bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial DNA and nuclear DNA from normal rat liver have a common sequence.
Authors: Hadler H I , Dimitrijevic B , Mahalingam R
Published: May 2006

Although Pst I does not cut the circular mitochondrial genome of the rat, BamHI generates from this genome two unequal fragments of DNA. Each of these fragments was cloned in pBR322. Nuclear DNA was digested from rat liver singly or doubly with Pst I and BamHI, and it was demonstrated that nuclear DNA shared a common sequence with the larger mitochondrial DNA BamHI fragment. The cloned larger mitochondrial DNA fragment was further subdivided with HindIII into four pieces that were labeled and then used to probe the double-digested nuclear DNA. The hybridization data showed that the common sequence is less than 3 kilobase pairs long and lies within the part of the mitochondrial genome containing the D-loop and a portion of the rRNA genes. It therefore appears that, as in lower eukaryotes, there are shared sequences between the nuclear and mitochondrial genomes in mammals.
Primary structure of the M subunit of the reaction center from
Rhodopseudomonas sphaeroides
Authors: Williams J. C. , Steiner L. A. , Ogden R. C. , Simon M. I. , Feher G.
Published: May 2006

The reaction center is a membrane-bound bacteriochlorophyll-protein complex that mediates the primary photochemical events in the photosynthetic bacterium
Rhodopseudomonas sphaeroides
. The previously determined amino-terminal sequences of the three subunits of the reaction center protein were used to design synthetic mixed oligonucleotide probes for the structural genes encoding the subunits. One of these probes was used to isolate and clone a fragment of DNA from
R. sphaeroides
that contained the gene encoding the M subunit. The nucleotide sequence of this gene was determined by the dideoxy method. In addition, a number of tryptic and chymotryptic peptides from the M protein were isolated and subjected to sequence analysis, and the sequence of the carboxyl terminus was determined. Together with the amino-terminal sequence, the data establish the primary structure of the M protein. The distribution of hydrophobic residues in the amino acid sequence suggests the presence of five membrane-spanning segments. A significant homology was found between the amino acid sequence of the M subunit and a thylakoid membrane protein (
M
r
32,000) from spinach that has been implicated in herbicide and quinone binding.
Complete amino acid sequence and predicted membrane topology of phenobarbital-induced cytochrome P-450 (isozyme 2) from rabbit liver microsomes.
Authors: Tarr G E , Black S D , Fujita V S , Coon M J
Published: May 2006

The complete amino acid sequence of phenobarbital-induced isozyme 2 of rabbit liver microsomal cytochrome P-450 (P-450LM2) is presented. The polypeptide consists of 491 residues with a calculated Mr of 55,755. The rabbit isozyme is 77% identical to the corresponding rat cytochrome, P-450b, as deduced from cDNA, with 96% of the hydrophobic, 88% of the anionic, and 83% of the cationic positions conserved. The secondary structure of isozyme 2 was predicted and a model was developed for the membrane topology of this cytochrome. Of the two highly conserved cysteinyl peptides in P-450LM2, P-450b, and bacterial P-450cam, we favor, on the basis of our model, the one nearer the NH2 terminus (Cys-152 in P-450LM2) as the source of the thiolate ligand to the heme iron atom. The recently reported sequence of the apparently identical protein [Heinemann, F. S. & Ozols, J. (1983) J. Biol. Chem. 258, 4195-4201] has two fewer residues and differs in 14 other amino acid assignments.
Homology of bone-inductive proteins from human, monkey, bovine, and rat extracellular matrix.
Authors: Sampath T K , Reddi A H
Published: May 2006

Allogeneic implantation of rat extracellular demineralized diaphyseal bone matrix in subcutaneous sites induces a sequence of events resulting in the local differentiation of endochondral bone. However, xenogenic subcutaneous implantation of human, monkey, and bovine extracellular bone matrix into rat showed that bovine matrix had only a weak capacity to induce bone, whereas human and monkey matrix had none at all. This suggested that extracellular matrix-induced bone differentiation is apparently species-specific. We recently reported that the extraction of matrix with 4 M guanidine X HCl resulted in complete removal of the ability to induce endochondral bone differentiation, with the biological activity of the matrix being again restored when the extracted active matrix components (less than 50,000 daltons) were reconstituted with the inactive residue. To define the possible biochemical basis of species specificity, human, monkey, and bovine extracellular bone matrices were extracted with 4 M guanidine X HCl and the extracts were reconstituted with biologically inactive rat residue and bioassayed. The results were similar to those obtained with intact matrices and showed that total extracts of bovine matrix had a weak capacity to induce bone, whereas corresponding extracts of human and monkey matrix did not induce bone. However, partial purification by gel filtration of 4 M guanidine X HCl extracts from each species followed by reconstitution of the different fractions with inactive rat residue resulted in bone induction by all species from fractions containing proteins of less than 50,000 daltons. These observations demonstrate that species specificity of xenogenic extracellular bone matrix is due to immunogenic or inhibitory components (or both) in the guanidine X HCl residue and solubilized extracellular matrix components of greater than 50,000 daltons. These results imply that there is homology in the bone inductive proteins from human, monkey, bovine, and rat extracellular bone matrices.
Composition and distribution of low density lipoprotein fractions in hyperapobetalipoproteinemia, normolipidemia, and familial hypercholesterolemia.
Authors: Teng B , Thompson G R , Sniderman A D , Forte T M , Krauss R M , Kwiterovich P O
Published: May 2006

