bluuebunny/crossref_metadata_2025 huggingface dataset:
Presence of abscisic acid, a phytohormone, in the mammalian brain.
Authors: Le Page-Degivry M T , Bidard J N , Rouvier E , Bulard C , Lazdunski M
Published: May 2006

This paper reports the presence of abscisic acid, one of the most important phytohormones, in the central nervous system of pigs and rats. The identification of this hormone in brain was made after extensive purification by using a radioimmunoassay that is very specific for (+)-cis-abscisic acid. The final product of purification from mammalian brain has the same properties as authentic abscisic acid: it crossreacts in the radioimmunoassay for the phytohormone and it has the same retention properties and the same gas chromatography/mass spectrometry characteristics. Moreover, like (+)-cis-abscisic acid itself, the brain factor inhibits stomatal apertures of abaxial epidermis strips of Setcreasea purpurea Boom (Commelinaceae). The presence of abscisic acid conjugates that are present in plants has also been identified in brain.
Isolation of the gene encoding yeast single-stranded nucleic acid binding protein 1.
Authors: Jong A Y , Campbell J L
Published: May 2006

A yeast gene encoding SSB-1, a single-stranded nucleic acid binding protein, has been isolated by screening a lambda gt11 genomic DNA library. The gene is located on a 1.84-kilobase chromosomal Bgl II-BamHI fragment. Yeast strains carrying the high-copy-number vector YEp24 with an SSB1 gene insert overproduce SSB-1 3-fold and SSB-1 mRNA 10-fold. A typical haploid cell contains about 20,000 molecules of SSB-1; thus, the cells can tolerate up to 60,000 copies. Yeast SSB-1 was expressed in Escherichia coli cells by using a phage T7 expression system. Spores containing the gene disrupted at a point within the coding sequence germinate and grow normally; thus, the gene is not essential. Protein blots show that no SSB-1 or novel immunologically related species that might retain SSB-1 activity are present in cells containing the disrupted SSB1 genes. Southern analysis and protein blots suggest the presence in yeast of a second, related, but nonidentical gene and two immunologically related proteins of 55 kDa and 75 kDa.
RNA splicing products formed with isolated fractions from HeLa cells are associated with fast-sedimenting complexes.
Authors: Perkins K K , Furneaux H M , Hurwitz J
Published: May 2006

Three fractions (designated Ia, Ib, and II) have been isolated from HeLa cell nuclear extracts that are required for splicing of adenovirus and human beta-globin RNA transcripts in vitro. The incubation of two of the fractions (Ib and II) in the presence of ATP resulted in cleavage of precursor mRNA at the 5' splice site and formation of the intron-exon lariat. Addition of fraction Ia to the combination of Ib and II resulted in the formation of spliced RNA and the intron lariat. When fraction II was incubated with precursor RNA in the presence of ATP and the resulting products were sedimented through sucrose gradients, a 30S complex was detected that contained precursor RNA. The combination of fractions Ib and II resulted in the production of a 55S complex that contained the 5' exon as a prominent RNA species. The combination of fractions I (containing Ia and Ib) and II resulted in the formation of the 55S complex and material sedimenting between 40 S and 20 S, in which the predominant RNA species was spliced RNA.
Structural features of the 5' noncoding region of the rabbit globin messenger RNAs engaged in translation.
Authors: Krowczynska A , Brawerman G
Published: May 2006

