bluuebunny/crossref_metadata_2025 huggingface dataset:
Alternative modes of enkephalin biosynthesis regulation by reserpine and cyclic AMP in cultured chromaffin cells.
Authors: Eiden L E , Giraud P , Affolter H U , Herbert E , Hotchkiss A J
Published: May 2006

Exposure of bovine chromaffin cells in primary culture to 5 microM reserpine or 25 microM forskolin results in an increase in enkephalin peptide levels within 24-48 hr; 25 microM forskolin (or cholera toxin at 50 micrograms/ml) causes a 1.5- to 2-fold increase in enkephalin peptide levels, which is maximal after 48 hr of exposure and is totally blocked by addition of cycloheximide (0.5 microgram/ml). Reserpine (5 microM) elicits a 1.5- to 2-fold increase in enkephalin peptide levels within 24 hr, which is only partially blocked by cycloheximide. Chromatographic analysis of cellular extracts shows that forskolin increases levels of both [Met]enkephalin pentapeptide and high molecular weight enkephalin-containing peptides, while reserpine causes an increase in [Met]enkephalin pentapeptide and a concomitant decrease in high molecular weight enkephalin-containing peptides, suggesting enhanced conversion of enkephalin precursor(s) to the mature polypeptide hormone. Measurement of preproenkephalin messenger RNA (mRNAenk) by RNA blot hybridization with a cDNA probe for mRNAenk reveals that forskolin and cholera toxin cause a relatively rapid (less than 17 hr) 3- to 5-fold increase in mRNAenk, while exposure to reserpine elicits a gradual decrease in enkephalin mRNA (a 50%-80% decline) beginning within 24 hr and continuing over a 72-hr period. These results suggest that forskolin and reserpine differentially regulate enkephalin biosynthesis in cultured chromaffin cells, the former by increasing, presumably via a cAMP-dependent mechanism, cellular mRNA coding for preproenkephalin and the latter by a post-translational increase in proenkephalin processing.
Illegitimate recombination generates a class switch from C mu to C delta in an IgD-secreting plasmacytoma.
Authors: Gilliam A C , Shen A , Richards J E , Blattner F R , Mushinski J F , Tucker P W
Published: May 2006

We present here the sequence characterization of a C mu----C delta immunoglobulin (Ig) heavy chain class switch. In the murine IgD-secreting plasmacytoma TEPC 1017, deletion of most of the mu switch recombination region (S mu) and the entire C mu gene occurred in the absence of switch region sequences 5' to C delta. This unique rearrangement resulted from an illegitimate recombination of sequences with only patchy homology to each other. The infrequent and variable nature of illegitimate recombination may explain the low frequency of IgD-secreting plasma cells in normal mouse tissues.
Role of T3 surface molecules in human T-cell activation: T3-dependent activation results in an increase in cytoplasmic free calcium.
Authors: Weiss A , Imboden J , Shoback D , Stobo J
Published: May 2006

The human T-cell leukemia, Jurkat, and a T3-negative mutant of Jurkat (S.5) were used to study the role of T3 in human T-cell activation. Incubation of Jurkat with phytohemagglutinin (PHA) resulted in the production of interleukin 2, which was markedly increased by the addition of phorbol 12-myristate 13-acetate (PMA). Antibodies reactive with T3 could activate Jurkat only if added together with PMA. However, S.5 cells failed to produce interleukin 2 in response to PHA and produced 1/16th the interleukin 2 activity that Jurkat produced in response to PHA and PMA. Incubation of S.5 cells with the calcium ionophore A23187 and PMA resulted in the production of interleukin 2 activity comparable to that produced by Jurkat. Like antibodies reactive with T3, A23187 demonstrated an obligate requirement for PMA in order to activate Jurkat or S.5. These observations suggested that T3 might participate in T-cell activation through mechanisms that increase intracellular Ca2+. This was examined by using the Ca2+ sensitive fluor, quin-2, to measure levels of cytoplasmic free Ca2+ [( Ca2+]i). Addition of PHA, A23187, or monoclonal antibodies reactive with T3 to Jurkat cells resulted in substantial increases of [Ca2+]i. In contrast, only A23187 could induce an increase in [Ca2+]i in S.5 cells. Three other monoclonal antibodies reactive with other membrane antigens expressed on Jurkat or S.5 did not increase [Ca2+]i. These results suggest that T3 and/or associated molecules participate in T-cell activation through mechanisms that lead to increases in [Ca2+]i and that their expression is a relative requirement for T-cell activation by PHA.
Striking similarities in amino acid sequence among nonstructural proteins encoded by RNA viruses that have dissimilar genomic organization.
Authors: Haseloff J , Goelet P , Zimmern D , Ahlquist P , Dasgupta R , Kaesberg P
Published: May 2006

The plant viruses alfalfa mosaic virus (AMV) and brome mosaic virus (BMV) each divide their genetic information among three RNAs while tobacco mosaic virus (TMV) contains a single genomic RNA. Amino acid sequence comparisons suggest that the single proteins encoded by AMV RNA 1 and BMV RNA 1 and by AMV RNA 2 and BMV RNA 2 are related to the NH2-terminal two-thirds and the COOH-terminal one-third, respectively, of the largest protein encoded by TMV. Separating these two domains in the TMV RNA sequence is an amber termination codon, whose partial suppression allows translation of the downstream domain. Many of the residues that the TMV read-through domain and the segmented plant viruses have in common are also conserved in a read-through domain found in the nonstructural polyprotein of the animal alphaviruses Sindbis and Middelburg. We suggest that, despite substantial differences in gene organization and expression, all of these viruses use related proteins for common functions in RNA replication. Reassortment of functional modules of coding and regulatory sequence from preexisting viral or cellular sources, perhaps via RNA recombination, may be an important mechanism in RNA virus evolution.
A beta-globin gene, inactive in the K562 leukemic cell, functions normally in a heterologous expression system.
Authors: Fordis C M , Anagnou N P , Dean A , Nienhuis A W , Schechter A N
Published: May 2006