Hyperapobetalipoproteinemia is defined as the combination of a normal low density lipoprotein (LDL) cholesterol in the face of an increased LDL apolipoprotein B (apoB) protein. To examine the physical basis for the apparent disproportion between LDL cholesterol and apoB characteristic of this syndrome, we used density gradient ultracentrifugation to isolate LDL fractions from 10 normal subjects, from 20 patients with hyperapobetalipoproteinemia (10 normotriglyceridemic and 10 hypertriglyceridemic), and from 7 patients with familial hypercholesterolemia. In familial hypercholesterolemia, more LDL was in fraction 1--"light" LDL--and this LDL was relatively enriched in cholesterol and poor in protein. By contrast, it was fraction 2--"heavy" LDL--that differed in hyperapobetalipoproteinemia, being denser, depleted of cholesterol (particularly cholesteryl ester), and relatively enriched in apoB. These findings were more pronounced in the hypertriglyceridemic patients than in the normotriglyceridemic patients with hyperapobetalipoproteinemia. Thus this study confirms that considerable heterogeneity exists between LDL subfractions within individuals but, in addition, indicates there are also marked--and apparently characteristic--differences in LDL composition amongst normal subjects and patients with hyperapobetalipoproteinemia or familial hypercholesterolemia.
Combination therapy of schistosomiasis by tubercidin and nitrobenzylthioinosine 5'-monophosphate.
Authors: el Kouni M H , Diop D , Cha S
Published: May 2006

Nitrobenzylthioinosine 5'-monophosphate (NBMPR-P) inhibits the transport of nucleosides, including tubercidin, in mammalian systems but not in Schistosoma mansoni. Administration of NBMPR-P with high doses of tubercidin (lethal doses if injected alone) by intraperitoneal injection into S. mansoni-infected mice was highly toxic to the parasite but not to the host. Combination therapy resulted in a striking decrease in the number and copulation of worms. The few worms that could be found were so stunted that it was difficult to identify their sex. Mice receiving the combination of tubercidin plus NBMPR-P appeared healthy and had normal-sized livers and spleens. Combination therapy also caused a drastic decrease in the number of eggs in the liver (from 32,500 to 1,800 eggs per liver) and in the intestine (from 1,295 to 2 eggs per cm2). All eggs found were dead, indicating the termination of oviposition. Very few granulomas were detected in livers of treated animals. Sections of these livers showed lesions containing dead worms and what appeared to be a process of regeneration of normal tissue around old granulomas. Thus, combination therapy reduced the number and the progress of the primary pathological lesions associated with schistosomiasis. These results demonstrate that through combination therapy, highly selective toxicity against a parasite can be achieved. The effectiveness, simplicity, and practicality of host protection afforded by this method may yield a promising chemotherapeutic approach for the treatment of schistosomiasis and other parasitic diseases.
Septal deafferentation increases hippocampal adrenergic receptors: correlation with sympathetic axon sprouting.
Authors: Morrow A L , Loy R , Creese I
Published: May 2006

Denervation of the hippocampal formation in adult rats through lesion of the medial septum and diagonal band or by transection of the fimbria/fornix elicits an increase in the number of putative alpha-adrenergic receptor binding sites labeled by the antagonist ligand [3H]WB4101 [2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane]. This increase in [3H]WB4101 binding is observable at 6 days postlesion, preceding the ingrowth of sympathetic axons into the partially denervated regions of the hippocampus. The receptor up-regulation is specific for lesions of the septal (primarily cholinergic) innervation of the hippocampus. Damage to noradrenergic, dopaminergic, or serotonergic afferents as well as kainate injections in the lateral septum had no effect on [3H]WB4101 binding levels. In vivo muscarinic/cholinergic-receptor blockade does not mimic the effects of the lesion on receptor binding levels or upon axonal sprouting of the sympathetic neurons. Although [3H]WB4101 binding consistently increased after septal deafferentation, there was no clear-cut effect upon the other adrenergic ligands, including [3H]prazosin, [3H]yohimbine, P-[3H]aminoclonidine, or [3H]dihydroalprenolol. These observations can be interpreted as demonstrating a unique and selective adrenergic receptor increase after a nonadrenergic denervation but accompanying the ingrowth of anomalous adrenergic fibers. We suggest several possible relationships between the new binding sites and the ingrowing axons.
Inhibition of transcription in somatic cells by microinjection of antibodies to chromosomal proteins.
Authors: Einck L , Bustin M
Published: May 2006

The in vivo function of defined chromosomal proteins was examined by microinjecting purified antibody and antibody fragments into living fibroblasts. The involvement of histones and chromosomal high mobility group proteins HMG-1, 2, and 17 in transcription was visualized by studying the [3H]uridine incorporation in KD human fibroblasts after microinjection of fluoresceinated antibodies to these proteins. Nuclear uridine incorporation was not affected by microinjection of control antibodies or by the presence of immune complexes formed after microinjection of antibodies to chromosomal proteins that are not involved in transcription. In contrast, injection of anti-histone IgG, F(ab')2, or Fab and anti-HMG-17 IgG causes a significant reduction in transcription. The reduction is proportional to the amount of antibody introduced into the cell. We conclude that histones and protein HMG-17 are present on transcribed regions of the genome and that passage of RNA polymerase along the chromatin fiber is prevented by antibody binding to these proteins.
Sequence-specific double-strand cleavage of DNA by penta-N-methylpyrrolecarboxamide-EDTA X Fe(II).
Authors: Schultz P G , Dervan P B
Published: May 2006

