bluuebunny/crossref_metadata_2025 huggingface dataset:
Lateral mobility of cytochrome c on intact mitochondrial membranes as determined by fluorescence redistribution after photobleaching.
Authors: Hochman J H , Schindler M , Lee J G , Ferguson-Miller S
Published: May 2006

Lateral mobility of an active fluorescent derivative of cytochrome c on the membranes of giant mitochondria was measured by fluorescence redistribution after photobleaching. A diffusion coefficient of 1.6 X 10(-10) cm2/sec was determined for the labeled cytochrome c on inner mitochondrial membranes under conditions where succinate oxidase activity was demonstrated. This relatively low rate of diffusion, together with results of other investigators, is explained in terms of a model involving a dynamic equilibrium between freely diffusing and associated forms of electron-transfer components.
Unusually long mRNA species coding for human alpha and beta interferons.
Authors: Sehgal P B , May L T , LaForge K S , Inouye M
Published: May 2006

Each of the several intronless interferon (IFN) alpha S genes is embedded in 4- to 5-kilobase (kb) DNA homology units which are arranged more or less in tandem in the human genome. In addition to the expected 0.8- to 1.4-kb-long IFN-alpha S mRNA species, we have detected polyadenylylated RNA molecules approximately 2-2.7, 3.5-4.5, and 7-8 kb long derived from IFN-alpha S genes in the cytoplasm of Sendai virus-induced bromodeoxyuridine-treated human lymphoblastoid (Namalwa) cells. These transcripts were detected by electrophoresis of cytoplasmic polyadenylylated RNA through agarose-CH3HgOH gels followed by blothybridization using an IFN-alpha 1 (a prototype alpha S) cDNA probe and also by translation of the eluted RNA into biologically active IFN in the Xenopus oocyte assay. At least some of these long translationally active RNA molecules appear to represent functional mRNA species because they can be detected in polysomes. In addition, the oocyte translation assay has revealed the presence of unusually long (2-8 kb) IFN-beta mRNA species both in Sendai virus-induced Namalwa cells and in poly(I) . poly(C)-induced diploid human fibroblasts (FS-4). The detection of very long IFN-alpha 1-related mRNA species suggests that some of the DNA sequence features observed in the flanking homology units may affect the transcription of the IFN-alpha S genes. Our hybridization analyses also indicate that the 1.8-kb human IFN-alpha L mRNA(s) shows no detectable cross-hybridization with an IFN-alpha 1 cDNA probe. Thus, this mRNA is derived from a gene(s) that is distinct from the IFN-alpha S set.
Evidence for a common ancestor sequence for the Balbiani ring 1 and Balbiani ring 2 genes in
Chironomus tentans
Authors: Wieslander Lars , Sümegi Janos , Daneholt Bertil
Published: May 2006

The Balbiani ring (BR) 1 and BR 2 genes in
Chironomus tentans
are functionally related and are only expressed in the salivary gland cells. Here we reveal the principal structure of the BR 1 gene and analyze the structural and evolutionary relationship between the BR 1 and BR 2 genes. The properties of the BR 1 gene, 37 kilobases in size, are derived from the analysis of a cloned cDNA sequence, pCt 21. A considerable part of the BR 1 gene consists of one or a few blocks of a tandemly repeated 246-base-pair (bp) major repeat unit. About half of this major repeat unit is in turn built from four tandem repeats of a 33-bp sequence. This hierarchic arrangement of repetitive sequences within the BR 1 gene suggests that the gene has evolved through two major amplification steps, starting from a short primordial sequence. A similar evolutionary model has been put forward for the BR 2 gene [Sümegi, J., Wieslander, L. & Daneholt, B. (1982)
Cell
30, 579-587]. The two putative primordial genes contain a similar, 102-bp-long sequence (86% nucleotide sequence homology), indicating that the BR 1 and BR 2 genes most likely arose from the same ancestor sequence. During the course of evolution the two genes diverged, mainly due to differences in the length and sequence of the gene segments involved in the two amplification steps. Moreover, at least one of the BR genes was translocated to another chromosomal locus.
Transcriptionally active DNA region that rearranges frequently in murine lymphoid tumors.
Authors: Adams J M , Gerondakis S , Webb E , Mitchell J , Bernard O , Cory S
Published: May 2006

A DNA region not associated with conventional immunoglobulin gene rearrangement is rearranged in many lymphoid tumors. This region, designated here as lymphoid rearranging (LyR) DNA, was cloned from plasmacytoma J558 in which it had recombined 5' to a constant (C) region of the alpha heavy (H) chain gene, C alpha, within a switch (S) region, S alpha, involved in the switching of CH genes. Sequence determination established that LyR DNA had recombined within a S alpha recombination unit. LyR DNA does not originate from the H chain locus, and discordance between LyR DNA and CH copy number in certain lines suggests that LyR DNA probably derives from another chromosome. LyR DNA rearrangement is a characteristic of tumors of mature B cells; it was detected in 24 of 28 plasmacytomas and B-cell lymphomas, usually as LyR-S alpha, but not in 11 Abelson retrovirus-induced lymphomas of B-cell precursors nor detectably in normal B cells. In contrast, rearrangement was observed in only 3 of 18 T-cell lymphomas, and none of seven nonlymphoid lines. Most tumor lines (49 of 52), whether lymphoid or not, contained a low level of polyadenylylated LyR transcript(s), but several new RNA species with differences in their 5' regions appeared in B-cell lines in which LyR DNA was rearranged, suggesting that rearrangement may activate a new promoter or mode of splicing. The results suggest that the LyR-S alpha rearrangement represents a translocation to chromosome 12 that alters expression of LyR-encoded genes; hence, it may have participated in lymphoid tumor oncogenesis.
Molecular cloning of translocations involving chromosome 15 and the immunoglobulin C alpha gene from chromosome 12 in two murine plasmacytomas.
Authors: Calame K , Kim S , Lalley P , Hill R , Davis M , Hood L
Published: May 2006