The K562 human leukemia cell is an erythroid-like cell that may serve as a model for the study of globin gene expression in transcriptionally active human erythroid cells. K562 cells express all globin genes with the exception of that for beta-globin; failure to produce beta-globin could result from an acquired mutation in each of the beta-globin genes or from an alteration in the regulatory factor environment of the beta-globin gene. To uncover a possible acquired mutation, restriction endonuclease analysis of genomic K562 DNA and expression studies of a cloned K562 beta-globin gene were carried out. Restriction endonuclease analysis revealed no structural alteration of the K562 beta-globin genes. Analysis of the polymorphic Ava II site in intervening sequence 2 of the beta-globin gene showed that K562 cells contain two different beta-globin alleles, both of which are inactive. A K562 beta-globin gene was cloned, ligated into the expression vector pLTN3B, and introduced into COS cells. Transcripts were analyzed by RNA blot, dot blot, S1 nuclease mapping, and primer extension assay. The cloned K562 beta-globin gene was transcribed in COS cells as efficiently as a normal beta-globin gene introduced into COS cells; the mRNA was 10 S and polyadenylylated; the 5' and 3' termini and the processing of transcripts were identical to that of mRNA transcribed from a normal gene. Based on these data we suggest that the absence of beta-globin gene expression results not from an alteration in the beta-globin gene, but from a quantitative or qualitative alteration in a trans-acting factor important in beta-globin gene expression.
Epstein-Barr virus receptor of human B lymphocytes is the C3d receptor CR2.
Authors: Fingeroth J D , Weis J J , Tedder T F , Strominger J L , Biro P A , Fearon D T
Published: May 2006

Identity of the Epstein-Barr virus (EBV) receptor with the complement receptor type 2 (CR2) was established in three sets of experiments using the monoclonal antibodies, HB-5 and anti-B2, which recognize a Mr 145,000 B-lymphocyte membrane protein that is CR2. First, the rank order for binding of fluoresceinated EBV to four lymphoblastoid cell lines (SB, JY, Raji, and Molt-4) was identical to the rank order for binding of HB-5 and anti-B2 by analytical flow cytometry. Second, pretreatment of cells with HB-5 followed by treatment with goat F(ab')2 fragments to mouse IgG blocked binding of fluoresceinated EBV on SB, a B-lymphoblastoid cell line. Virus attachment was not inhibited by HB-5 alone, second antibody alone, rabbit anti-C3b receptor, or UPC10 (an irrelevant monoclonal antibody). Third, transfer of CR2 from SB to protein A-bearing Staphylococcus aureus particles, to which HB-5 had been absorbed, conferred on them the specific ability to bind 125I-labeled EBV. We conclude that CR2 is the EBV receptor of human B lymphocytes.
Internal complementarities in the immune system: regulation of the expression of helper T-cell idiotypes.
Authors: Martinez C , Pereira P , Bernabé R , Bandeira A , Larsson E L , Cazenave P A , Coutinho A
Published: May 2006

More than half of BALB/c helper T lymphocytes specific for 2,4,6-trinitrophenyl (TNP)-modified syngeneic spleen cells are inhibited in their proliferative responses to antigen-presenting cells and in their cooperation with B lymphocytes by monoclonal anti-idiotypic antibodies directed to a TNP-binding BALB/c myeloma protein (MOPC 460). This inhibition is specific for anti-TNP-self helper cells of BALB/c origin and is controlled by IgCh-linked genes, as it is not observed with CB.20 helper cells of the same specificity. In contrast, anti-TNP-self helper cells prepared from BALB/c mice that were chronically suppressed with anti-mu chain antibodies and possessed no B lymphocytes were not inhibited by anti-idiotypic antibodies. We conclude that the B-cell antibody repertoires contribute to the selection of the (idiotypic) T-helper-cell repertoires.
Influence of the beta-adrenergic receptor concentration on functional coupling to the adenylate cyclase system.
Authors: Severne Y , Coppens D , Bottari S , Riviere M , Kram R , Vauquelin G
Published: May 2006

Only part of the beta-adrenergic receptors can undergo functional coupling to the adenylate cyclase regulatory unit. This receptor subpopulation shows an increased affinity for agonists in the presence of Mg2+ and undergoes rapid "inactivation" (locking-in of the agonist) by the alkylating reagent N-ethylmaleimide in the presence of agonists. Several experimental conditions, known to modify the total receptor concentration without alteration of the other components of the adenylate cyclase system, do not affect the percentage of receptors that can undergo functional coupling: (i) homologous regulation of beta 1 receptors in rat brain by noradrenaline (through antidepressive drug or reserpine injections); (ii) up- and down-regulation of the beta 2 receptors in Friend erythroleukemia cells by, respectively, sodium butyrate and cinnarizine treatment; and (iii) dithiothreitol-mediated inactivation of receptors in turkey erythrocytes, Friend erythroleukemia cells, and rat brain. Our findings argue against a stoichiometric limitation in the number of regulatory components, genetically different receptor subpopulations, bound guanine nucleotides, or reduced accessibility of part of the receptors to the agonists as the cause for functional receptor heterogeneity. Differences in either the receptor conformation or its membrane microenvironment are more plausible explanations.
An evolutionary tree relating eight alphaviruses, based on amino-terminal sequences of their glycoproteins.
Authors: Bell J R , Kinney R M , Trent D W , Strauss E G , Strauss J H
Published: May 2006