In the presence of O2 and 5 mM dithiothreitol, penta-N-methylpyrrolecarboxamide-EDTA X Fe(II) [P5E X Fe(II)] at 0.5 microM cleaves pBR322 plasmid DNA (50 microM in base pairs) on opposite strands to afford discrete DNA fragments as analyzed by agarose gel electrophoresis. High-resolution denaturing gel electrophoresis of a 32P-end-labeled 517-base-pair restriction fragment containing a major cleavage site reveals that P5E X Fe(II) cleaves 3-5 base pairs contiguous to a 6-base-pair sequence, 5'-T-T-T-T-T-A-3' (4,323-4,328 base pairs). The major binding orientation of the pentapeptide occurs with the amino terminus at the adenine side of this sequence. In the presence of 5 mM dithiothreitol, 0.01 microM P5E X Fe(II) converts form I pBR322 DNA at 0.22 microM plasmid (1.0 mM in base pairs) to 40% form II, indicating the cleavage reaction is catalytic, turning over a minimum of nine times. This synthetic molecule achieves double-strand cleavage of DNA (pH 7.9, 25 degrees C) at the 6-base-pair recognition level and may provide an approach to the design of "artificial restriction enzymes".
Isolation of a genomic clone for bovine pancreatic trypsin inhibitor by using a unique-sequence synthetic DNA probe.
Authors: Anderson S , Kingston I B
Published: May 2006

Unique-sequence synthetic DNA probes, based on the known amino acid sequence of bovine pancreatic trypsin inhibitor, were constructed from oligodeoxynucleotides. In genomic Southern blot experiments, these probes were shown to hybridize specifically to discrete restriction fragments. A synthetic probe also was used to isolate a cloned BPTI gene from a bovine genomic library. DNA sequence analysis of this clone indicated that the BPTI coding region was neither preceded by a start codon nor immediately followed by a termination codon. This suggests that the mature form of BPTI may be produced through proteolytic processing from a larger polypeptide precursor.
Gene conversion and associated reciprocal recombination are separable events in vegetative cells of Saccharomyces cerevisiae.
Authors: Roman H , Fabre F
Published: May 2006

Mitotic gene conversion occurs in both the G1 and G2 phases of cell growth. We postulate that the DNA strand or strands that connect the duplexes in G1 as a consequence of strand transfer are cleaved by an endonuclease. When this takes place in G1, crossing-over occurs in G2 at a frequency that is higher than would be expected from independent events. Conversion and associated reciprocal recombination are therefore separable with respect to the stage in the cell cycle when each occurs and possibly with respect to mechanism.
Transformasomes: specialized membranous structures that protect DNA during Haemophilus transformation.
Authors: Kahn M E , Barany F , Smith H O
Published: May 2006

The mechanism by which Haemophilus protects donor DNA from cellular restriction and degradative enzymes during transformation is unclear. In this report, we demonstrate that donor DNA enters Haemophilus influenzae through specialized membranous extensions, which we have termed "transformasomes." DNA within transformasomes is in a protected state--resistant to external DNase and cellular restriction enzymes, although remaining unmodified and double-stranded. The ability of donor DNA to exit from transformasomes is dependent on its topological conformation. Circular DNA remains intact within transformasomes, while linear DNA rapidly exits and undergoes homologous recombination. Protected donor DNA can be preferentially removed from the surface of competent cells by extraction with organic solvents. Structurally intact transformasomes containing donor DNA could be partitioned into the organic layer and can be further purified by density centrifugation.
7S-K nuclear RNA from simian virus 40-transformed cells has sequence homology to the viral early promoter.
Authors: Sohn U , Szyszko J , Coombs D , Krause M
Published: May 2006

Previous findings in our laboratory have identified a specific small nuclear RNA (7S-K) that promotes transcription initiation by RNA polymerase II in isolated mammalian nuclei. The present study was designed to investigate the homology between 7S-K RNAs and host and viral sequences in simian virus 40 (SV40)-transformed and in untransformed mouse 3T3 cells, with the object of testing the hypothesis that these RNAs take part in the transcription initiation complex by base pairing to promoter/enhancer regions of active genes. DNA . RNA hybridization experiments, using either Southern or RNA blotting techniques, indicated that both 7S-K and 7S-L RNAs hybridize to midrepetitive fractions of the mouse genome. However, only 7S-K RNA from transformed cells hybridized to SV40 DNA. Restriction mapping and nuclease S1 treatment of the hybridized region of SV40 yielded a 45-nucleotide-long hybrid duplex. Partial sequence analysis of the 5' end of the DNA in this duplex revealed sequence homology with the 21-base-pair repeat sequence, identified as the SV40 early promoter. Because the viral early gene is expressed in transformed cells, we conclude that 7S-K RNAs in these cells contain a species that has sequence homology to a promoter of an active gene. Taking these results together with previous ones, we postulate that the observed stimulatory activity of 7S-K RNA on transcription initiation is due to its recognition of promoter sequences, either to facilitate the formation or as part of the transcription initiation complex.
Identification of a primosome assembly site in the region of the ori 2 replication origin of the Escherichia coli mini-F plasmid.
Authors: Imber R , Low R L , Ray D S
Published: May 2006