Accessible sites in the 5' noncoding region of the rabbit alpha- and beta-globin mRNAs were identified and compared in deproteinized RNA and in the mRNAs engaged in translation in the reticulocyte lysate. Preparations of RNA and lysate were subjected to limited nuclease digestion by RNase T1 and Neurospora endonuclease, and the cleavage sites were analyzed by a nuclease S1 mapping procedure. The free alpha-globin mRNA contained few nuclease-sensitive sites and its initiation codon AUG was masked. The free beta-globin mRNA contained a larger number of accessible sites and its AUG was highly exposed. The distribution of sensitive sites differed considerably in the lysate. In both mRNA species, a site near the 5' terminus became the one most accessible to Neurospora endonuclease. Also the accessibility of the AUG in beta-globin mRNA decreased considerably. The distribution of accessible sites in the lysate was the same when the mRNAs were undergoing rapid initiation and when initiation became limited after prolonged incubation. Inhibition of initiation by the cap analogue 7-methylguanosine 5'-triphosphate was accompanied by increased sensitivity of some of the sites in both mRNA species. One of the accessible sites in each mRNA species had a sequence complementary to the 3'-terminal portion of the 18S ribosomal RNA.
Involvement of the 5'-leader sequence in coupling the stability of a human H3 histone mRNA with DNA replication.
Authors: Morris T , Marashi F , Weber L , Hickey E , Greenspan D , Bonner J , Stein J , Stein G
Published: May 2006

Two lines of evidence derived from fusion gene constructs indicate that sequences residing in the 5'-nontranslated region of a cell cycle-dependent human H3 histone mRNA are involved in the selective destabilization that occurs when DNA synthesis is terminated. The experimental approach was to construct chimeric genes in which fragments of the mRNA coding regions of the H3 histone gene were fused with fragments of genes not expressed in a cell cycle-dependent manner. After transfection in HeLa S3 cells with the recombinant plasmids, levels of fusion mRNAs were determined by S1 nuclease analysis prior to and following DNA synthesis inhibition. When the first 20 nucleotides of an H3 histone mRNA leader were replaced with 89 nucleotides of the leader from a Drosophila heat-shock (hsp70) mRNA, the fusion transcript remained stable during inhibition of DNA synthesis, in contrast to the rapid destabilization of the endogenous histone mRNA in these cells. In a reciprocal experiment, a histone-globin fusion gene was constructed that produced a transcript with the initial 20 nucleotides of the H3 histone mRNA substituted for the human beta-globin mRNA leader. In HeLa cells treated with inhibitors of DNA synthesis and/or protein synthesis, cellular levels of this histone-globin fusion mRNA appeared to be regulated in a manner similar to endogenous histone mRNA levels. These results suggest that the first 20 nucleotides of the leader are sufficient to couple histone mRNA stability with DNA replication.
Specificity and mechanism of protein kinase C activation by sn-1,2-diacylglycerols.
Authors: Ganong B R , Loomis C R , Hannun Y A , Bell R M
Published: May 2006

The specificity of protein kinase C activation by sn-1,2-diacylglycerols and analogues was investigated by using a Triton X-100 mixed micellar assay [Hannun, Y. A., Loomis, C. R. & Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043]. Analogues containing acyl or alkyl chains eight carbons in length were synthesized because sn-1,2-dioctanoylglycerol is an effective cell-permeant activator of protein kinase C. These analogues were tested as activators and antagonists of rat brain protein kinase C to determine the exact structural features important for activity. The analogues established that activation of protein kinase C by diacylglycerols is highly specific. Several analogues established that both carbonyl moieties of the oxygen esters are required for maximal activity and that the 3-hydroxyl moiety is also required. None of the analogues were antagonists. These data, combined with previous investigations, permitted formulation of a model of protein kinase C activation. A three-point attachment of sn-1,2-diacylglycerol to the surface-bound protein kinase C-phosphatidylserine-Ca2+ complex is envisioned to cause activation. Direct ligation of diacylglycerol to Ca2+ is proposed to be an essential step in the mechanism of activation of protein kinase C.
Mutations activating human c-Ha-ras1 protooncogene (HRAS1) induced by chemical carcinogens and depurination.
Authors: Vousden K H , Bos J L , Marshall C J , Phillips D H
Published: May 2006