The NH2-terminal amino acid sequences of both structural glycoproteins of each of eight alphaviruses have been obtained. These sequences demonstrate that the alphaviruses are all closely related and have in all probability descended from a common ancestor. Cysteines are conserved as well as several other residues important for secondary structure, suggesting that the three-dimensional conformations of the alphavirus glycoproteins are conserved while considerable variation in the primary sequence has evolved. Secondary structure predictions based upon the amino acid sequences are consistent with this hypothesis. An evolutionary tree for these eight alphaviruses has been constructed from the amino acid sequence data and, at many positions in the sequence, the amino acids present in the ancestral glycoproteins have been deduced.
Different joining region J elements of the murine kappa immunoglobulin light chain locus are used at markedly different frequencies.
Authors: Wood D L , Coleclough C
Published: May 2006

In order to assess whether DNA rearrangement occurs with equal frequency at each of the several J (joining region) elements in the mouse kappa light chain locus, we set out to determine the relative frequency of usage of J kappa segments in populations of B lymphocytes unperturbed by antigenic selection or cloning. To obtain such a population, we exposed a suspension of spleen cells to a mixture of mitogens capable of activating most B cells independently of their specificity for antigen. We estimated the relative usage of the J kappa elements in unspliced kappa chain gene transcripts in total and poly(A)-containing nuclear RNA, using an S1 nuclease protection assay, and in mature kappa chain mRNA, using a specifically primed cDNA hybridization assay. Both types of assay reveal a marked difference in the frequency of J kappa elements and indicate that their relative usage is: J1 approximately J2 much greater than J4 approximately J3. Comparison of the 5' flanking regions of the mouse J kappa elements, including the conserved putative recombination target sequences, shows no obvious differences consistent with the variation in recombinational efficiency, so we conclude that, although the consensus heptamer and nonamer signals may be sufficient to identify a recombination site, the probability that that site will be used depends also on other determinants. A review of published data suggests a nonequivalence of usage also among human J kappa elements and between mouse J lambda I and J lambda III loci. Extending the comparison to include these sets of sequences indicates that one of the determinants of frequency of J use may be the proximity of the consensus heptamer to a T-G dinucleotide within the J coding sequence, perhaps revealing an Escherichia coli gyrase-like substrate preference within a recombination enzyme.
Structural model of human ceruloplasmin based on internal triplication, hydrophilic/hydrophobic character, and secondary structure of domains.
Authors: Ortel T L , Takahashi N , Putnam F W
Published: May 2006

A molecular model for the structure of human ceruloplasmin is proposed that is based on the determination of the complete amino acid sequence, studies of the products of limited proteolytic cleavage, calculations of the hydrophilic/hydrophobic character (hydropathy profile), and predictions of the local secondary structure. This multicopper oxidase (Mr approximately 132,000) consists of a single polypeptide chain (1046 amino acid residues) with four attached glucosamine oligosaccharides. Computer-assisted statistical analysis of the internal repetition in the amino acid sequence confirms that the entire polypeptide chain is divided into three contiguous homology units, each containing about 350 amino acid residues. Each homology unit is subdivided into three domains, designated A1, A2, and B, that differ in structure and probably in function. Calculations of the hydropathy profile and predictions of the secondary structure support a molecular model based on internal repetition of three homology units and help to identify characteristic features of the interdomain junctions. The alignment scores for internal duplication of pairings of the three homology units of ceruloplasmin exceed the scores yet reported for contiguous internal duplication of any other protein. This highly significant evidence for intragenic repetition suggests that the ceruloplasmin molecule evolved by tandem triplication of ancestral genes coding for a primordial copper oxidase.
Identification of a cloned cytokinin biosynthetic gene
Authors: Barry G. F. , Rogers S. G. , Fraley R. T. , Brand L.
Published: May 2006

A small region of the Ti plasmid (the
tmr
locus), thought to be involved in phytohormone metabolism in
Agrobacterium tumefaciens
-transformed plant tissue, was cloned and expressed in
Escherichia coli
. By enzyme assay, the
tmr
locus was shown to encode isopentenyltransferase, an enzyme that catalyzes the first step in cytokinin biosynthesis.
Epidermal growth factor inhibits the synthesis of the nuclear protein cyclin in A431 human carcinoma cells.
Authors: Bravo R
Published: May 2006

The effect of epidermal growth factor (EGF) on the synthesis of the nuclear protein cyclin and its relationship with cell proliferation has been studied in human carcinoma A431 cells. Quantitative two-dimensional gel electrophoretic analysis of [35S]methionine-labeled polypeptides from EGF-treated and untreated A431 cells showed that the synthesis of cyclin (0.07% of total labeled protein) could be decreased to 20% (less than 0.01% of total labeled protein) by EGF at a concentration of 100 ng/ml. A good correlation between the effect of different concentrations of EGF on cyclin synthesis and A431 cell proliferation was found. A431-derived cells resistant to the growth-inhibitory effect of EGF showed no significant changes in cyclin synthesis after EGF treatment. Taken together, these results support the idea that cyclin may be an important protein that is involved in the control of cell proliferation.
Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts.
Authors: Collins T , Korman A J , Wake C T , Boss J M , Kappes D J , Fiers W , Ault K A , Gimbrone M A , Strominger J L , Pober J S
Published: May 2006