A primosome assembly site for F plasmid DNA replication has been identified. This site, which we term rriA (F), is localized to one strand of a 385-base-pair Sau3A restriction fragment very close to ori 2 and within the 2.25-kilobase DNA sequence required for replication and incompatibility of the entire F plasmid. rriA (F) was isolated by cloning into the deletion phage vector M13 delta Elac. This phage forms very faint plaques due to a deletion of the M13 complementary strand origin but forms large wild-type plaques when DNA single-strand initiation determinants are inserted. The single-stranded viral DNA of the Sau3A F-M13 delta Elac recombinant provides an effector site of dATP hydrolysis by the primosomal protein n'. It also provides an assembly site for the Escherichia coli primosome protein complex that directs the in vitro conversion of the single-stranded DNA to a double-stranded form by the same mechanism as that used by phi X174. Homologies of the nucleotide sequence between this F DNA sequence and the previously identified primosome assembly sites in phi X174 phage DNA and in ColE1 plasmid DNA (rriA and rriB) have been found. The sequences 5' G-T-G-A-G-C-G 3' and 5' G-N-G-G-A-A-G-C 3' or variations of these sequences occur from two to five times within each assembly locus. In addition, two distinct 15-base-pair sequences in rriA (F) are perfectly homologous to corresponding sequences in rriA (ColE1).
Construction and characterization of an infectious vaccinia virus recombinant that expresses the influenza hemagglutinin gene and induces resistance to influenza virus infection in hamsters.
Authors: Smith G L , Murphy B R , Moss B
Published: May 2006

A DNA copy of the influenza virus hemagglutinin gene, derived from influenza virus A/Jap/305/57 (H2N2) was inserted into the genome of vaccinia virus under the control of an early vaccinia virus promoter. Tissue culture cells infected with the purified recombinant virus synthesized influenza hemagglutinin, which was glycosylated and transported to the cell surface where it could be cleaved with trypsin into HA1 and HA2 subunits. Rabbits and hamsters inoculated intradermally with recombinant virus produced circulating antibodies that inhibited hemagglutination by influenza virus. Furthermore, vaccinated hamsters achieved levels of antibody similar to those obtained upon primary infection with influenza virus and were protected against respiratory infection with the A/Jap/305/57 influenza virus.
Deuterium NMR as a structural probe for micelle-associated carbohydrates: D-mannose.
Authors: Prestegard J H , Miner V W , Tyrell P M
Published: May 2006

Residual quadrupole splittings for deuterons on perdeuterated alpha- and beta-D-mannose oriented in a potassium laurate liquid crystal have been measured. Multiple quantum two-dimensional NMR spectroscopy at high magnetic field is used to gain sufficient resolution to assign splittings to specific sites within these molecules. The assigned splittings are interpreted in terms of molecular geometries and preferred orientations of the molecules relative to the surface of the potassium laurate micelles that make up the liquid crystalline phase.
Detection of lymphocytes producing a human retrovirus associated with adult T-cell leukemia by syncytia induction assay.
Authors: Hoshino H , Shimoyama M , Miwa M , Sugimura T
Published: May 2006

Recently 10 T-cell lines were established from patients with adult T-cell leukemia (ATL). During establishment of these cell lines, it was found that when T-cell lines expressing the ATL-associated retroviral antigen were cocultivated with 8C cat cells, multinucleated syncytia were formed. Retroviral antigen-negative T-cell lines did not induce syncytia. Peripheral blood lymphocytes obtained from ATL patients did not express the retroviral antigen before cultivation in vitro but became positive for the retroviral antigen after cultivation for a short period; these retroviral antigen-positive lymphocytes, but not retroviral antigen-negative lymphocytes, induced syncytia upon cocultivation with 8C cells. Peripheral blood lymphocytes isolated from patients with chronic lymphocytic leukemia of T-cell origin or Sézary syndrome or from normal adults and lymph node cells from a patient with immunoblastic lymphadenopathy-like T-cell lymphoma did not express the retroviral antigen even after cultivation in vitro and did not induce syncytia upon cocultivation with 8C cells. Thus, there was complete agreement between the presence of the retroviral antigen in established T-cell lines or freshly isolated peripheral blood lymphocytes and their ability to induce syncytia. Syncytia formation was enhanced 5- to 20-fold by the presence of Polybrene and inhibited by addition of plasma of ATL patients to the cocultures. Syncytia were detected within 4 hr on cocultivation of 8C cells with the retroviral antigen-positive T-cells, indicating that most syncytia were formed by early polykaryocytosis. After cocultivation, a clone of 8C cells that harbored the ATL virus genome and had syncytia-inducing activity was isolated. These findings indicate that the retrovirus associated with ATL has syncytia-inducing activity. Syncytia induction assay using 8C cells will be useful for detection and characterization of human retrovirus associated with T-cell malignancies.
Associative long-term potentiation in hippocampal slices.
Authors: Barrionuevo G , Brown T H
Published: May 2006

