeptide, according to electrophoretic transfer immunoblot analyses. Most of the 17,500-dalton polypeptide is extracted at 0.65 ionic strength, the remainder at 1.2, and none at 2.0, nor thereafter in 8 M urea. In addition, short-term digestion of purified liver nuclei with micrococcal nuclease solubilizes the 17,500-dalton polypeptide. All three protocols also solubilize low levels of intermediate 17,500- to 14,000-dalton species, the latter size being the same as that of the cytosolic protein target of the carcinogen. The presence of protease inhibitors during the isolations and extractions of the nuclei and chromatin reduces the amounts of these smaller polypeptides. In normal rat liver only nuclei and cytoplasm of hepatocytes contain reactive antigen according to peroxidase-antiperoxidase immunohistochemistry, staining most intensely perilobularly, less in the lobular midzone, and least centrilobularly. The nuclei of the perilobular hepatocytes constitute the strongest staining compartment within all of normal liver. Of 22 nonhepatic tissues of normal rats, 16 contain relatively few cells with immunoreactive cytoplasm. Nonhepatic nuclear antigen is present only in villar crest cells of duodenum (which are normally exposed to liver bile), also having cytoplasmic antigen as well. Five kinds of evidence appear to connect the chromatin-bound 17,500-dalton polypeptide of normal liver nuclei to the cytosolic 14,000-dalton polypeptide that is the principal target of the carcinogen early during hepatocarcinogenesis in rats. The present findings indicate a direct connection between a chromosomal protein and the immediate principal cytosolic protein target of a carcinogen.
Growth control in cultured 3T3 fibroblasts: neutralization and identification of a growth-inhibitory factor by a monoclonal antibody.
Authors: Hsu Y M , Barry J M , Wang J L
Published: May 2006

A fibroblast growth regulator, which inhibits the growth and division of proliferating fibroblasts, has been isolated from medium conditioned by exposure to density-inhibited mouse 3T3 cells. This partially purified preparation of the growth-inhibitory activity, termed FGR-s, contained two major polypeptides (Mrs, 10,000 and 13,000). Using FGR-s as the immunogen, we have carried out in vitro immunization of rat splenocytes and have generated hybridoma lines, each secreting an antibody directed against components of the FGR-s preparation. One such monoclonal antibody, designated 2A4, specifically bound the Mr 13,000 polypeptide of FGR-s. Antibody 2A4 also neutralized the growth-inhibitory effect of FGR-s in a concentration-dependent fashion. These results strongly suggest that the Mr 13,000 polypeptide carries growth-inhibitory activity.
Osteonectin, bone proteoglycan, and phosphophoryn defects in a form of bovine osteogenesis imperfecta.
Authors: Termine J D , Robey P G , Fisher L W , Shimokawa H , Drum M A , Conn K M , Hawkins G R , Cruz J B , Thompson K G
Published: May 2006

Bovine osteogenesis imperfecta is a congenital disease in Holstein cattle having several characteristics in common with human osteogenesis imperfecta syndromes. In particular, affected calves have multiple bone fractures and friable teeth. Bone collagen isolated from the affected animals (Texas variant) showed slightly decreased alpha 1(I) and alpha 2(I) chain electrophoretic mobility and increased hydroxylysine content. Overall collagen was present in the affected bones at 80-90% of normal values. However, osteonectin, a 32,000 Mr bone-specific protein found previously to promote collagen mineralization in vitro and present in abundance (approximately equal to 3% of total protein) in normal calf bone, was severely depleted (less than 2% of normal levels) in the osteogenesis imperfecta bone and dentin. The bone proteoglycan was similarly depleted. In contrast, the bone sialoprotein was not as severely affected. Further, the diseased teeth lacked (less than 10% of normal values) phosphophoryn, a dentin-specific protein normally present as 4-5% of the total calf dentin matrix. The data suggest multiple hard tissue matrix protein deletions, perhaps due to impaired cell development.
C6 glioma cell-conditioned medium induces neurite outgrowth and survival of rat chromaffin cells in vitro: comparison with the effects of nerve growth factor.
Authors: Unsicker K , Vey J , Hofmann H D , Müller T H , Wilson A J
Published: May 2006