Immune interferon (IFN-gamma) increases the surface expression of HLA-A,B antigens and induces the surface expression of HLA-DR antigens on vascular endothelial cells and dermal fibroblasts. Here we report that IFN-gamma induces parallel expression of two other class II major histocompatibility complex (MHC) antigens, SB and DC. Maximal surface expression of all three antigens is reached in 4-6 days, and HLA-DR and -SB are induced to a higher level of expression than HLA-DC. For all three class II antigens, induction is marked by the de novo appearance of detectable transcripts of class II heavy and light chains and of the non-MHC-encoded invariant chain, suggestive of the transcription of multiple previously silent genes. Class I message levels and antigen expression are also increased by IFN-gamma at similar rates but from initial levels that are 50% of maximal. After removal of IFN-gamma, class II antigen expression persists for at least 4 days, while mRNA levels decrease rapidly. The parallel induction and persistence of the several class II MHC antigens may be important in conferring immune accessory function on vascular and stromal cells.
Cloning and physical mapping of a gene fragment coding for a 64-kilodalton major late antigen of human cytomegalovirus.
Authors: Pande H , Baak S W , Riggs A D , Clark B R , Shively J E , Zaia J A
Published: May 2006

We have isolated a clone containing a gene fragment coding for a 64-kilodalton glycoprotein that is the major late antigen of human cytomegalovirus (HCMV). Based upon the amino acid sequence of a tryptic peptide of this glycoprotein (HCMVgp64), two sets of mixed-sequence probes, one consisting of a mixture of 16 heptadecadeoxyribonucleotides and the other a mixture of 32 icosadeoxyribonucleotides, were synthesized. A subgenomic library of HCMV (Towne strain) DNA was constructed in plasmid pBR327 and transformants were screened with 32P-labeled aliquots of these synthetic oligodeoxyribonucleotide probes. Two clones among 15,000 gave strong positive signals. Plasmid DNA was isolated from the positive clones and characterized by restriction mapping and Southern blot analysis using both probes. The plasmid DNA contained a 2.3-kilobase insert, which yielded an 800-base-pair and a 1500-base-pair fragment after Sau3A digestion. Only the 800-base-pair fragment hybridized to the mixed probes, and DNA sequence analysis revealed that it contains nucleotide sequences compatible with amino acid sequences of tryptic peptides of HCMVgp64. Restriction mapping studies of HCMV DNA using this 32P-labeled 800-base-pair cloned DNA have allowed us to locate this gene fragment in the long unique region of HCMV (Towne strain) genome at approximately equal to 0.5-0.51 map unit.
Conversion of proglucagon in pancreatic alpha cells: the major endproducts are glucagon and a single peptide, the major proglucagon fragment, that contains two glucagon-like sequences.
Authors: Patzelt C , Schiltz E
Published: May 2006

It has previously been shown by biosynthetic labeling studies that glucagon is synthesized in mammalian islets via an 18-kDa precursor, proglucagon, that during processing gives rise to glucagon and a secreted peptide of 10 kDa (the major proglucagon fragment, MPGF). We have now developed a simple procedure for the isolation of this peptide from rat pancreatic islets and have characterized it more fully. On the basis of its amino acid composition, MPGF is identified as the COOH-terminal portion of proglucagon that contains two glucagon-related sequences. These sequences do not appear to be liberated from MPGF in alpha cells of the islets of Langerhans but MPGF may be processed further elsewhere in the body or in other cells of the gastrointestinal tract that produce glucagon precursors.
Constitutive production of human interferons by mouse cells with bovine papillomavirus as a vector.
Authors: Fukunaga R , Sokawa Y , Nagata S
Published: May 2006

Mouse C127I cells were transformed with a chimeric plasmid consisting of bovine papillomavirus and human interferon (HuIFN) gene (either IFN-gamma or IFN-alpha 5) placed under the control of simian virus 40 early promoter. The transformed cells retained 30-50 copies each of the hybrid plasmid extrachromosomally and secreted a high level of HuIFNs constitutively up to 3-4 X 10(5) international units/ml. The secreted HuIFN-gamma had no detectable activity on mouse cells, whereas HuIFN-alpha 5 was slightly active on mouse cells. Each IFN was neutralized either by anti-HuIFN-gamma or anti-HuIFN-alpha antiserum. The partially purified 35S-labeled HuIFN-gamma produced by the transformed cells showed heterogeneous bands with apparent Mrs of 22,000-25,000 on NaDodSO4/polyacrylamide gel electrophoresis. This value is similar to that of natural IFN-gamma and larger than the molecular weight calculated from the amino acid sequence, which suggests that HuIFN-gamma secreted by transformed mouse cells was glycosylated. The 35S-labeled IFN-alpha 5, immunoprecipitated with anti-HuIFN-alpha antiserum from the supernatant of the transformed cells, had a molecular weight of mature protein. These results suggest that the bovine papillomavirus vector can be used to produce a large quantity of foreign proteins in mammalian cells.
Na+ movement in a single turnover of the Na pump.
Authors: Forbush B
Published: May 2006