In vitro modification of plasmids containing the human c-Ha-ras1 protooncogene (HRAS1) with the ultimate carcinogens N-acetoxy-2-acetylaminofluorene and r-7, t-8-dihydroxy-t-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (anti-BPDE) generated a transforming oncogene when the modified DNA was transfected into NIH 3T3 cells. The protooncogene was also activated by heating the plasmid at 70 degrees C, pH 4, to generate apurinic/apyrimidinic sites in the DNA. DNA isolated from transformed foci was analyzed by hybridization with 20-mer oligonucleotides designed to detect single point mutations within two regions of the gene commonly found to be mutated in tumor DNA. Of 23 transformants studied, 7 contained a mutation in the region of the 12th codon, whereas the remaining 16 were mutated in the 61st codon. Of the codon-61 mutants, 6 were mutated at the first base position (C X G), 5 at the second (A X T), and 5 at the third (G X C). The point mutations induced by anti-BPDE were predominantly G X C----T X A and A X T----T X A base substitutions, whereas four N-acetoxy-2-acetylaminofluorene-induced mutations were all G X C----T X A, and a single depurination-induced activation that was analyzed contained an A X T----T X A transversion. Together, these methods provide a useful means of determining point mutations produced by DNA-damaging agents in mammalian cells.
Poly(riboadenylic acid) preferentially inhibits in vitro translation of cellular mRNAs compared with vaccinia virus mRNAs: possible role in vaccinia virus cytopathology.
Authors: Bablanian R , Banerjee A K
Published: May 2006

Vaccinia virus-induced inhibition of host protein synthesis seems to be mediated by viral transcripts based on their differential inhibition of cellular mRNA translation in a rabbit reticulocyte lysate system. In this study, we demonstrated that the removal of poly(riboadenylic acid) [poly(A)] from the in vitro viral transcripts abolished this inhibition in the same cell-free system. This observation led us to the finding that less than 1 microM poly(A) completely inhibited HeLa cell mRNA translation in the reticulocyte lysate, whereas only 50% inhibition of vaccinia virus mRNA translation was observed at the same concentration. Similar results were also obtained in a wheat germ protein-synthesizing system. This inhibitory effect of poly(A) was totally abrogated by the addition of polydeoxythymidylate. This selective inhibition was highly specific for poly(A) since other homopolymers, including poly(G), poly(C), and poly(dA), were not capable of causing such an inhibition. Poly(U), however, had a moderate selective inhibitory effect. Among the several mRNAs tested, the translation of L-cell, encephalomyocarditis virus, and reovirus RNAs was also sensitive to poly(A). However, vesicular stomatitis virus mRNA translation was strikingly more resistant. These results suggest that poly(A), which is also synthesized by the virion-associated poly(A) polymerase may be involved in vaccinia virus-mediated host cell shutoff.
Studies of the radical species in compound ES of cytochrome c peroxidase altered by site-directed mutagenesis.
Authors: Goodin D B , Mauk A G , Smith M
Published: May 2006

Yeast cytochrome c peroxidase reacts with hydrogen peroxide to form an intermediate, compound ES, in which the heme iron atom is converted to a ferryl function (Fe4+ = O) and a radical center is generated on a reversibly oxidizable amino acid residue of uncertain identity. As methionine-172 is a possible site of this radical, we have constructed specific variants of cytochrome c peroxidase in which methionine-172 is replaced by serine or cysteine. These mutants and the wild-type enzyme have been expressed in Saccharomyces cerevisiae, purified, and crystallized. Both mutant enzymes are fully active. A stable, reversible, peroxide-induced intermediate with optical properties characteristic of compound ES is observed for the three forms of the enzyme. The electron paramagnetic resonance spectrum of this intermediate at 93 K for the serine mutant exhibits the narrow free-radical signal and hyperfine structure observed for the wild-type enzyme. However, a broader component of the signal that is observed for the wild-type enzyme at this temperature is absent from the spectrum observed with the serine mutant. These results demonstrate that the narrow component of the free-radical signal observed at 93 K cannot reside at methionine-172. The absence of the broader component of the signal for the serine mutant may reflect the loss of spin density on methionine or, alternatively, could arise from conformationally induced changes in the properties of the radical. The results are consistent with a heterogeneity of radical species in the ES complex.
Direct measurements of intramolecular electron transfer rates between cytochrome c and cytochrome c peroxidase: effects of exothermicity and primary sequence on rate.
Authors: Cheung E , Taylor K , Kornblatt J A , English A M , McLendon G , Miller J R
Published: May 2006