Interactions between two excitatory monosynaptic inputs to hippocampal neurons of the CA1 region were examined in the in vitro slice. By adjusting the strengths of the electrical stimuli delivered to the two input pathways, one was made to generate a weak and the other a strong synaptic response. Simultaneous tetanic stimulation of both input pathways resulted in a subsequent long-term enhanced synaptic efficacy in the weak input under conditions in which the same tetanic stimulation of either input alone failed to have this effect. This form of long-term synaptic potentiation (LTP), known as associative LTP, was shown in some cases to last hours without decrement. The plastic changes were localized within the CA1 region and appear to reside in the pre- or postsynaptic elements of the monosynaptic excitatory input to the pyramidal neurons. The increased synaptic efficacy could not be accounted for by any of several measured postsynaptic passive membrane properties.
Metorphamide: isolation, structure, and biologic activity of an amidated opioid octapeptide from bovine brain.
Authors: Weber E , Esch F S , Böhlen P , Paterson S , Corbett A D , McKnight A T , Kosterlitz H W , Barchas J D , Evans C J
Published: May 2006

Acid acetone extracts of caudate nucleus from bovine brain were found to contain an amidated opioid octapeptide with the following structure: Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-NH2. The peptide has been named metorphamide. Bovine metorphamide appears to be derived by proteolytic cleavage from proenkephalin, the common precursor to [Met5]enkephalin and [Leu5]enkephalin. The cleavage within the precursor giving rise to the carboxyl terminus of metorphamide occurs at a single arginine residue and is followed by transformation of a carboxyl-terminal glycine into an amide group. Metorphamide was detected in bovine caudate nucleus extracts by radioimmunoassay, and it was purified to homogeneity by gel filtration and reversed-phase high performance liquid chromatography. Amino acid composition analysis and automated Edman degradation in the gas-phase sequencer confirmed the postulated amino acid sequence. Carboxyl-terminal amidation of bovine metorphamide was shown by stability to carboxypeptidase A digestion and full crossreactivity in a radioimmunoassay that required the carboxyl-terminal amide as part of the recognition site. A synthetic replicate of metorphamide as well as several synthetic analogs were tested for opioid activity in several bioassays and binding assays, and metorphamide was found to have a high mu-binding activity. Metorphamide is the only known naturally occurring opioid peptide that has a high mu-binding activity. The kappa-binding activity is approximately equal to 50% that of the mu-binding activity, but delta-binding activity is negligible.
Structural organization and expression of human DNA sequences related to the transforming gene of avian myeloblastosis virus.
Authors: Franchini G , Wong-Staal F , Baluda M A , Lengel C , Tronick S R
Published: May 2006

Bacteriophage libraries of human DNA were screened for sequences homologous to the transforming gene (v-myb) of avian myeloblastosis virus. The three overlapping clones isolated were shown to contain a total of 1.0 kilobase pair (kbp) of sequence related to v-myb distributed over 6.2 kbp. Restriction enzyme mapping and heteroduplex analysis revealed the presence of five myb-related domains interrupted by four stretches of non-homology. To study the extent of human DNA coding sequences that constitute the myb gene homologue, c-myb (human), probes spanning about 30 kbp were prepared from the clones and used to study transcription in a human hematopoietic cell line (MOLT-4). Each of the probes hybridized a 4.5-kilobase transcript, which suggests that either the c-myb (human) gene encompasses 30 kbp or it contains two or more transcription units that each give rise to a mRNA of 4.5 kilobases.
Purification and characterization of the unusual deoxynucleoside, alpha-N-(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)glycinamide, specified by the phage Mu modification function.
Authors: Swinton D , Hattman S , Crain P F , Cheng C S , Smith D L , McCloskey J A
Published: May 2006

Bacteriophage Mu encodes a protein that modifies approximately equal to 15% of DNA adenine residues to a new and unusual form. Modified DNA was enzymatically digested to deoxynucleosides, and the products were fractionated by HPLC. A modified adenine nucleoside, designated dA'x, was purified and its molecular structure was established by mass spectrometry. We show that dA'x is alpha-N-(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)-glycinamide. The dA'x obtained from DNA was indistinguishable from the synthetic product with respect to its chromatographic behavior (HPLC and gas chromatography) and mass spectrum. Acid hydrolysis degrades dA'x to produce N6-carboxymethyladenine; this compound corresponds to the base Ax observed in earlier studies.
Activation of the c-myc gene by translocation: a model for translational control.
Authors: Saito H , Hayday A C , Wiman K , Hayward W S , Tonegawa S
Published: May 2006

We have shown that the human cellular oncogene c-myc is composed of three exons and is transcribed from two initiation sites separated by 175-base-pair DNA in HeLa cells. For both resulting mRNA species, exon 1 composes the 5' untranslated region and the initiator methionine is located 16 base pairs down-stream from the 5' splice acceptor of exon 2. In a non-Hodgkin lymphoma, Manca, harboring a t(8; 14) translocation, c-myc gene is broken within intron 1, and its exons 2 and 3 are translocated to a site between the heavy chain joining region cluster and C mu-coding DNA segment of the immunoglobulin heavy chain locus. The translocated c-myc gene is transcribed from points within intron 1 but is apparently still translated from the same methionine codon as the mRNA from the unrearranged c-myc gene. The nucleotide sequence of the c-myc gene shows that a region of exon 1 is highly complementary to a region of exon 2. Thus the mRNA from the untranslocated c-myc gene, as opposed to that of the translocated c-myc gene, could form a stable stem-loop structure (delta Go = -90 kcal/mol; 1 cal = 4.184 J) where the initiator AUG would be located within the loop. In view of the bind-and-scan model for the initiation of eukaryotic translation, we propose that such a secondary structure will severely hinder the translation. We further propose that the c-myc gene is often activated by translocation through the escape from such a translational suppression.
31P NMR spectroscopy of rat organs, in situ, using chronically implanted radiofrequency coils.
Authors: Koretsky A P , Wang S , Murphy-Boesch J , Klein M P , James T L , Weiner M W
Published: May 2006