The effects of medium conditioned by rat C6 glioma cells (C6-CM) on the survival, neurite formation, and catecholamine content of adrenal medullary cells in culture were investigated and compared with the effects of nerve growth factor (NGF). Adrenal medullary cells were isolated from 10-day-old rats and the proportions of surviving and neurite-extending cells were determined after 8 days in culture. In the presence of C6-CM virtually all seeded cells survived and 50% developed neuritic processes. In contrast, NGF did not support survival above control levels (30%) and induced neurite formation from approximately one-third of the surviving cells. C6-CM and NGF had no additive effects on neurite outgrowth. C6-CM-mediated fiber outgrowth was not inhibited by physiological concentrations of glucocorticoids which abolished the NGF-induced neurite formation. Both C6-CM and NGF increased the catecholamine content of the cultures and reduced the relative content of epinephrine. However, in view of its substantial effect on cell survival as compared to NGF, C6-CM caused a reduction of the catecholamine content per cell. We conclude that adrenal medullary cells, like other members of the sensory-sympathetic cell lineage of the neural crest, respond to glial-conditioned medium. This response differs both quantitatively and qualitatively from that mediated by NGF.
Cloning and sequence analysis of cDNA for rat spleen thymosin beta 4.
Authors: Wodnar-Filipowicz A , Gubler U , Furuichi Y , Richardson M , Nowoswiat E F , Poonian M S , Horecker B L
Published: May 2006

Molecular cloning of a cDNA has established the sequence of the translated portion of the mRNA for rat spleen thymosin beta 4. The presence of a methionyl initiator codon immediately preceding the codon for the first seryl residue of mature thymosin beta 4 is consistent with previous results indicating the absence of a signal peptide in the product translated in vitro from rat spleen mRNA. The cDNA sequence analysis also established the presence of two terminator codons immediately following the codon for the COOH-terminal seryl residue. Thymosin beta 4 is thus synthesized as a 5100-dalton peptide containing 44 amino acid residues. Removal of the initiator methionyl residue and acetylation of the NH2-terminal serine residue would yield mature thymosin beta 4 containing 43 amino acids. The absence of a signal peptide makes it unlikely that thymosin beta 4 is a secreted peptide.
Potential basis for regulation of the coordinately expressed fibrinogen genes: homology in the 5' flanking regions.
Authors: Fowlkes D M , Mullis N T , Comeau C M , Crabtree G R
Published: May 2006

The three chains of fibrinogen are encoded by three separate genes whose transcription is coordinately regulated. The breakdown of fibrinogen during the acute-phase reaction leads to a simultaneous increase in alpha-, beta-, and gamma-fibrinogen mRNA in the liver. In a search for the basis of this coordinate increase in transcription, we have determined the sequences of the regions surrounding the points of transcriptional initiation of the three rat fibrinogen genes, 1490 nucleotides upstream and 730 nucleotides downstream. Two unique regions of homology have been found. One region consists of 15 nucleotides that have a common 6-nucleotide core lying between -116 and -160; the other is approximately equal to 100 nucleotides long and is in the -165 to -472 region. In this region, the beta- and gamma-fibrinogen genes are approximately equal to 65% homologous. alpha-Fibrinogen has somewhat less homology with both beta- and gamma-fibrinogen. In addition, the beta-fibrinogen gene has 22 nucleotides at position -480 that are homologous to sequences that have been noted to occur in glucocorticosteroid-regulated genes in a similar position. We feel that these areas of conserved sequences play a role in the regulation of the transcription of fibrinogen. The fibrinogen chains are synthesized as precursor peptides, and the amino-terminal portion, the so-called signal peptide, is removed during the translocation of the peptide chain across the endoplasmic reticulum. We have determined those sequences that encode the signal peptides. Homology in the amino acid sequence between the rat and human signal peptides varies between 52% for alpha-fibrinogen and 66% for beta-fibrinogen. This homology implies that there has been strong selective pressure on this portion of these genes.
Interaction of the 1 alpha,25-dihydroxyvitamin D3 receptor with RNA and synthetic polyribonucleotides.
Authors: Franceschi R T
Published: May 2006