Rapid mixing of ferrocytochrome c peroxidase [cyt c peroxidase(II)] and ferricytochrome c [cyt c(III)] results in the reduction of cyt c(III) by cyt c peroxidase(II). In 10 mM phosphate, pH 7.0, the rate of decay of cyt c peroxidase(II) and the rate of accumulation of cyt c(II) give equal first-order rate constants: k = 0.23 +/- 0.02 s-1. Equivalent results are obtained by pulse radiolysis using isopropanol radical as the reducing agent. This rate is independent of the initial cyt c(III):cyt c peroxidase(II) ratios. These results are consistent with unimolecular electron transfer occurring within a cyt c(III)-cyt c peroxidase(II) complex. When cyt c is replaced by porphyrin cyt c (iron-free cyt c), a complex still forms with cyt c peroxidase. On radiolysis, using e-aq as the reducing agent, intracomplex electron transfer occurs from the porphyrin cyt c anion radical to cyt c peroxidase(III) with k = 150 s-1. This large rate increase with increasing delta G degrees suggests that the barrier for intracomplex electron transfer is large. Finally, we have briefly investigated how the cyt c peroxidase(II)----cyt c(III) rate depends on the primary structure of cyt c(III). We find the reactivity order to be as follows: yeast (k = 3.4 s-1) greater than horse (k = 0.3 s-1) greater than tuna (k = 0.2 s-1). These results mirror a report [Ho, P. S., Sutoris, C., Liang, N., Margoliash, E. & Hoffman, B. M. (1985) J. Am. Chem. Soc. 107, 1070-1071] on excited state reactions of the cyt c/cyt c peroxidase couple.
Adhesion and erythrophagocytosis of human senescent erythrocytes by autologous monocytes and their inhibition by beta-galactosyl derivatives.
Authors: Vaysse J , Gattegno L , Bladier D , Aminoff D
Published: May 2006

Senescent human erythrocytes (RBC) are able to adhere to and be phagocytized by autologous monocytes in vitro to a greater extent than are young RBC. This adhesion and erythrophagocytosis of senescent RBC is inhibited by D-galactose, N-acetyl-D-galactosamine, their corresponding derivatives of bovine serum albumin, and lactose. On the other hand, D-glucose, D-mannose, L-fucose, N-acetyl-D-glucosamine, and their corresponding derivatives of bovine serum albumin are noninhibiting. The glycopeptides released by tryptic digestion of senescent RBC and purified on immobilized peanut agglutinin are the most effective inhibitors of both RBC adhesion and phagocytosis by autologous monocytes obtained from peripheral blood.
Frequent lambda light chain gene rearrangement and expression in a Ly-1 B lymphoma with a productive kappa chain allele.
Authors: Hardy R R , Dangl J L , Hayakawa K , Jager G , Herzenberg L A , Herzenberg L A
Published: May 2006

We describe here a murine Ly-1-bearing pre-B-cell tumor that, when induced for kappa light chain expression with bacterial lipopolysaccharide, also gives rise spontaneously to a few percent of cells expressing surface lambda light chains. These lambda-positive cells have undergone DNA rearrangements involving either V lambda 1 or V lambda 2 genes. Nearly all clones of lambda-bearing cells express mu and lambda on their surface (but not kappa). However, all these lambda-positive clones continue to transcribe kappa mRNA and synthesize internal kappa chains. Further, surface lambda-positive clones show JH rearrangements on one or both heavy chain chromosomes.
Only high-affinity receptors for interleukin 2 mediate internalization of ligand.
Authors: Weissman A M , Harford J B , Svetlik P B , Leonard W L , Depper J M , Waldmann T A , Greene W C , Klausner R D
Published: May 2006