A technique for making 31P NMR spectroscopic measurements in rat kidney, heart, and liver in vivo is presented. Two-turn solenoid coils were surgically implanted around the organ sufficiently in advance of NMR experiments to allow recovery of the animal. These chronically implanted coils allowed acquisition of high-resolution spectra at 40.5 and 97.3 MHz. No resolution improvement occurred at the higher field. Spectra were stable for up to 24 hr, during which time a variety of experiments could be performed. By accumulating spectra at 10-min intervals, the effects of intraperitoneal fructose injections were monitored; in kidney and liver, a rapid increase in sugar phosphates at the expense of Pi and ATP resulted. Fructose had no effect on heart metabolite levels. Spectra from the heart in vivo were obtained at systole and diastole by gating the spectrometer to the aortic pressure wave; no differences in phosphate metabolites were detected. Finally, saturation transfer techniques were used to monitor the rate of ATP synthesis in the kidney. The unidirectional rate constant for the conversion of Pi to ATP was 0.12 +/- 0.03 sec-1.
Human natural killer cells, activated lymphocyte killer cells, and monocytes possess similar cytotoxic mechanisms.
Authors: Burns G F , Triglia T , Bartlett P F , Mackay I R
Published: May 2006

The relationship between the killing mechanisms of human natural killer (NK) cells, mitogen- and mixed-lymphocyte-culture-induced activated lymphocyte killer (ALK) cells, and monocytes was investigated with a monoclonal antibody. The IgG2 antibody 9.1C3 was prepared from mice immunized with purified human large granular lymphocytes and selected from clones that inhibited NK cell killing. The 9.1C3 antibody bound to all mononuclear cells but not to granulocytes or K562 cells, and it selectively blocked killing of K562 targets by both NK and ALK cells without affecting the binding of effector to target cells. The antibody blocked killing when present from time zero and it still inhibited partially even when added 1 hr after initiation of the lytic reaction. Killing of Epstein-Barr virus-transformed B lymphoblasts by classical cytotoxic T lymphocytes was not inhibited. Of interest, 9.1C3 did block the killing of K562 target cells by cultured peripheral blood monocytes. Other monoclonal antibodies that bound to monocytes did not block killing, and a nonspecific effect of the antibody on monocytes was excluded. These data suggest that NK cells, ALK cells, and monocytes can kill tumor cell targets by using similar lytic mechanisms.
Role of mRNA competition in regulating translation: further characterization of mRNA discriminatory initiation factors.
Authors: Ray B K , Brendler T G , Adya S , Daniels-McQueen S , Miller J K , Hershey J W , Grifo J A , Merrick W C , Thach R E
Published: May 2006

Host and reovirus mRNAs compete with one another for translation in infected cells. Kinetic analysis has suggested that the site of competition is a message discriminatory initiation factor which must bind to the mRNA before it can interact with the 40S ribosomal subunit. The present communication describes an in vitro assay which can detect message discriminatory activities. A competitive situation is established by using reovirus and globin mRNAs, and then the specificity with which this competition is relieved by added components is measured. Among the various initiation factors surveyed with this assay, two have the properties expected of the mRNA discriminatory factor. These are eukaryotic initiation factor 4A and a "cap binding protein" complex. Inasmuch as the cap binding protein complex contains a subunit similar or identical to the initiation factor eIF-4A, it seems likely that only one form of the latter factor may be active in vivo. In vitro, both factors relieve competition among both capped and uncapped reovirus mRNAs according to similar hierarchies. These results suggest that some feature other than the m7G cap, such as nucleotide sequence or secondary structure, is recognized by the discriminatory factor.
Phosphorylation of parathyroid secretory protein.
Authors: Bhargava G , Russell J , Sherwood L M
Published: May 2006

The phosphorylation of proteins released into the medium of bovine parathyroid gland slices or isolated cells incubated with 32Pi has been investigated. The primary protein phosphorylated had a Mr of 68,000 and coeluted with newly synthesized parathyroid secretory protein (PSP) on Bio-Gel chromatography and on polyacrylamide gel electrophoresis. Isoelectric focusing of double-labeled samples ([35S]methionine and 32Pi) revealed comigration of the two radioactive markers at a pH of 4.6, which was similar to that of purified PSP. Phosphorylation of the Mr 68,000 protein was also demonstrated in cell homogenates incubated with [gamma-32P]ATP; the Mr 68,000 protein was the predominant labeled protein. Increasing quantities of calcium, with and without added EGTA, caused a progressive decrease in phosphorylation of the protein. These studies demonstrate that PSP is readily phosphorylated in parathyroid cells, that the degree of phosphorylation is inversely proportional to calcium concentration, and that PSP is the major phosphorylated protein released from the gland. The relationship of phosphorylation to the potential physiologic importance of PSP remains to be determined.
Pro-dynorphin peptides are found in the same neurons throughout rat brain: immunocytochemical study.
Authors: Watson S J , Khachaturian H , Taylor L , Fischli W , Goldstein A , Akil H
Published: May 2006