Expression of IgA by plasmacytomas occurs as a result of a DNA rearrangement that brings the variable region gene, VH, a few kilobases 5' to the constant region gene, C alpha. In this study, we show that the allelic nonexpressed C alpha gene also is rearranged in most plasmacytomas. Cloning, restriction mapping, heteroduplex analyses, and sequence analyses of the nonproductively rearrange C alpha genes from two plasmacytomas, M603 and M167, have demonstrated that the nonproductive rearrangement occurs within the alpha switching region, S alpha. In each case, the same DNA sequence has been joined to the 5' side of C alpha and we have termed this DNA "NIRD" (for nonimmunoglobulin rearranged DNA). Southern blotting analyses of genomic DNAs from various IgG-, IgM-, or IgA-producing plasmacytomas suggest that NIRD is rearranged in almost all plasmacytomas. However, NIRD rearranges to the S alpha region only in IgA-producing cells, not in IgM or IgG producers. Cytogenetic evidence has shown that T(12;15) translocations are common in murine plasmacytomas. Immunoglobulin heavy chain genes are located on chromosome 12, and the translocation breakpoint in plasmacytomas occurs near the immunoglobulin genes. NIRD has been mapped to chromosome 15 by Southern blotting analysis of mouse-hamster cell lines, suggesting that the nonproductively rearranged C alpha clones represent the T(12;15) translocations identified cytogenetically. Therefore, we have identified a region of DNA on chromosome 15 that is commonly rearranged in transformed mouse lymphocytes. We speculate on the significance of NIRD in neoplastic transformation of mouse lymphocytes.
Origin of the right-handed twist of beta-sheets of poly(LVal) chains.
Authors: Chou K C , Scheraga H A
Published: May 2006

The energies of three- and five-chain antiparallel and parallel beta-sheets were minimized. Each chain consisted of six L-valine residues with CH3CO and NHCH3 end groups; the chains were considered to be equivalent, but all dihedral angles of a given chain were allowed to vary independently during energy minimization. The minimum-energy structures had a considerable right-handed twist, as observed in globular proteins. This right-handed twist is due primarily to intrachain nonbonded interactions. Such interactions between the C gamma 1H3 group of the ith residue and the C gamma 2H3 group of the (i + 2)th residue of the same chain favor a twist of either handedness over the flat structure. However, many small intrastrand pair-wise interatomic interactions involving the C gamma 1H3 and C gamma 2H3 groups, especially the interactions of these groups with the O and amide H atoms of the neighboring peptide groups, make the right-handed twisted structure energetically more favorable than the left-handed one. The intrastrand side-chain torsional energy plays a small additional role in favoring the right-twisted structure over both the flat and the left-twisted structures. The interstrand interactions favor flat structures, but they are not strong enough to overcome the intrastrand interactions that favor the twisted structure; they only decrease somewhat the extent of the right-handed twist of the beta-sheets.
Isolation and analysis of genomic DNA clones encoding the third component of mouse complement.
Authors: Wiebauer K , Domdey H , Diggelmann H , Fey G
Published: May 2006

A gene library was constructed with DNA from strain A mice by using the phage lambda vector lambda 1059. By screening with cloned cDNA for the third component of mouse complement, C3, four different C3 genomic clones were isolated from this library. Two of the recombinant phages carry insertions of 14 and 18 kilobase pairs, respectively, which together cover one complete copy of the C3 gene and several hundred nucleotides of its 5' and 3' flanking sequences. The distance from the 5' end of the gene, which includes the hexanucleotide T-A-T-A-A-A and a translation initiation codon, to its 3' end as defined by the poly(A) attachment site is 24 kilobase pairs. From the genomic DNA sequence, a signal peptide of 24 amino acid residues is predicted at the NH2 terminus of the initial translation product. The signal peptide and the next two amino acids are encoded by the first exon of this gene.
Sequences upstream from the T-A-T-A box are required in vivo and in vitro for efficient transcription from the adenovirus serotype 2 major late promoter.
Authors: Hen R , Sassone-Corsi P , Corden J , Gaub M P , Chambon P
Published: May 2006

We show that sequences located upstream from the T-A-T-A box, between positions -97 and -34, are necessary for efficient in vivo transcription from the adenovirus serotype 2 major late promoter. The effect of these upstream sequences was also investigated in vitro using a whole cell or an S100 extract and circular or linear templates. With the whole cell extract, the in vivo effect of the upstream sequences was reproduced in vitro. With the S100 extract, some effect of the upstream sequences was observed with circular, but not with linear, templates.
Protein D1 preferentially binds A + T-rich DNA in vitro and is a component of Drosophila melanogaster nucleosomes containing A + T-rich satellite DNA.
Authors: Levinger L , Varshavsky A
Published: May 2006