Ouabain-sensitive 22Na efflux from right-side-out membrane vesicles prepared from dog kidney has been examined with a time resolution of 30 msec. The vesicles are preloaded with 22Na and caged ATP [P3-1-(2-nitro)phenylethyl adenosine triphosphate], so that transport by the Na pump can be initiated by light. After a brief illumination, which releases less ATP than the number of catalytic sites, a burst of 22Na extrusion is observed corresponding to a single turnover of the Na pump. By the use of a rapid filtration apparatus, with which a continuous record of the rate of efflux is obtained, it has been possible to resolve the efflux burst in the time range of 20-1500 msec. The rate of efflux rises rapidly, but not instantaneously, to a peak and then decays, with a time constant of approximately equal to 6 sec-1 at 15 degrees C. The time course of Na efflux is unaffected by extracellular K+, as predicted by models of the Na pump in which Na is released early in the cycle. Unphotolyzed caged ATP is found to bind to the catalytic site of Na,K-ATPase, in competition with ATP that is produced in the flash, and the possibility has not been excluded that dissociation of unphotolyzed caged ATP and binding of ATP are involved in the Na efflux time course. It seems most likely that binding of ATP and translocation of 22Na are involved in the increase in the 22Na efflux rate in the single turnover and that the release of transported 22Na from extracellular pump sites limits the slow decay.
Isolation and partial molecular characterization of pituitary fibroblast growth factor.
Authors: Böhlen P , Baird A , Esch F , Ling N , Gospodarowicz D
Published: May 2006

Fibroblast growth factor (FGF) has been purified to homogeneity from bovine pituitaries by two methods. Starting material for both methods was an FGF preparation partially purified as described by Gospodarowicz [Gospodarowicz, D. (1975) J. Biol. Chem. 250, 2515-2520]. Purification procedure I involved cation-exchange and reversed-phase HPLC, while procedure II employed gel filtration and ion-exchange chromatography. Isolation was monitored by testing column fractions for their capacity to stimulate the proliferation of vascular endothelial cells in vitro. The growth factor has an approximate molecular weight of 16,000. Its amino-terminal sequence was determined as Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly-Ala-Phe-Pro-Pro-Gly. Sequence and amino acid composition indicate that the structure of pituitary FGF is different from that of other known growth factors. Pituitary FGF, as isolated under nonacidic conditions (procedure II), has high potency and intrinsic activity to stimulate adult bovine aortic endothelial cells (half-maximal proliferation at 2 pM). Acidic conditions as in procedure I, however, lead to about 90% loss of potency while the intrinsic activity remains intact (identical maximal stimulation values). By all other criteria (molecular weight, amino acid composition, amino-terminal sequence), the two preparations are indistinguishable. Antibodies were raised in rabbits against a synthetic peptide representing the first nine residues of the amino-terminal sequence of the pituitary FGF. The polyclonal antibodies recognize the synthetic peptide and the purified growth factor on an equimolar basis and are capable of inhibiting mitogenic activity in vitro. This report describes a partial chemical characterization of a pituitary FGF and demonstrates rigorously that the characterized protein possesses the mitogenic activity commonly referred to as "basic pituitary FGF."
Nature and origin of the RNA associated with simian virus 40 large tumor antigen.
Authors: Darlix J L , Khandjian E W , Weil R
Published: May 2006

Simian virus 40 (SV40) large tumor (T) antigen isolated from mammalian cells undergoing lytic or transforming infection is associated with small RNA fragments ("T-antigen RNA") that are protected from nuclease digestion. The rather high complexity of the ribonuclease T1 fingerprints of T-antigen RNA suggested that it is mainly derived from cellular heterogeneous nuclear RNAs. In the present study, 5'-32P-labeled T-antigen RNA was hybridized to monkey, mouse, and human Alu and SV40 DNA, and the nucleotide sequence of 37 T1 oligonucleotides was determined. The results suggest that the bulk of T-antigen RNA is derived from noncoding, double-stranded, ordered regions of cellular heterogeneous nuclear RNAs that exhibit sequence homologies with interspersed repetitive elements of the cellular genome. The possible biological implications of these results are discussed.
Demonstration of the production of frameshift and base-substitution mutations by quasipalindromic DNA sequences.
Authors: de Boer J G , Ripley L S
Published: May 2006

The in vivo production of frameshift and base-substitution mutations predicted as a consequence of the metabolic processing of misaligned quasipalindromic DNA sequences has been confirmed. Spontaneous frameshift mutations of the T4 rII gene that had been genetically mapped to quasipalindromic DNA sequences were sequenced. Some of the mutant sequences are exactly those predicted by a mutational model based on misaligned quasipalindromes. Furthermore, these sequences are distinct from those predicted by the classical frameshift model based on misaligned repeated sequences. The rII frameshift mutant sequences reported here result from the deletion of a specific base or bases that would remain looped out should the quasipalindromes assume a hairpin secondary structure. One hairpin predicted not only the deletion of two bases (a frameshift) but the concomitant production of nearby but noncontiguous base substitutions. The substitution of as many as three bases as well as the frameshift were predicted to arise as a consequence of a single mutational event in the palindrome. Two independent examples of the predicted deletion frameshift were found among the small sample of sequenced spontaneous frameshifts examined and both were associated with the predicted transversion and transition base substitutions.
Clones from the human gene complex coding for salivary proline-rich proteins.
Authors: Azen E , Lyons K M , McGonigal T , Barrett N L , Clements L S , Maeda N , Vanin E F , Carlson D M , Smithies O
Published: May 2006