Interleukin 2 (IL-2) receptors are expressed on activated T cells and in select T-cell leukemias. Recently, it has been demonstrated that at least two classes of receptor for IL-2 exist with markedly different affinities for ligand. All known biological actions of IL-2 have been correlated with occupancy of high-affinity sites; the function of the low-affinity sites remains unknown. Receptor-mediated endocytosis is the primary means of internalization of cell-surface receptors and their ligands. The internalization of IL-2 bound to high- and low-affinity receptor sites was studied in a human T-cell lymphotrophic virus type 1 (HTLV-1)-infected human T-cell leukemia cell line and in a cloned murine cytotoxic T-cell line (CTLL). Internalization of IL-2 occurred only when bound to high-affinity sites. In addition, an anti-receptor antibody (anti-Tac), which binds equally well to high- and low-affinity sites, demonstrated no detectable internalization. The implications of these findings as they relate to IL-2 receptor structure and function are discussed.
Spin glass model of learning by selection.
Authors: Toulouse G , Dehaene S , Changeux J P
Published: May 2006

A model of learning by selection is described at the level of neuronal networks. It is formally related to statistical mechanics with the aim to describe memory storage during development and in the adult. Networks with symmetric interactions have been shown to function as content-addressable memories, but the present approach differs from previous instructive models. Four biologically relevant aspects are treated--initial state before learning, synaptic sign changes, hierarchical categorization of stored patterns, and synaptic learning rule. Several of the hypotheses are tested numerically. Starting from the limit case of random connections (spin glass), selection is viewed as pruning of a complex tree of states generated with maximal parsimony of genetic information.
Excitation trapping and primary charge stabilization in
Rhodopseudomonas viridis
cells, measured electrically with picosecond resolution
Authors: Deprez J. , Trissl H. W. , Breton J.
Published: May 2006

The transmembrane primary charge separation in the photosynthetic bacterium
Rhodopseudomonas viridis
was monitored by electric measurements of the light-gradient type [Trissl, H. W. & Kunze, U. (1985)
Biochim. Biophys. Acta
806, 136-144]. Excitation of whole cells with 30-ps laser pulses at either 532 nm or 1064 nm gave rise to a biphasic increase of the photovoltage. The fast phase, contributing about 50% of the total, rose with an exponential time constant ≤40 ps and was independent of the redox state of the quinone electron acceptor. It is assigned to the migration of the excitation energy in the antenna and its subsequent trapping by the reaction center, monitored by the ultrafast charge separation between the primary electron donor and the bacteriopheophytin intermediary acceptor. The slower phase (125 ± 50 ps) only occurred when the quinone was oxidized and disappeared when it was reduced (either chemically or photochemically). It is assigned to the forward electron transfer from the bacteriopheophytin to the quinone. The relative amplitudes of these two electrogenic steps demonstrate that the bacteriopheophytin intermediary acceptor is located halfway between the primary donor and the quinone.
Fetal characteristics of small intestinal crypt cells.
Authors: Quaroni A
Published: May 2006