Our previous work [Levinger, L. & Varshavsky, A. (1982) Cell 28, 375-385] has shown that D1, a 50-kilodalton chromosomal protein of Drosophila melanogaster, is specifically associated with isolated nucleosomes that contain a complex A + T-rich satellite DNA with buoyant density of 1.688 g/ml. We show here that D1 is also a component of nucleosomes containing a simple-sequence, pure A + T satellite DNA, buoyant density 1.672 g/ml. Furthermore, using a modification of a protein blotting technique in which proteins are not exposed to dodecyl sulfate denaturation, we have found that D1 preferentially binds to A + T-rich double-stranded DNA in vitro, and it is apparently the only abundant nuclear protein in cultured D. melanogaster cells that possesses this property. Synthetic poly[d(A-T)].poly[d(A-T)] and poly(dA).poly(dT) duplexes effectively compete in vitro with A + T-rich D. melanogaster satellite DNAs for binding to D1, whereas total Escherichia coli DNA is an extremely poor competitor. These findings strongly suggest that D1 is a specific component of A + T-rich, tandemly repeated, heterochromatic regions, which constitute up to 15-20% of the total D. melanogaster genome. Possible functions of D1 protein include compaction of A + T-rich heterochromatin and participation in microtubule-centromere interactions in mitosis. In addition, D1 may prevent nonspecific binding to A + T-rich satellite DNA of other nuclear proteins that have a preference for AT-DNA, such as RNA polymerase or regulatory proteins, and may also participate in the higher-order chromatin organization outside tandemly repetitive regions by binding to nonrandomly positioned stretches of A + T-rich DNA.
Crystallization of mitochondrial cytochrome oxidase.
Authors: Ozawa T , Tanaka M , Wakabayashi T
Published: May 2006

Cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was purified from beef heart mitochondria. By washing the oxidase with detergent on a hydrophobic interaction column, phospholipids were depleted to the level of 1 mol of cardiolipin per mol of heme a. Hydrophobic impurities and partially denatured oxidase were separated from the intact oxidase on an affinity column with cytochrome c as the specific ligand. The final preparation of the oxidase contained seven distinct polypeptides. The molecular weight of the oxidase was estimated to be 130,000 from its specific heme a and copper content and from the subunit composition. Crystals of the oxidase were obtained by slow removal of the detergent from the buffer in which the oxidase was dissolved. The needle-shaped crystals were 100 microns in average length and 5 microns in width, and they strongly polarized visible light. Electron diffraction patterns were obtained with an unstained glutaraldehyde-fixed single crystal by electron microscopy using 1,000-kV electrons. From electron micrographs and the diffraction patterns of the crystal, it was concluded that the crystal is monoclinic in the space group P21, with unit cell dimensions a = 92 A, b = 84 A, and c = 103 A, and alpha = beta = 90 degrees, gamma = 126 degrees.
Local mutagenesis within deletion loops of DNA heteroduplexes.
Authors: Peden K W , Nathans D
Published: May 2006

An efficient method has been developed to generate base substitution mutations within deletion loops of DNA heteroduplexes. This method utilizes a heteroduplex formed between a deletion mutant cloned in a plasmid vector and its wild-type counterpart from which two restriction sites had been removed from the vector. The heteroduplex is exposed to sodium bisulfite to deaminate cytosine residues in the single-stranded loop, and the mutagenized plasmid DNA is used to transform a strain of bacteria lacking the enzyme uracil N-glycosylase. Pooled progeny DNA is digested with the two restriction enzymes, whose sites had been mutated in the wild-type plasmid, to eliminate the original deletion mutant DNA. Point mutants with C . G-to-T . A transitions are obtained at high frequency after a second transformation. To test the feasibility of the approach, the tetracycline resistance gene of pBR322 was chosen as the target sequence. It was found that the proportion of tetracycline-sensitive transformants increased as both the size of the heteroduplex loop and the time of incubation with the mutagen increased and this varied from 20% up to 70%. Nucleotide sequence analysis of several tetracycline-sensitive mutants confirmed that C-to-T transitions had been produced in the segment of DNA corresponding to the deletion loop.
Purified Epstein-Barr virus Mr 340,000 glycoprotein induces potent virus-neutralizing antibodies when incorporated in liposomes.
Authors: North J R , Morgan A J , Thompson J L , Epstein M A
Published: May 2006

The purified Mr 340,000 glycoprotein component of Epstein-Barr (EB) virus-induced membrane antigen complex incorporated into liposomes was shown to be a potent immunogen in mice. High-titer antisera were induced that (i) are specific for membrane antigen components without absorption, (ii) bind the antigens induced by three different EB virus isolates, and (iii) neutralize the ability of the virus to transform fetal cord blood lymphocytes in vitro. The development of this immunogenic form of purified antigen provides an important step towards a potential subunit vaccine against Epstein-Barr virus infection.
Abnormal adenosine-induced immunosuppression and cAMP metabolism in T lymphocytes of patients with systemic lupus erythematosus
Authors: Mandler Raya , Birch Ruth E. , Polmar Stephen H. , Kammer Gary M. , Rudolph Stephen A.
Published: May 2006