The salivary protein gene complex consists of a series of loci coding for related but distinct proline-rich proteins (PRPs) found chiefly in saliva. We have screened a library of human genomic DNA fragments in bacteriophage lambda Charon 4A with a PRP cDNA synthesized and cloned from rat parotid gland mRNA. Two phages (PRP1 and PRP2) hybridizing to the rat probe under moderately stringent conditions contain related but not identical DNAs. Preliminary nucleotide sequence data indicate that both DNAs include regions comprised of nearly identical tandemly repeated sequences, each able to code for about 21 amino acids. The decoded consensus repeat sequence is homologous to the repeating amino acid units found by others in human PRPs. This and other features demonstrate that these two clones are members of the PRP gene family. Polymorphic differences between the DNAs of different individuals were observed after probing digests of human genomic DNA with a HinfI fragment from PRP1. These DNA polymorphisms reflect size differences, possibly caused by frequent unequal crossing-over between the repeated units in the PRP genes.
Site-specific carcinogen binding to DNA.
Authors: Boles T C , Hogan M E
Published: May 2006

Benzo[alpha]pyrene diol epoxide (BPDE) is a well-studied environmental carcinogen that binds covalently to DNA. Here we describe a photochemical technique that allows us to map BPDE-binding sites within cloned gene sequences. The technique is based upon our observation that, when irradiated with laser light at 355 nm, one single-strand DNA cut is produced at each BPDE binding site. In initial experiments we have studied the distribution of such cuts in cloned DNA from the chicken adult beta-globin gene. We find that BPDE binding in this gene sequence is distinctly nonrandom. While several prominent BPDE-binding sites are evident, a 300-base-pair sequence immediately 5' to the RNA cap site is most strongly attacked by the carcinogen. This region is believed to contain important transcriptional control sequences. We discuss the possibility that sequence-specific binding to such regulatory elements may be an important feature of the mechanism of the carcinogen.
The complete cDNA and deduced amino acid sequence of a type II mouse epidermal keratin of 60,000 Da: analysis of sequence differences between type I and type II keratins.
Authors: Steinert P M , Parry D A , Racoosin E L , Idler W W , Steven A C , Trus B L , Roop D R
Published: May 2006

We present the complete nucleotide and deduced amino acid sequences of a mouse epidermal keratin subunit of 60,000 Da. The keratin possesses a central alpha-helical domain of four tracts (termed 1A, 1B, 2A, and 2B) that can form coiled-coils, interspersed by short linker sequences, and has non-alpha-helical terminal domains. This pattern of secondary structure is emerging as common to all intermediate filament subunits. The alpha-helical sequences conform to the type II class of keratins. Accordingly, this is the first type II keratin for which complete sequence information is available, and thus it facilitates elucidation of the fundamental distinctions between type I and type II keratins. It has been observed that type I keratins are acidic and type II keratins are neutral--basic in charge. We suggest that the basis for this empirical correlation between type and charge resides in the respective net charges of the 1A and 2B tracts. Calculations on interchain interactions between charged residues in the alpha-helical domains indicate that this keratin prefers to participate in dimers according to an in-register parallel arrangement. The terminal domains of this keratin possess characteristic glycine-rich sequences, and the carboxyl-terminal domain is highly homologous to that of a human epidermal keratin of 56,000 Da. According to the hypothesis that end-domains are located on the periphery of keratin filaments, we conclude that the corresponding mouse and human keratins are closely related, both structurally and functionally.
Expression of hepatitis B virus S gene by herpes simplex virus type 1 vectors carrying alpha- and beta-regulated gene chimeras.
Authors: Shih M F , Arsenakis M , Tiollais P , Roizman B
Published: May 2006

The domain of the hepatitis B virus (HBV) S gene specifying the HBV surface antigen (HBsAg) and comprising 25 base pairs of the 5'-transcribed noncoding region, the structural gene sequences, and the 3'-noncoding gene sequences including the polyadenylylation site was fused to the promoter-regulatory regions of the beta-thymidine kinase and of the alpha 4 gene of herpes simplex virus type 1 (HSV-1). The chimeric constructs were then inserted into the HSV-1 genome and specifically into the thymidine kinase gene by homologous recombination through flanking sequences. Cells infected with recombinants carrying the chimeric genes produced and excreted the HBsAg into the extracellular medium for at least 12 hr concurrently with the multiplication of the HSV-1 vector. The temporal patterns of expression and the observation that HBV S gene linked to the HSV-1 alpha promoter-regulatory region was regulated as an HSV-1 alpha gene indicate that the HBsAg gene chimeras inserted into the virus were regulated as viral genes. The HBsAg banded in isopycnic CsCl density gradients at a density of 1.17 g/cm3. Electron microscopic studies revealed that HBsAg harvested from the extracellular medium and banded in CsCl density gradients contained spherical particles 15-22 nm in diameter, characteristic of empty HBV envelopes. The results indicate that HSV-1 is a suitable vector for the expression of foreign genes placed under the control of HSV promoter-regulatory regions.
Cytoplasmic acidosis as a determinant of flooding intolerance in plants.
Authors: Roberts J K , Callis J , Jardetzky O , Walbot V , Freeling M
Published: May 2006

We present evidence that cytoplasmic acidosis is a cause of meristematic death in hypoxic root tips of maize and pea seedlings. Usually, leakage of acid from the vacuole is responsible for cytoplasmic acidosis. Leakage of acid, which occurs earlier during hypoxia in pea root tips than in maize root tips, appears to account for the lower tolerance of peas for hypoxia. Cytoplasmic acidosis is accelerated in maize root tips that are either (i) deficient in alcohol dehydrogenase, so that lactic acid production continues throughout hypoxia, or (ii) exposed to external CO2 during hypoxia, or (iii) perfused slowly so that escape of CO2 produced during ethanolic fermentation is retarded. All three conditions decrease the length of time maize root tips can tolerate hypoxia; more rapid cytoplasmic acidosis is associated with more rapid death under hypoxia. Possible mechanisms by which cytoplasmic acidosis leads to death are suggested; the mechanism does not involve inhibition of glycolysis by low pH.
Determinants for protein localization: beta-lactamase signal sequence directs globin across microsomal membranes.
Authors: Lingappa V R , Chaidez J , Yost C S , Hedgpeth J
Published: May 2006