Nine monoclonal antibodies were prepared against luminal membranes purified from rat intestinal cells at day 19 of gestation, and seven of them were found to define antigens common to adult crypt cells and fetal or embryonic intestinal epithelial cells. The FBB 2/29 antigen was first detected over the entire intestinal epithelial population at days 14-15 of gestation, a period of development characterized by formation of a stratified intestinal epithelium and differentiation of the surrounding mesenchyme. This antigen, identified as a set of high molecular mass proteins, became restricted to the crypt and lower villus cells after birth and was exclusively expressed by the crypt cells in adult intestine. It also was found to be expressed by the epithelial cells of the distal tubuli in the kidney of adult rats and by cultured human tumor colonic cells. The FBB 1/54/1, FBB 3/46, and FBB 3/78/9 antibodies stained only the epithelial cells present at the base of the villi in fetal intestine, starting at days 20-21 of gestation (about 1-2 days before birth), and stained the crypt and lower villus cells in newborn and adult intestine; these antigens may be regarded as specific markers for the developing crypt cells in fetal intestine shortly before birth. The FBB 1/20 and FBB 4/2 antigens were first detected on the fetal intestinal cells at day 18 of gestation; they were located over the entire epithelium in newborn rats and became restricted to the crypts after weaning. The FBB 2/28 antigen was expressed by the entire intestinal epithelium at all stages of development, starting from days 18-19 of gestation in the fetus. Two antibodies, FBB 3/4 and FBB 3/24, were found to be specific for lactase. These results have demonstrated the expression of cell- and tissue-specific components in rat intestine during early embryonic development and revealed a marked similarity in surface membrane antigens between fetal intestinal epithelial cells and adult crypt cells.
Transcription regulation of peroxisomal fatty acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase in rat liver by peroxisome proliferators.
Authors: Reddy J K , Goel S K , Nemali M R , Carrino J J , Laffler T G , Reddy M K , Sperbeck S J , Osumi T , Hashimoto T , Lalwani N D
Published: May 2006

The structurally diverse peroxisome proliferators ciprofibrate, clofibrate, and bis(2-ethylhexyl) phthalate [(EtHx)2 greater than Pht] increase the activities of hepatic catalase and peroxisomal fatty acid beta-oxidation enzymes in conjunction with profound proliferation of peroxisomes in hepatocytes. In order to delineate the level at which these enzymes are induced in the liver, the transcriptional activity of specific genes for fatty acyl-CoA oxidase (FAOxase) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (PBE), the first two enzymes of the peroxisomal beta-oxidation system, and for catalase were measured in isolated hepatocyte nuclei obtained from male rats following a single intragastric dose of ciprofibrate, clofibrate, or (EtHx)2 greater than Pht. All three peroxisome proliferators rapidly increased the rate of FAOxase and PBE gene transcription in liver, with near maximal rates (9-15 times control) reached by 1 hr and persisting until at least 16 hr after administration of the compound. FAOxase and PBE mRNA levels, measured by blot-hybridization analysis and FAOxase and PBE protein content, analyzed by immunoblotting, increased concurrently up to at least 16 hr following a single dose of peroxisome proliferator. The catalase mRNA level increased about 1.4-fold, but the transcription rate of the catalase gene was not significantly affected. The results show that the peroxisome proliferators clofibrate, ciprofibrate, and (EtHx)2 greater than Pht selectively increase the rate of transcription of peroxisomal fatty acid beta-oxidation enzyme genes. Whether the transcriptional effects are mediated by peroxisome proliferator-receptor complexes remains to be elucidated.
Heterogeneity of chromosome 22 breakpoint in Philadelphia-positive (Ph+) acute lymphocytic leukemia.
Authors: Erikson J , Griffin C A , ar-Rushdi A , Valtieri M , Hoxie J , Finan J , Emanuel B S , Rovera G , Nowell P C , Croce C M
Published: May 2006

In chronic myelogenous leukemias (CML) with the t(9;22)(q34;q11) chromosome translocation the breakpoints on chromosome 22 occur within a 5.8-kilobase segment of DNA referred to as "breakpoint cluster region" (bcr). The same cytogenetically indistinguishable translocation occurs in approximately 10% of patients with acute lymphocytic leukemias (ALL). In this study we have investigated the chromosome breakpoints in several cases of ALL carrying the t(9;22) translocation. In three of five cases of ALL we found that the bcr region was not involved in the chromosome rearrangement and that the 22q11 chromosome breakpoints were proximal (5') to the bcr region at band 22q11. In addition, we observed normal size bcr and c-abl transcripts in an ALL cell line carrying the t(9;22) translocation. We conclude, therefore, that if c-abl is inappropriately expressed in ALL cells without bcr rearrangements, the genetic mechanism of activation must be different from that reported for CML.
Isolation and structure of a C-terminally amidated nonopioid peptide, amidorphin-(8-26), from bovine striatum: a major product of proenkephalin in brain but not in adrenal medulla.
Authors: Liebisch D C , Weber E , Kosicka B , Gramsch C , Herz A , Seizinger B R
Published: May 2006