Normal human T lymphocytes incubated with adenosine (10 μM) for 30 min at 37°C show an increase in the percentage of cells expressing receptors for the Fc portion of IgG (RFc
γ
) and the OKT8 antigen, while the proportion of OKT4
+
cells decreases. These effects occur exclusively in a subset of T cells with theophylline-resistant sheep erythrocyte receptors (T
R
cells) that is enriched for OKT4
+
cells. Untreated normal T
R
cells express helper/inducer cell activity for T-cell-dependent B-cell differentiation, while adenosine-treated T
R
cells suppress B-cell differentiation. In contrast, in T
R
cells isolated from patients with systemic lupus erythematosus (SLE), adenosine fails to induce immunosuppressor activity or to increase the percentage of OKT8
+
and RFc
γ
+
cells. In addition, although incubation of normal T
R
cells with adenosine causes a transient increase in cAMP levels (up to 160% of control within 5 min), in SLE T
R
cells, cAMP levels fall by 50% within 10 min. The photoaffinity label 8-azidoadenosine cyclic [
32
P]monophosphate has been used to show that human T lymphocytes have a single cAMP receptor site that appears to be the regulatory subunit of type I protein kinase. In normal T
R
cells, this receptor becomes occupied in response to adenosine. In contrast, in SLE T
R
cells, no change in cAMP receptor occupancy is detected. Although adenosine has a differential effect on normal and SLE T
R
cells, cAMP derivatives that can traverse the cell membrane (8-bromo- and 8-azidoadenosine cyclic monophosphates) induce an increase in the RFc
γ
+
cell subset in both normal and SLE T
R
cells. These results suggest that cAMP mediates the effects of adenosine on cell surface markers of T lymphocytes. The lack of an adenosine receptor-coupled adenylate cyclase activity in SLE T
R
cells may account, in part, for their lack of immunosuppressive activity.
Use of a monoclonal antibody to purify the tetrodotoxin binding component from the electroplax of Electrophorus electricus.
Authors: Nakayama H , Withy R M , Raftery M A
Published: May 2006

The tetrodotoxin binding component of the voltage-sensitive sodium channel from Electrophorus electricus electroplax was purified by using a monoclonal antibody. An impure preparation of tetrodotoxin binding component was mixed with the pure monoclonal antibody, and the immune complex so formed was isolated by affinity chromatography on a protein A-Sepharose column. Excess antibody was removed by ion-exchange chromatography. The purified material has a specific activity of over 1,800 pmol of [3H]tetrodotoxin bound per mg of protein. By assuming that the immune complex has a stoichiometry of 1:1, this specific activity then represents an actual specific activity of 3,000 pmol of [3H]tetrodotoxin bound per mg of eel electroplax protein, or 75% of the theoretical specific activity expected for a pure toxin binding component of Mr 250,000. The peptide composition of the purified material was simple with the predominant species present being of Mr approximately equal to 250,000. Minor components were also present with Mrs of approximately equal to 95,000, approximately equal to 44,000, and approximately equal to 23,000.
Translational control of catalase synthesis by hemin in the yeast
Saccharomyces cerevisiae
Authors: Hamilton Barbara , Hofbauer Reinhold , Ruis Helmut
Published: May 2006

mRNA-dependent cell-free protein synthesis systems were prepared from a heme-deficient
ole3
mutant of the yeast
Saccharomyces cerevisiae
grown either in the absence or in the presence of the heme precursor δ-aminolevulinate. When supplemented with total yeast mRNA, the two systems—from heme-deficient and from heme-containing cells—translate most mRNAs with comparable efficiencies. mRNAs coding for the hemoproteins catalase T and catalase A, however, are translated at a low rate by the system from heme-deficient cells whereas their translation in the system from heme-containing cells is comparable to that observed in a heterologous
in vitro
system from wheat germ. Addition of 10 μM hemin to the system from heme-deficient cells stimulates translation of catalase mRNAs significantly. Control experiments showed that the results obtained cannot be explained by specific proteinase or nuclease action. Together with previous findings indicating lack of translation of catalase T mRNA in heme-deficient cells
in vivo
, the results demonstrate that specific control of yeast catalase formation occurs at the level of translation.
Monoclonal antibody against angiotensin-converting enzyme: its use as a marker for murine, bovine, and human endothelial cells.
Authors: Auerbach R , Alby L , Grieves J , Joseph J , Lindgren C , Morrissey L W , Sidky Y A , Tu M , Watt S L
Published: May 2006

A monoclonal antibody has been prepared against rat angiotensin-converting enzyme (ACE). By selection for antibody binding to endothelial cells of bovine rather than rat origin we have obtained a reagent that has broad cross-species binding properties and that can at the same time serve as a useful marker for the surface of endothelial cells. The IgM-producing clone that we have established, alpha-ACE 3.1.1, has been grown in ascites form to yield ascites fluid that binds selectively to immobilized ACE at a greater than 1:10,000 dilution. By use of enzyme-linked immunosorbent assays, immunofluorescence histology, and flow cytometry, we have demonstrated the presence of ACE on endothelial cells of murine, bovine, and human origin. By means of a fluorescence-activated cell sorter (FACS-IV) we have been able to selectively isolate viable endothelial cells from a mixture of endothelial cells and fibroblasts. We believe the antibody will be useful not only for the selection and in vitro cultivation of endothelial cells but also as a tool for the identification and pharmacological study of ACE.
Chemotherapy by intravenous administration of conjugates of daunomycin with monoclonal and conventional anti-rat alpha-fetoprotein antibodies.
Authors: Tsukada Y , Hurwitz E , Kashi R , Sela M , Hibi N , Hara A , Hirai H
Published: May 2006

Monoclonal antibodies to rat alpha-fetoprotein (AFP) were produced by hybridization of mouse myeloma cells with spleen cells from mice immunized with rat AFP. The monoclonal antibodies as well as horse anti-rat AFP were coupled via a dextran bridge to daunomycin. Both types of conjugates were tested in vitro and in vivo for their anti-tumor activity. They were equally cytotoxic to rat AH66 hepatoma cell line in culture. Rats challenged with hepatoma cells were treated with the conjugates either by intraperitoneal or intravenous injections. Daunomycin conjugates with horse anti-AFP and monoclonal mouse anti-AFP were capable of delaying the tumor development more efficiently than the controls of antibodies or free drug, mixtures of drug with antibodies, and a conjugate of drug and normal immunoglobulin. The specific conjugates were considerably more effective when the treatments were given intravenously. The specific conjugates produced 60% long-term survival, whereas the controls delayed only slightly tumor development.
Localization of genes for the double-stranded RNA killer virus of yeast.
Authors: Welsh J D , Leibowitz M J
Published: May 2006