It is known that the opioid peptide dynorphin A has a broad distribution throughout the neuraxis. Recent biochemical studies have extended the sequence of dynorphin A by 15 amino acids to include another [Leu]enkephalin-containing peptide known as dynorphin B. These sequence data have been validated by the elucidation of the structure of the hypothalamic mRNA coding for alpha- and beta-neo-endorphin, dynorphin A, and dynorphin B. Using specific antisera directed against each of the three opioid peptides, we have studied their cellular distribution in rat brain. Their distribution patterns are extremely similar, if not identical. Furthermore, all three peptide immunoreactivities can be localized to the same cells in five nuclear groups throughout the brainstem--the supraoptic nucleus, the paraventricular nucleus, a group of cells in the lateral hypothalamic area, the nucleus parabrachialis, and the nucleus tractus solitarius. The sequence of a common precursor for dynorphin A, B, and alpha- and beta-neo-endorphin was deduced from hypothalamic mRNA. The ability to localize all three peptides together within cells in widely placed nuclei strongly supports the use of the same biosynthetic precursor for the neo-endorphin and dynorphin peptides in other parts of the central nervous system as well.
Immunological crossreactivity of Paramecium surface antigens: conformational differences account for cell surface specificity.
Authors: Bolivar I
Published: May 2006

Surface antigens (i-Ags) G and D of Paramecium primaurelia, reported earlier to be completely unrelated on the basis of peptide mapping and immunological tests, were compared and found to be similar in molecular weight (Mr 235,000), isoelectric point (ca. 4.5), and amino acid composition. They are tightly folded proteins with more than 100 intramolecular disulfide bonds. Native i-Ags and their CNBr fragments were blotted onto nitrocellulose and labeled with a battery of homologous and heterologous antibodies fractionated on native and reduced i-Ag immunoadsorbents. It was found the i-Ags retained part of their antigenic structure upon cleavage and reduction. Analysis of labeling revealed that the two proteins share many of their antigenic sites, though they are completely distinct in their native conformation. Immunofluorescence and immunoadsorption on fixed cells confirmed these results. Antigenic crossreactivity also was found with surface antigens of a ciliate outside the Paramecium species complex. It is concluded that surface specificity of paramecia is due, in this case, to the expression of related proteins in different conformations.
Characterization of L-CAM, a major cell adhesion molecule from embryonic liver cells
Authors: Gallin Warren J. , Edelman Gerald M. , Cunningham Bruce A.
Published: May 2006

We have developed a method for purifying L-CAM, the cell adhesion molecule from embryonic chicken liver cells, and have compared its properties with those of N-CAM, the neural cell adhesion molecule. L-CAM was released from membranes with trypsin, purified by a series of chemical techniques, and used to generate monoclonal antibodies which allowed the identification of the intact L-CAM molecule from membranes. The monoclonal antibodies were used to isolate trypsin-released L-CAM in a single step by affinity chromatography. Material purified by either technique was predominantly a component of
M
r
81,000 on NaDodSO
4
/polyacrylamide gel electrophoresis with a pI of 4.0-4.5. Rabbit antibodies to this component and to the
M
r
81,000 species that had been further purified on NaDodSO
4
/polyacrylamide gel electrophoresis displayed all of the activities of anti-L-CAM. Some of the trypsin-released L-CAM bound specifically to lentil lectin, suggesting that L-CAM is a glycoprotein. The apparent molecular weight of material having L-CAM antigenic determinants depended upon the procedures used to extract membranes; this appears to account for the various values reported previously in the literature. Both the rabbit serum antibodies and the monoclonal antibodies detected the
M
r
81,000 species on immunoblots of unfractionated trypsin-released material. Immunoblots of whole liver cell membranes with the same antibodies revealed a major
M
r
124,000 component, with minor components of
M
r
94,000 and 81,000. Active L-CAM derivatives released by trypsin in the presence of EGTA were detected as a species of
M
r
40,000. L-CAM derivatives obtained by extraction of membranes with EDTA alone appeared as species of
M
r
53,000, 62,000, and 81,000. The combined results suggest that L-CAM on the cell surface is an acidic glycoprotein of
M
r
124,000. In the presence of calcium, the molecule can be released from membranes by trypsin as a soluble
M
r
81,000 fragment; in the absence of calcium, it is released by either endogenous proteases or by trypsin as a variety of smaller fragments.
Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene.
Authors: Zukowski M M , Gaffney D F , Speck D , Kauffmann M , Findeli A , Wisecup A , Lecocq J P
Published: May 2006

A method to isolate fragments of DNA that promote gene expression in Bacillus subtilis is described. The system is based on production of catechol 2,3-dioxygenase [CatO2ase; catechol:oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.2] encoded by the Pseudomonas putida TOL plasmid gene xylE. The gene was transferred to aB. subtilis/Escherichia coli plasmid vector to construct pTG402. Although xylE is functionally expressed in E. coli, CatO2ase is not detected in B. subtilis unless a fragment of DNA capable of promoting gene expression is ligated into a cleavage site on pTG402 upstream from xylE. Fragments of chromosomal DNA from B. subtilis, Bacillus licheniformis, Bacillus pumilus, and E. coli are shown to promote xylE gene expression in B. subtilis. The special feature of the system is the method of detection: colonies of cells that express xylE become yellow within seconds after selection plates are sprayed with catechol, a colorless substrate that is converted by CatO2ase to the yellow product, 2-hydroxymuconic semialdehyde. The complete nucleotide sequence of xylE is presented. Strong complementarity between the ribosome binding site and 16S rRNA suggests that xylE mRNA translation in B. subtilis may commence at the same site as that recognized by P. putida. Identity of CatO2ase produced in B. subtilis, E. coli, and P. putida support the hypothesis. Our sensitive color assay offers an approach to develop plasmid gene expression vectors for a wide variety of host organisms.
Association of RNA with thymidylate synthase from methotrexate-resistant Streptococcus faecium.
Authors: Rao K N , Kisliuk R L
Published: May 2006

Thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) from methotrexate-resistant Streptococcus faecium has a UV absorbance peak at 259 nm and stains with acridine orange because of the presence of RNA on the protein. Material having an absorbance peak at 254 nm, obtained from the enzyme by phenol extraction, is degraded by treatment with pancreatic RNase, T1 RNase, and alkali but is stable to DNase. Dowex-1 chromatography of the pure enzyme yields two polynucleotide fragments in addition to the apoenzyme. As estimated from their absorbance, these fragments contain 4 and 11 mononucleotide residues per mole of enzyme, respectively. In crude extracts, thymidylate synthase is associated with rapidly sedimenting material that is sensitive to RNase. Treatment of crude extracts with RNase, as is done routinely during thymidylate synthase purification, most likely results in the formation of the small polynucleotides found on the enzyme. The RNA is not required for enzyme activity.
Ordered phosphorylation of 40S ribosomal protein S6 after serum stimulation of quiescent 3T3 cells.
Authors: Martin-Pérez J , Thomas G
Published: May 2006

The amino acids and tryptic peptides that become phosphorylated in 40S ribosomal protein S6 after serum stimulation of quiescent 3T3 cells were examined by two-dimensional thin-layer electrophoresis. In the maximally phosphorylated form of the protein, most of the phosphate was incorporated into serine and a small amount, into threonine. Digestion of this form of the protein with trypsin revealed 10 major phosphopeptides. All 10 contained phosphoserine and 2 of the 10 also contained phosphothreonine. Next, the five forms of increasingly phosphorylated S6 were individually separated on two-dimensional polyacrylamide gels or total S6 was isolated from cells that were stimulated for only a short time and their phosphotryptic maps were analyzed. The results showed that, as larger amounts of phosphate were added to S6, the phosphopeptides appeared in a specific order.
Unmasking of magnesium-dependent high-affinity binding sites for [dAla2, dLeu5]enkephalin after pretreatment of brain membranes with guanine nucleotides.
Authors: Chang K J , Blanchard S G , Cuatrecasas P
Published: May 2006

The regulation of mu- and delta-opiate receptors by guanine nucleotides and cations was studied by examining the binding of [3H][DAla2, DLeu5]enkephalin to rat brain membranes. The binding to mu-opiate receptors could be suppressed by 1 microM [DPro4]morphiceptin, a highly specific mu-agonist, thus permitting separate assessment of delta-opiate receptor binding. GTP, GDP, and the nonhydrolyzable analogs 5'-guanylyl imidodiphosphate (Gpp[NH]p) and guanosine 5'-O-(2-thiodiphosphate) (GDP-S) effectively decreased the binding to both receptor types. This inhibitory effect was potentiated by Na+. The inhibitory effect of GTP and GDP, but not of the nonhydrolyzable analogs, was reversed by Mg2+. Pretreatment of membranes with GDP or GTP increased substantially the subsequently measured high-affinity binding, and this effect required the presence of Mg2+ in the binding assay. Similar pretreatment with GDP-S resulted in only a partial increase of binding compared to GTP or GDP, and Gpp[NH]p was relatively ineffective. Similar results were observed for both mu- and delta-receptors, although the effects on mu-receptors were quantitatively more profound. These data suggest that guanine nucleotides play a dual function in regulating opiate receptor binding in a manner dependent upon the presence of Na+ or Mg2+.
Isolation of cDNA clones for the light subunit of rat liver ferritin: evidence that the light subunit is encoded by a multigene family.
Authors: Brown A J , Leibold E A , Munro H N
Published: May 2006

The shell of the iron-storage protein ferritin consists of two types of subunit (heavy, Mr = 21,000; light, Mr = 19,000). To study the structure and expression of the ferritin subunit genes, recombinant plasmids containing ferritin cDNA have been isolated. A cDNA library was constructed in the vector pBR322 from rat liver polysomal mRNA and screened by using I125-labeled antibody to rat liver ferritin. Six positive clones were identified and were shown to contain cDNA inserts ranging in length from 800 to 950 base pairs. When these cDNA clones were used for hybrid selection of rat liver mRNA and the selected mRNAs were translated in vitro, the products from each clone migrated on denaturing gels in a position similar to that of the light subunit of ferritin. No evidence of translation of the heavy subunit was obtained, indicating that the two subunits are encoded by separate mRNAs. RNA blot analysis gave a length of 1,100 nucleotides for the light-subunit mRNA. One of the cDNA inserts was fractionated into four fragments by using the restriction enzyme Sau3A. When the fragments were hybridized with Southern blots of rat spleen DNA, each fragment yielded similar patterns of hybridization to that obtained with the intact cDNA. Therefore, all regions of the cDNA sequence contain homologous sequences to similar genomic restriction fragments. This is consistent with the existence of a family of genes that encode the light subunit of rat ferritin.