We have isolated and sequenced a C-terminally amidated peptide from bovine striatum. The peptide was purified to homogeneity by adsorption to XAD-2 resins and four different HPLC steps. Amino acid composition analysis and gas-phase sequence analysis revealed identity of this peptide with residues 8-26 of the proenkephalin-derived opioid peptide amidorphin, which we have recently isolated from bovine adrenal medulla. C-terminal amidation of amidorphin-(8-26) from bovine striatum was demonstrated by its stability to carboxypeptidase A digestion and full crossreactivity in a radioimmunoassay that required the C-terminal amide group as part of the recognition site. The nonopioid peptide amidorphin-(8-26), which lacks the N-terminal [Met]enkephalin sequence of amidorphin, is a major product of the opioid peptide precursor proenkephalin in the brain. In the adrenal medulla, however, where amidorphin occurs in remarkably high concentrations, amidorphin-(8-26) could not be detected. This is indicative of differential post-translational processing of proenkephalin in different tissues. In the brain, as opposed to the adrenal medulla, amidorphin is further processed at the typical cleavage signals of two basic residues, giving rise to the nonopioid peptide amidorphin-(8-26) and, possibly, to the opioid peptide [Met]enkephalin. Thus, proenkephalin in the brain might be considered as a precursor in which an opioid peptide is linked with a nonopioid peptide of possibly different biological function.
Nerve growth factor receptor molecules in rat brain.
Authors: Taniuchi M , Schweitzer J B , Johnson E M
Published: May 2006

We have developed a method to immunoprecipitate rat nerve growth factor (NGF) receptor proteins and have applied the method to detect NGF receptor molecules in the rat brain. Crosslinking 125I-labeled NGF to either PC12 cells or cultured rat sympathetic neurons yielded two radiolabeled molecules (90 kDa and 220 kDa) that were immunoprecipitated by monoclonal antibody 192-IgG. Further, 192-IgG precipitated two radiolabeled proteins, with the expected sizes (80 kDa and 210 kDa) of noncrosslinked NGF receptor components, from among numerous surface-iodinated PC12 cell proteins. These results demonstrate the specific immunoprecipitation of NGF receptor molecules by 192-IgG. We applied the 125I-NGF crosslinking and 192-IgG-mediated immunoprecipitation procedures to plasma membrane preparations of the following areas of rat brain: medial septum, cerebellum, brainstem, hippocampus, cerebral cortex, thalamus, and olfactory bulb. NGF receptor molecules of the same molecular masses as the peripheral receptor components were consistently detected in all of these regions and in preparations from whole brains. Removal of the peripheral sympathetic innervation of the brain did not eliminate these NGF receptor proteins, indicating that the receptor is endogenous to central nervous system tissues. We also observed retrograde transport of 125I-labeled 192-IgG from the parietal cortex to the nucleus basalis and from the hippocampus to the nucleus of the diagonal band of Broca and the medial septal nucleus. These findings demonstrate the presence in brain of NGF receptor molecules indistinguishable from those of the peripheral nervous system.
Tissue-specific expression of glucokinase: identification of the gene product in liver and pancreatic islets.
Authors: Iynedjian P B , Möbius G , Seitz H J , Wollheim C B , Renold A E
Published: May 2006