The M double-stranded RNA (ds RNA) genome segment of the cytoplasmically inherited killer virus of yeast codes for two polypeptides when denatured and translated in vitro: a previously known 32,000-dalton peptide and a newly discovered 19,000-dalton peptide (NaDodSO4/polyacrylamide gel electrophoresis). An internal 190-base-pair region of the ds RNA is selectively degraded by S1 nuclease treatment at 65 degrees C, resulting in two ds RNA fragments which contain the termini of the original ds RNA. The larger fragment codes for the 32,000-dalton polypeptide and the smaller fragment codes for the 19,000-dalton polypeptide. Thus, the two gene products of M are encoded by distinct regions of this ds RNA.
Variable-sized free episomes of Shope papilloma virus DNA are present in all non-virus-producing neoplasms and integrated episomes are detected in some.
Authors: Wettstein F O , Stevens J G
Published: May 2006

The state of rabbit (Shope) papilloma virus DNA in virus-induced nonproducing tumors on domestic rabbits was investigated. Virus-specific sequences were resolved into many distinct bands by one-dimensional agarose gel electrophoresis. Two-dimensional gel electrophoresis, CsCl/propidium iodide density equilibrium centrifugation, partial digestion with a restriction endonuclease, and S1 nuclease digestion permitted us to identify the bands as free viral episomes representing circular molecules of increasing size. In some tumors (both papillomas and carcinomas), up to 25% of the virus-specific DNA was linear and comigrated with cellular DNA. Integration of at least some of these sequences was suggested by the detection of viral-cellular junction bands in one tumor after digestion of DNA with EcoRI and Sal I, enzymes that cut Shope DNA once. Finally, the physical states of viral DNA in papillomas and carcinomas were found to be similar, although free episomes were generally larger in carcinomas.
Isolation and chromosomal localization of unique DNA sequences from a human genomic library.
Authors: Kao F T , Hartz J A , Law M L , Davidson J N
Published: May 2006

Recombinant bacteriophage lambda from a human genomic library were screened to indentify human DNA inserts having only unique sequences. Unique human inserts were found in about 1% of the phage screened. One recombinant phage, P3-2, was studied in detail. It contains a human insert of 14.7 kilobases with four internal EcoRI cleavage sites. A restriction map was constructed for EcoRI and BamHI sites. Hybridization of the 32P-labeled P3-2 probe to a Southern blot of EcoRI-digested total human DNA yielded distinct bands at positions corresponding to the human insert fragments contained in P3-2. By using a series of human-Chinese hamster somatic cell hybrids containing unique combinations of human chromosomes, the human DNA segment in phage P3-2 was assigned to human chromosome 22 by blot hybridization and synteny analysis. In addition, another human DNA segment, 11.4 kilobases, in phage P3-10 was assigned to human chromosome 10 by similar procedures. With this approach, more unique DNA sequences can be isolated, assigned to specific human chromosomes, and used as genetic markers for gene mapping and linkage, polymorphism, and other genetic studies in the human genome.
Photobleaching through glass micropipettes: sodium channels without lateral mobility in the sarcolemma of frog skeletal muscle.
Authors: Stühmer W , Almers W
Published: May 2006

Sodium currents were recorded from frog skeletal muscle by using fire-polished micropipettes to electrically isolate and voltage clamp a small patch of sarcolemma. Sodium current amplitude served as an assay for the number of functional sodium channels in the patch. With the pipette as a light guide, these channels were irradiated with ultraviolet (UV) light directed through a quartz fiber into the back end of the pipette. The UV light emerging from the pipette tip caused localized destruction of the sodium channels in the patch, reducing sodium current 3- to 5-fold during a 30-90 s irradiation. If sodium channels could diffuse laterally in the membrane, current from the patch should recover with time as fresh channels enter from neighboring areas. No such recovery was observed during observation for 1 hr after irradiation. Our results set an upper limit of 10(-12) cm2/s for the diffusion coefficient--1/1000th that of rhodopsin, a membrane protein in the cell membrane of retinal rods. It is suggested that sodium channels are anchored in the sarcolemma.
Acetylcholinesterase of human erythrocytes and neuromuscular junctions: homologies revealed by monoclonal antibodies.
Authors: Fambrough D M , Engel A G , Rosenberry T L
Published: May 2006

Human erythrocyte acetylcholinesterase was used to immunize mice, and hybridomas were generated by fusion of mouse spleen cells with cells of the Sp 2/0 myeloma cell line. Five independently derived hybridoma clones produced antibodies that bound to purified erythrocyte acetylcholinesterase. All of these antibodies crossreacted with human and monkey neuromuscular junctions; immunocytochemical staining patterns corresponded to the distribution of junctional acetylcholinesterase. The monoclonal antibodies fell into at least four categories based on differences in crossreactivity with neuromuscular acetylcholinesterase of rabbit, dog, calf, and guinea pig, and competition tests indicated that the antibodies defined five different antigenic sites on the acetylcholinesterase molecule. It is concluded that there is a high level of homology between the acetylcholinesterases of erythrocytes and neuromuscular junctions.
Simian virus 40 large tumor antigen: a "RNA binding protein"?
Authors: Khandjian E W , Loche M , Darlix J L , Cramer R , Türler H , Weil R
Published: May 2006