The interaction of the 1 alpha,25-dihydroxyvitamin D3 receptor with RNA and synthetic polynucleotides has been examined by using receptor from rachitic chicken intestine. Total intestinal RNA inhibited the binding of receptor to calf thymus DNA-cellulose with an efficiency equivalent to single-stranded DNA. A comparison of the inhibitory activity of several polyribonucleotides gave the following order of activities: poly(I) = poly(G) greater than or equal to double-stranded DNA greater than single-stranded DNA = poly(U). Poly(A), poly(C), and the ribonucleoside monophosphates AMP, GMP, CMP, and UMP had minimal activity. A preference for single-stranded homopolymers was observed [i.e., poly(I) and poly(U) were active, whereas poly(I):poly(C) and poly(U):poly(A) were not]. The ability of nucleic acids to displace receptor from DNA-cellulose was also measured. Both poly(G) and poly(I) were more active than double-stranded DNA in this assay. Furthermore, differences were noted between intestinal RNA fractions separated on the basis of poly(A) content. Receptor also bound to immobilized intestinal RNA and polynucleotides. The KCl concentration necessary to disrupt binding to a given polynucleotide generally paralleled the activity of that molecule in DNA-cellulose inhibition and displacement assays. These results suggest that the 1 alpha,25-dihydroxyvitamin D3 receptor can interact with RNA as well as DNA.
Immunity and protection against influenza virus by synthetic peptide corresponding to antigenic sites of hemagglutinin.
Authors: Shapira M , Jibson M , Muller G , Arnon R
Published: May 2006

Four peptides have been synthesized, corresponding to different regions of the H3 influenza hemagglutinin, that are related to antigenic sites "A" and "B" of the molecule. The peptides consisted of the following sequences: 139-146, which forms the "loop" in the native hemagglutinin molecule, with either glycine or aspartic acid at position 144; 147-164, which contains part of antigenic determinant "B"; and 138-164, which comprises both the loop and the area 147-164. The peptides were conjugated to tetanus toxoid and used for immunization of rabbits and mice. All four conjugates elicited an immune response against the homologous peptides, but only the peptides 138-164 and 147-164 gave rise to antibodies that recognized and bound to the intact virus. Protection of mice against challenge infection with A/Eng/42/72 virus was achieved only by immunization with the conjugate (138-164)-TT, which led to partial protective effect. These data emphasize the role of molecular structure in determining the antigenic properties of synthetic peptides and indicate that the length of the peptide could be crucial for enforcing the right folding required to mimic the native structure.
Monoclonal integration of human T-cell leukemia provirus in all primary tumors of adult T-cell leukemia suggests causative role of human T-cell leukemia virus in the disease.
Authors: Yoshida M , Seiki M , Yamaguchi K , Takatsuki K
Published: May 2006

The genome of human T-cell leukemia virus (HTLV) was surveyed in fresh tumor cells of 163 patients with lymphoma and leukemia from the southwest part of Japan where adult T-cell leukemia (ATL) is endemic. Leukemic cells of all 88 cases of ATL tested so far were found to contain the provirus genome and also found to be monoclonal with respect to the integration site of provirus genome. In most cases of ATL, leukemic cells contained one or two copies of the complete HTLV provirus genome, and it was shown that the single species of HTLV with a fully determined sequence is typical in ATL. Some cases of T-cell malignancies, diagnosed as chronic lymphocytic leukemia or non-Hodgkin lymphoma, also had the provirus genome in their tumor cells, whereas some cases with the same diagnosis did not. No cases of other types of lymphoma or leukemia contained the provirus genome in their tumor cells. Monoclonal integration of the HTLV provirus genome in all primary tumor cells of ATL not only indicates that HTLV directly interacts with target cells, which become leukemic, and that integration of the provirus genome is a prerequisite for development of ATL and possibly other related diseases but also indicates that the virus is not associated with other types of lymphoma or leukemia.
Insulin and insulin-like growth factor II permit nerve growth factor binding and the neurite formation response in cultured human neuroblastoma cells.
Authors: Recio-Pinto E , Lang F F , Ishii D N
Published: May 2006