A hybrid gene containing 182 codons of Escherichia coli beta-lactamase at the amino terminus of the corresponding protein and 141 codons of alpha-globin at the carboxyl terminus was generated by inserting chimpanzee alpha-globin cDNA into the Pst I site of plasmid pBR322. RNA transcribed in vitro from this plasmid gave a corresponding hybrid protein in a wheat germ cell-free translation system. The hybrid protein was protected from tryptic digestion and the pre-beta-lactamase signal peptide was removed when dog pancreas membrane vesicles were present during translation. A deletion mutant containing 23 codons of pre-beta-lactamase signal sequence and 5 codons of mature beta-lactamase fused to the alpha-globin cDNA gave a shorter hybrid protein that behaved similarly. However, a mutation that removed essentially all of the pre-beta-lactamase sequence gave a protein that was neither protected nor processed. Hence, at most, only the signal peptide and the first 5 amino acids of beta-lactamase were necessary to convert alpha-globin (a cytoplasmic protein) into a secretory protein.
Changes in local cerebral glucose utilization during rewarding brain stimulation.
Authors: Esposito R U , Porrino L J , Seeger T F , Crane A M , Everist H D , Pert A
Published: May 2006

The quantitative 2-deoxy[14C]glucose method was used to determine local cerebral glucose utilization in unrestrained rats responding (lever-press) for rewarding electrical stimulation to area A10 (ventral tegmental area) and in similarly implanted inactive controls. Self-stimulation was associated with significant increases in metabolic activity, highly circumscribed in the ventral tegmental area, that continued rostrally within a rather compact zone of activity through the medial forebrain bundle, extending via the diagonal band of Broca to the level of the preoptic area. In the forebrain terminal areas bilateral increases in local cerebral glucose utilization were noted in the nucleus accumbens, lateral septum, hippocampus, and the mediodorsal nucleus of the thalamus. Ipsilateral (i.e., side of stimulation) increases in glucose utilization were noted in the bed nucleus of the stria terminalis, the basolateral and central amygdaloid nuclei, and the medial prefrontal cortex. Caudal to the stimulation site, increases in glucose utilization were found in the midline dorsal raphe, the ipsilateral pontine gray, medial parabrachial nucleus, and the locus coeruleus. Significant bilateral increases were noted in various sensory and motor areas. These results indicate that rather than a diffuse pattern of activity, rewarding brain stimulation is associated with discrete activation of specific neuronal projection fibers and selective terminal sites.
Cross-polarization/magic-angle sample-spinning
13
C NMR spectroscopic study of chlorophyll
a
in the solid state
Authors: Brown Charles Eric , Spencer Robert B. , Burger Vern T. , Katz Joseph J.
Published: May 2006

Solid-state cross-polarization/magic-angle sample-spinning
13
C NMR spectra have been recorded on chlorophyll
a
-water aggregates, methyl pyrochlorophyllide
a
, and methyl pyropheophorbide
a
(derivatives that lack a phytyl chain). Spectra have also been collected under a decoupling regime in which resonances of certain hydrogen-bearing carbon atoms are suppressed. These observations are used to assign the solid-state spectra.
Laplace operators of infinite-dimensional Lie algebras and theta functions
Authors: Kac Victor G.
Published: May 2006

Until recently, the generalized Casimir operator constructed by Kac [Kac, V. G. (1974)
Funct. Anal. Appl.
8, 68-70] has been the only known element of the center of a completion of the enveloping algebra of a Kac-Moody algebra. It has been conjectured [Deodhar, V. V., Gabber, O. & Kac, V. G. (1982)
Adv. Math.
45, 92-116], however, that the image of the Harish-Chandra homomorphism contains all theta functions defined on the interior of the complexified Tits cone and hence separates the orbits of the Weyl group. Developing the ideas of Feigin and Fuchs [Feigin, B. L. & Fuchs, D. B. (1983)
Dokl. Akad. Nauk SSSR
269, 1057-1060], I prove this conjecture. Another application of this method is the Chevalley type restriction theorem for simple finite-dimensional Lie superalgebras.
U1 RNA-protein complex preferentially binds to both 5' and 3' splice junction sequences in RNA or single-stranded DNA.
Authors: Tatei K , Takemura K , Mayeda A , Fujiwara Y , Tanaka H , Ishihama A , Ohshima Y
Published: May 2006

We have investigated factors that recognize the splice junctions for mRNA by means of a rapid and sensitive filter binding assay using chemically synthesized single-stranded (ss) DNA (16-21 nucleotides) that includes a splice junction sequence or using RNA transcribed from the DNA. When small nuclear RNA-protein complexes from HeLa cells or rat liver were separated by a DEAE-Sepharose column, U1 RNA-protein complex fractions showed strong binding to ss DNA including a 5' or 3' consensus splice junction sequence. This binding took place in the presence of a large excess of Escherichia coli denatured DNA or RNA, but it was significantly reduced when conserved G-T or A-G within the splice junction was altered. In contrast, the U2 RNA-protein complex fractions did not show significant binding. We also have prepared RNA carrying the splice junction sequence by in vitro transcription of double-stranded splice junction DNA, which was linked to the E. coli lac promoter. By using this RNA, preferential binding to both 5' and 3' splice junction sequences has been confirmed with the partially purified U1 RNA-protein complex fraction described above. When the U1 RNA-protein complex is highly purified, it always retains a strong binding activity for a 5' splice junction. The binding activity for a 3' splice junction is partly or mostly lost during purification. These results strongly suggest that the U1 RNA-protein complex and/or an associated factor participates in the recognition of both 5' and 3' splice junctions.
Activation of pS2 gene transcription is a primary response to estrogen in the human breast cancer cell line MCF-7.
Authors: Brown A M , Jeltsch J M , Roberts M , Chambon P
Published: May 2006