Simian virus 40 large tumor antigen was isolated by immunoaffinity chromatography from monkey or mouse cell cultures undergoing lytic or transforming infection. RNase-treated gel-purified large tumor antigen, on hydrolysis with alkali, gave about equimolar amounts of AMP, GMP, CMP, and UMP. Furthermore, RNA fragments of approximately 45 nucleotides could be isolated from large tumor antigen purified by the same procedure. Mapping of the T1 oligonucleotides showed a high complexity, as indicated by the presence of unique sequences of 15-30 nucleotides and of poly(A). This is compatible with the hypothesis that these RNA fragments are derived from cellular pre-mRNAs or mRNAs. Our results suggest that Simian virus 40 large tumor antigen is a RNA-binding protein and might possibly be involved in regulation of synthesis, maturation, or translation of cellular mRNAs.
Introduction of a human X-6 translocation chromosome into a mouse teratocarcinoma: investigation of control of HLA-A, B, C expression.
Authors: Goodfellow P N , Banting G , Trowsdale J , Chambers S , Solomon E
Published: May 2006

We have developed an approach to human developmental biology which exploits somatic cell genetics. With this system we have examined the production of the HLA-A,B,C antigens, A human-mouse somatic cell hybrid was constructed which contained a human X-7 chromosome translocation carrying the HLA region; this hybrid was used as a donor of the X-6 translocation in the technique of microcell transfer. The X-6 chromosome recipient was the mouse embryonal carcinoma cell line PCC4. The microcell hybrid MCP-6 retained the embryonal carcinoma phenotype as judged by shape and absence of H-2 expression. Nonetheless, the expression of the HLA-A,B,C genes was not extinguished. HLA-A,B,C antigen production of the cell surface, however, was not detected because this hybrid apparently could not make beta 2-microglobulin.
Region-specific effects on chromosome integrity of mutations at essential loci in
Drosophila melanogaster
Authors: Baker Bruce S. , Smith David A. , Gatti Maurizio
Published: May 2006

Two mutagen-sensitive loci of
Drosophila melanogaster, mus-105
and
mus-109
, previously identified by viable alleles, are shown to specify essential functions. Lethal alleles at the loci produce larvae that have degenerate imaginal discs and die at the larva-pupa boundary. Our data suggest that the causes of lethality are intolerable levels of cell death produced by high frequencies of chromosome aberrations (in excess of 0.5 aberration per cell per cycle). The pattern of aberrations is a locus-specific character. In
mus-105
mutants the most common aberrations are breaks and exchanges in euchromatic portions of the genome whereas in
mus-109
mutants the most common aberrations are breaks at heterochromatin-euchromatin junctions. The sensitivity of these junctions to breakage in
mus-109
mutants is a property of all such junctions whether natural or produced by a rearrangement that juxtaposes heterochromatin and euchromatin. Larvae carrying the combination of two viable mutants,

mus-105
A1
mus-109
D1

, die at the larva-pupa boundary and display a high frequency of aberrations (0.7 per cell vs. 0.075 for either mutant alone) clustered at euchromatin-heterochromatin junctions. This synergistic interaction suggests there is a class of lesions that can be repaired by both
mus-105
+
and
mus-109
+
. Thus, the apparent euchromatic specificity of
mus-105
+
, which was inferred from the pattern of predominantly euchromatic breakage observed in
mus-105 mus-109
+
flies, is in fact generated by the wild-type function of
mus-109
+
masking an effect of
mus-105
in the heterochromatin. The fact that lethal mutants at the
mus-105
and
mus-109
loci have small imaginal discs coupled with the observation of a maternal effect of
mus-105
suggests a paradigm for the control of cell division during the life cycle of
Drosophila
.
Purification and characterization of a monoclonal T-cell suppressor factor specific for poly(LGlu60LAla30LTyr10).
Authors: Krupen K , Araneo B A , Brink L , Kapp J A , Stein S , Wieder K J , Webb D R
Published: May 2006

A monoclonal T-cell-derived suppressor factor specific for the terpolymer poly(LGlu60LAla30LTyr10) produced by the T-cell hybridoma 258 C4.4, was purified to homogeneity. This was accomplished by fractionation of the culture medium by using a combination of affinity chromatography and reverse-phase and ion-exchange high-performance liquid chromatography. The purified factor is composed of a single Mr 24,000 polypeptide chain, and the homogeneous protein maintains the ability to suppress antibody and T-cell proliferative responses to poly(LGlu60LAla30LTyr10) specifically. The specific activity of pure suppressor factor is calculated to be 8 X 10(7) units/micrograms.
Rate constant of vibrational redistribution in molecules using adiabatic approximation model
Authors: Lin S. H. , Xing-Guo Zhang , Zhi-Ding Qian , Xing-Wen Li , Eyring H.
Published: May 2006

By means of the proposed adiabatic approximation model for vibrational redistribution [Lin, S. H. (1980)
Chem. Phys. Lett.
70, 492-499], the calculations of various rate constants caused by different couplings have been given in detail. By two methods it has been demonstrated that the rate constant caused by the Born-Oppenheimer mechanism, corrected by the non-Condon approximation, has the same order of magnitude as that contributed by the anharmonic mechanism. Furthermore, the important effect of molecular rotation on the rate constant based on the Coriolis coupling has been demonstrated.
Intracellular receptor for somatostatin in gastric mucosal cells: decomposition and reconstitution of somatostatin-stimulated phosphoprotein phosphatases.
Authors: Reyl F J , Lewin M J
Published: May 2006