In serum-free medium, SH-SY5Y human neuroblastoma cells specifically and reversibly lost the capacity to bind 125I-labeled nerve growth factor (NGF) to the high-affinity sites (slow sites) and to respond by neurite outgrowth, unless physiological concentrations of insulin or insulin-like growth factor II were present. In serum-containing medium, anti-insulin antiserum decreased the neurite formation response to NGF, and insulin supplementation increased the number of available NGF slow sites. The low-affinity NGF fast sites are absent from SH-SY5Y cells and did not emerge on treatment with insulin. Insulin potentiated the induction of neurites by NGF in rat pheochromocytoma PC12 cells also. These results implicate a wider role for insulin and its homologs in the nervous system.
Structure of the 5' ends of immunoglobulin genes: a novel conserved sequence.
Authors: Parslow T G , Blair D L , Murphy W J , Granner D K
Published: May 2006

Recent investigations have suggested that tissue-specific regulatory factors are required for immunoglobulin gene transcription. Cells of the mouse lymphocytoid pre-B-cell line 70Z/3 contain a constitutively rearranged immunoglobulin kappa light chain gene; the nucleotide sequence of this gene exhibits all the known properties of a functionally competent transcription unit. Nevertheless, transcripts derived from this gene are detectable only after exposure of the cells to bacterial lipopolysaccharide, implying that accurate DNA rearrangement is not sufficient to activate expression of the gene. Comparison of the sequence of the 70Z/3 kappa light chain gene with those encoding other immunoglobulin heavy and light chains has revealed that a distinctive promoter region structure is characteristic of this multigene family. The sequence A-T-T-T-G-C-A-T lies approximately 70 base pairs upstream from the site of transcriptional initiation in every light chain gene examined; in heavy chain genes, the corresponding location is occupied by the precise inverse (A-T-G-C-A-A-A-T) of this sequence. Although adjacent regions of DNA have diverged extensively in evolution, these octanucleotide sequences are stringently conserved at this location among diverse immunoglobulin genes from at least two mammalian species. The proximity of this conserved octanucleotide block to the site of transcriptional initiation suggests that it may serve as a recognition locus for factors regulating immunoglobulin gene expression in a tissue-specific fashion.
Cellular ADP-ribosyltransferase with the same mechanism of action as diphtheria toxin and Pseudomonas toxin A.
Authors: Lee H , Iglewski W J
Published: May 2006

An ADP-ribosyltransferase was found in elongation factor 2 (EF-2) preparations from polyoma virus-transformed baby hamster kidney (pyBHK) cells. Like fragment A of diphtheria toxin and Pseudomonas toxin A, this eukaryotic cellular enzyme transfers [14C]adenosine from NAD+ to EF-2. However, the cellular transferase is immunologically distinct from fragment A. The transferase also can be distinguished from fragment A and Pseudomonas toxin A by the inhibition of the activity of the former by cytoplasmic extracts and by histamine. Snake venom phosphodiesterase digestion of the [14C]adenosine-labeled EF-2 product of the cellular transferase reaction yielded [14C]AMP, indicating that the cellular enzyme is a mono(ADP-ribosyl)transferase. The forward ADP-ribosylation reaction catalyzed by the cellular enzyme is reversed by fragment A, yielding [14C]NAD+. The results strongly suggest that the cellular transferase is a mono(ADP-ribosyl)transferase, which ADP-ribosylates the same diphthamide residue of EF-2 as does fragment A and Pseudomonas toxin A.