We have shown previously that an increase in the level of accumulated pS2 mRNA is first detectable in MCF-7 cells after 3 hr of estradiol treatment. Using in vitro nuclear run-on transcription with nuclei prepared from MCF-7 cells grown in the presence of estradiol or in estradiol-stripped medium, we demonstrate here that expression of the pS2 gene is controlled by estrogen at the transcriptional level. Induction of transcription is a very early event that is already apparent within 15 min after addition of estradiol to the culture medium. In addition, pretreatment of the cells with the protein synthesis inhibitor cycloheximide does not prevent induction of pS2 gene transcription, indicating that it corresponds to a primary effect of estrogen. The pS2 gene in MCF-7 cells represents a unique example of a human gene whose transcription is directly controlled by estrogen.
Nitrendipine block of cardiac calcium channels: high-affinity binding to the inactivated state.
Authors: Bean B P
Published: May 2006

Block of Ca2+ currents by the dihydropyridine drug nitrendipine was studied in single canine ventricular cells by using the whole-cell variant of the patch clamp technique. When cells were held at depolarized membrane potentials at which Ca2+ currents were approximately equal to 70% inactivated, nitrendipine blocked Ca2+ currents very potently, with half-block by subnanomolar concentrations. The concentration dependence of block had the form expected for 1:1 binding, with an apparent dissociation constant (Kd) of 0.36 nM. In contrast, when cells were held at hyperpolarized potentials, nitrendipine blocked Ca2+ currents much less potently (Kd approximately equal to 700 nM). The results can be explained if nitrendipine binds very tightly to the inactivated state of the Ca2+ channel and only weakly to the normal resting state. The Kd estimated for binding to the inactivated state is very similar to the dissociation constants previously found for high-affinity [3H]nitrendipine binding to membrane fragments from heart, smooth muscle, brain, and other tissues; moreover, the concentration-dependent kinetics of binding to the inactivated state are similar to those reported for [3H]nitrendipine binding to membranes. These results make it seem very likely that the high-affinity [3H]nitrendipine binding site is an inactivated state of the Ca2+ channel.
Antigen-specific helper T-cell clone supernatant is sufficient to induce both polyclonal proliferation and differentiation of small resting B lymphocytes.
Authors: Leclercq L , Bismuth G , Thèze J
Published: May 2006

Gradient-purified resting B lymphocytes can be polyclonally stimulated by antigen-specific major histocompatibility complex (MHC)-restricted helper T lymphocytes as well as by antigen-activated helper T-cell supernatant. In contrast to what has been described so far, we show that helper T-cell supernatant (in the absence of any other added stimulus, such as that provided by anti-mu antibodies) is sufficient to induce both proliferation of resting B cells and their differentiation into IgM-secreting cells. The stimulation induced by the helper T-cell supernatant takes place in serum-free medium and is not MHC-restricted. Our findings strongly support the existence of a B-cell activating factor acting on the resting B cell and causing it to enter the G1 phase of the cell cycle in a MHC-unrestricted manner.
Specific in vitro adenylylation of the simian virus 40 large tumor antigen.
Authors: Bradley M K , Hudson J , Villanueva M S , Livingston D M
Published: May 2006

Incubation of the simian virus 40 (SV40) large tumor antigen (T) from either transformed or lytically infected cells with adenosine [8-3H]-, [alpha-32P]-, or [alpha-[35S]thio]-triphosphate in the presence of Mg2+ resulted in its labeling as defined by the appearance of an intact, appropriately immunoreactive band in NaDodSO4/polyacrylamide gels. Radioactivity remained associated with the protein after boiling in buffer containing 3% NaDodSO4, and 2-mercaptoethanol as well as after heating in 0.1 M HCl, 0.1 M NH4OH, or hydroxylamine, but it was dissociated after incubation in 0.1 M NaOH at 37 degrees C. After limited boiling of gel-purified [alpha-32P] ATP + T complex in 5.6 M HCl, o-[32P]phosphoserine was released, and snake venom phosphodiesterase or 0.5 M piperidine treatment of such a complex resulted in the liberation of [alpha-32P]AMP. The reaction proceeded when either purified, soluble T or insoluble, specifically immunoprecipitated antigen was used as substrate. ATP and dATP were the preferred nucleotide substrates by comparison with the other six standard ribonucleoside or deoxynucleoside triphosphates. Partial tryptic digests of T + [alpha-32P]ATP complexes revealed the presence of a single labeled peptide of Mr approximately equal to 12 - 14 X 10(3), and after exhaustive digestion, there was a single radioactive spot in the fingerprint. These data indicate that T can be adenylylated at a specific seryl residue(s) in a limited portion of the protein surface. Furthermore, adenylylation appears to be reversible and to proceed by a pyrophosphorylytic mechanism, since the nucleotide was released from the protein following incubation of adenylylated T with Mg2+, sodium pyrophosphate, and poly(dT).