Using 32P-labeled histone as exogenous substrate, we showed a potent stimulatory effect of somatostatin on cytosolic phosphoprotein phosphatases (PPPases; phosphoprotein phosphohydrolase, EC 3.1.3.16) in rat gastric mucosal cells. Partial purification of cytosolic fraction in DEAE-Sephadex ion-exchange chromatography and further gel filtration on Sephadex C-75 and Sephadex G-100 separated somatostatin-dependent PPPases into three distinct molecular species. One corresponding to Mr 130,000 was devoid of any PPPase activity but specifically bound [Tyr1]somatostatin 125I-labeled on the Tyr ([125I-Tyr1]somatostatin) with an apparent equilibrium dissociation constant of 3 x 10(-10) M. The two other molecular species corresponded to Mrs 64,000 and 13,000. They produced catalytic dephosphorylation of 32P-labeled histone, but they were not sensitive to somatostatin and did not show any specific binding to radiolabeled hormone. Mixing of the larger with either of the two smaller molecular species resulted in concentration -dependent inhibition of PPPase activity. However this inhibition was reversed by increased concentrations of somatostatin, with the concentration for half-maximal reactivation on being close to 0.1 nM. Furthermore somatostatin stimulation in reconstituted materials developed according to a rapid time course (t1/2, less than 5 sec), consistent with that observed for binding of [125I-Tyr1]somatostatin. These results strongly argue for the presence of an intracellular somatostatin receptor in gastric mucosal cells and characterize this receptor as a PPPase regulatory subunit. Thus, substrate dephosphorylation could be the primary event triggering physiological effects of somatostatin in stomach and perhaps other organs of the digestive tract [Reyl, F. & Lewin, M. J.l M. (1981) Biochim. Biophys. Acta 675, 297-300].
Site-directed mutagenesis of a tRNA gene: base alterations in the coding region affect transcription.
Authors: Ciampi M S , Melton D A , Cortese R
Published: May 2006

Point mutations have been introduced in vitro in a cloned nematode tRNAPro gene. Four different mutant clones altered in the DNA that codes for tRNAPro have been isolated. Studies on the expression of the mutant genes by microinjection into Xenopus oocyte nuclei reveal that their activities as transcription templates are reduced. The results show that DNA sequences that code for the extra arm and the stem of the T-psi-C-G arm of the tRNAPro have an important role in the initiation of tRNA gene transcription.
Assembly of nuclear ribonucleoprotein particles during in vitro transcription.
Authors: Economidis I V , Pederson T
Published: May 2006

The assembly of heterogeneous nuclear RNA (hnRNA) into ribonucleoprotein (RNP) particles has been investigated during in vitro transcription in isolated nuclei. Approximately 80% of the in vitro transcription observed in mouse Friend erythroleukemia cell nuclei is attributable to the activity of RNA polymerase II. In vitro hnRNA transcripts are assembled into particles having the same properties as the nuclear ribonucleoprotein (hnRNP) particles in which hnRNA is found in vivo. Direct contact of hnRNP proteins with newly transcribed hnRNA was demonstrated by nuclease protection experiments and by the covalent transfer of 32P-labeled nucleotides from [alpha-32P]UTP-labeled hnRNA transcripts to specific proteins by RNA--protein crosslinking followed by nuclease digestion and electrophoresis of the nucleotide-bearing proteins. The availability of an in vitro system for hnRNP assembly opens a new route for investigating the functional relationship between nuclear structure and mRNA processing.
Polyoma mutants that productively infect F9 embryonal carcinoma cells do not rescue wild-type polyoma in F9 cells
Authors: Fujimura Frank K. , Linney Elwood
Published: May 2006

Mouse embryonal carcinoma cells are refractory to infection by wild-type polyoma virus, the infection process apparently being blocked at a stage after adsorption and penetration but before early protein synthesis. Polyoma virus mutants capable of productive infection of mouse embryonal carcinoma cells have been isolated and these mutants all have DNA sequence alterations in a noncoding region near the origin of replication of the viral genome. PyF101 and PyF441 are two mutants selected for their ability to infect the embryonal carcinoma cell line F9. Here we show that these PyF mutants do not rescue replication of wild-type polyoma during a mixed infection of F9 cells. The mutant and wild-type DNAs were distinguished on the basis of restriction fragments obtained by digestion with
Msp
I or
Bst
NI, and no wild-type DNA was detected in F9 cells coinfected with wild-type polyoma and with either PyF101 or PyF441. The mutant viruses do not appear to inhibit wild-type replication during a mixed infection because both mutant and wild-type DNAs can replicate efficiently in coinfected 3T6 cells which are permissive for both mutant and wild-type viruses. A double mutant having the PyF101 mutation and the ts-25E temperature-sensitive mutation in polyoma large tumor antigen was constructed and found to be temperature-sensitive for replication in F9 cells. This double mutant, designated PyFts-1, can be rescued in F9 cells at the restrictive temperature by coinfection with PyF441. These results suggest that the PyF mutations affect two processes in F9 cells, one involving expression of polyoma early genes and a second involving viral DNA replication.
A monkey Alu sequence is flanked by 13-base pair direct repeats by an interrupted alpha-satellite DNA sequence.
Authors: Grimaldi G , Singer M F
Published: May 2006

A member of the Alu family, the dominant family of short interspersed repeated DNA sequences in primates, interrupts a cloned repeat unit of African green monkey alpha-satellite DNA. The Alu is immediately flanked by 13-base-pair duplications of the known sequence of the satellite at the site of insertion. These observations support the idea that Ala family members may be moveable elements.
Cellular location affects protein stability in Escherichia coli.
Authors: Talmadge K , Gilbert W
Published: May 2006

We study the biosynthesis of preproinsulin and proinsulin in Escherichia coli by pulse-chase experiments. When E. coli is transformed with plasmids bearing a gene consisting of a fusion of preproinsulin DNA to part or to all of the DNA encoding a bacterial signal sequence, a variety of hybrid preproinsulin molecules can be made. Molecules with largely complete hybrid signal sequences are secreted into the periplasm. Molecules with defective signal sequences, lacking the hydrophobic core, are not secreted and remain in the cytoplasm. Such molecules are degraded with a 2-min half-life, whereas the molecules transported to the periplasm are at least 10 times more stable. A full-length preproinsulin precursor appears transiently before the signal sequence is cleaved off by the bacterial signal peptidase, and we propose a model to account for this.
Transfer of an antigen-specific immediate hypersensitivity-like reaction with an antigen-binding factor produced by T cells.
Authors: Ptak W , Askenase P W , Rosenstein R W , Gershon R K
Published: May 2006

The fact that T cell-dependent activation of mast cells occurs in delayed-type hypersensitivity led us to investigate whether a T cell product could mimic some of the functions of IgE. We report that 24- or 48-hr cultures of T cells from mice immunized optimally for delayed-type hypersensitivity resulted in release of an antigen-binding factor that transferred the ability to elicit an antigen-specific immediate hypersensitivity-like skin reaction in normal recipients. The responsible factor was concentrated and purified by affinity chromatography on antigen columns and was distinguished from immunoglobulin by several criteria: (i) it was released by purified T cells (anti-immunoglobulin plate depletion of B cells); (ii) it expressed no known antigenic markers of immunoglobulins (enzyme-linked immunosorbant direct binding assay); (iii) it had a molecular weight of 70,000 or less (sucrose gradient ultracentrifugation); and (iv) it had serological markers associated with antigen-specific T cell factors from other experimental systems. We suggest that, at sites of delayed-type hypersensitivity, antigen-reactive T cells may release antigen-specific factors that lead to mast cell activation and release of vasoactive amines, which is required for elicitation for these responses.
Single amino acid substitution altering antigen-binding specificity.
Authors: Rudikoff S , Giusti A M , Cook W D , Scharff M D
Published: May 2006

S107, a phosphocholine-binding myeloma protein, has been cloned in soft agar, and an antigen-binding variant has been isolated and characterized. The variant does not bind phosphocholine attached to carrier or as free hapten in solution but does retain antigenic determinants (idiotypes) of the parent. Chain recombination experiments suggest that the defect in binding is entirely in the heavy chain. Amino acid sequence analysis showed a single substitution--glutamic acid to alanine at position 35--in the first hypervariable or complementarity-determining region. In terms of the three-dimensional model of the phosphocholine-binding site, glutamic acid-35 provides a hydrogen bond to tyrosine-94 of the light chain that appears to be critical for stability of this portion of the binding site. The removal of this bond and the presence of the smaller alanine side chain is thus consistent with the loss in binding activity. These results suggest that small numbers of substitutions in antibodies, such as those presumably introduced by somatic mutation, may in some situations be effective in altering antigen-binding specificity.
Identification of the 48-base-long primordial building block sequence of mouse immunoglobulin variable region genes.
Authors: Ohno S , Matsunaga T , Wallace R B
Published: May 2006

Mouse immunoglobulin heavy-chain variable region (Ig VH) genes apparently arose from the approximately 600-base-pair-long (approximately 12 tandem repeats of the 48-base-pair-long primordial building block sequence TTC-AGC-AGC-CTG-ACT-GGA-TAT-GAC-CTG-GAG-TGG-ACT-TAC-TGC-GCA-AGA) that in the original reading frame specified the amino acid sequence Phe-Ser-Ser-Leu-Thr-Gly-Tyr-Asp-Leu-Glu-Trp-Thr-Tyr-Cys-Ala-Arg. The previously identified, shorter prototype building blocks merely represented particular portions of the above primordial sequence. Even today, the direct descendant in toto of this primordial sequence specifies the last one-sixth of each VH coding sequence: the 83rd to 98th amino acid residues. Furthermore, its four truncated derivatives specify the 4th to 14th, 17th to 23rd, 29th to 37th, and 38th to 48th amino acid residues. Accordingly, all three relatively invariant--therefore, conserved--framework regions (FW-1, FW-2, and FW-3) of VHs are specified by recognizable--therefore, conserved--descendants of the primordial sequence.
Parathyroid hormone receptors in avian bone cells.
Authors: Pliam N B , Nyiredy K O , Arnaud C D
Published: May 2006

We have demonstrated binding of synthetic bovine parathyroid hormone (1-34) [bPTH-(1-34)] to embryonic avian bone cells in monolayer culture. The binding sites have qualitative and quantitative characteristics of a physiologically important parathyroid hormone (PTH) receptor. At apparent steady state (60 min at 24 degrees C), 5-10% of electrolytically labeled, receptor-purified 125I-labeled bPTH-(1-34) bound specifically to the cells whereas nonspecific binding was less than 1% of the added labeled hormone. Scatchard analysis showed a single order of PTH binding sites (Kd = 0.6 nM) with approximately 10,000 sites per cell. In this bone cell system, PTH bound to its binding site and stimulated cAMP accumulation over the same concentration range. Bovine PTH-(1-84) bound to the cells with the same apparent affinity as bPTH-(1-34).