The tissue distribution of glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was examined by protein blotting analysis. Antibodies raised against rat liver glucokinase recognized a single protein subunit with an apparent Mr of 56,500 on nitrocellulose blots of cytosol protein from liver, separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A protein of identical electrophoretic mobility was detected by immunoblotting of cytosol protein from pancreatic islets. Hepatic glucokinase and the immunoreactive islet product bound to and were eluted from DEAE-cellulose at the same ionic strength. Glucokinase was displayed as a set of two spots with apparent pI values of 5.54 and 5.64 by immunoblotting after two-dimensional gel electrophoresis. The two isoforms appeared equally abundant in liver extract, whereas the component with a pI of 5.64 was predominant in islets. By quantitative immunoblotting, glucokinase was estimated to represent 0.1% of total cytosol protein in liver and 1/20th as much in islets. The glucokinase activity of both liver and islet cytosols was suppressed by the antibodies to hepatic glucokinase. Immunoblotting of cytosol protein from intestinal mucosa, exocrine pancreas, epididymal adipose tissue, kidney, brain, and spleen failed to reveal the glucokinase protein. Thus, significant expression of the glucokinase gene appears restricted to the liver and pancreatic islets.
Differential degradation of [35S]methionine polypeptides in Duchenne muscular dystrophy skin fibroblasts in vitro.
Authors: Rodemann H P , Bayreuther K
Published: May 2006

Rates of protein turnover have been measured in three normal and three Duchenne muscular dystrophy (DMD) skin fibroblast cell lines. Cell populations were analyzed at identical states with regard to cell number, state of topoinhibition, and cumulative population doublings (CPD). Net protein synthesis measured by the incorporation of [35S]methionine in an 18-hr pulse was reduced by an average of 34%; degradation of total cellular protein measured after an 18-hr pulse with [35S]methionine and a 24-hr chase was enhanced by an average of 50% in DMD fibroblasts. Two-dimensional gel electrophoresis analyses revealed that the breakdown of the majority of [35S]methionine polypeptides was markedly increased in DMD fibroblasts. Quantitative determinations of the differential degradation rates of 10 selected proteins in the tropomyosin region of two-dimensional gels were undertaken by scintillation counting and computer analyses. In three series of experiments, the degradation of the 10 proteins in DMD fibroblasts was enhanced by individual polypeptides between 12.0% and 151.2% as measured by scintillation counting or between 0.8% and 128% as determined by computer analyses.
Biogenesis of the nuclear lamina: in vivo synthesis and processing of nuclear protein precursors.
Authors: Lehner C F , Fürstenberger G , Eppenberger H M , Nigg E A
Published: May 2006

Utilizing antibodies against lamins A, B1, and B2, we have studied the biogenesis of the nuclear lamina in chicken embryo fibroblasts. (Lamins B1 and B2 have been identified recently as structurally distinct "lamin B" proteins.) We demonstrate that, unique among the nuclear proteins studied to date, lamin A is synthesized as a higher molecular mass precursor. A short-lived higher molecular mass variant (t 1/2 approximately equal to 3 min) accompanying the mature-size protein was also detected in the case of lamin B2 biosynthesis, but no precursor was found for lamin B1. By combining pulse-chase experiments with subcellular fractionation, we provide evidence that synthesis of lamin proteins occurs on free polysomes; subsequently, the newly synthesized proteins become rapidly associated with a crude nuclear fraction. The lamin A precursor is processed within the nucleus with a half-time of about 30 min. Concomitantly, lamin proteins acquire a characteristic resistance to detergent extraction, suggesting their insertion into a submembraneous protein network. The described biogenetic pathway involving precursor synthesis and processing is very unusual for nuclear proteins; it may have interesting implications for the mechanisms of transport and assembly of poorly soluble nuclear proteins.
Overreplication and recombination of DNA in higher eukaryotes: potential consequences and biological implications.
Authors: Schimke R T , Sherwood S W , Hill A B , Johnston R N
Published: May 2006

We propose that a fundamental problem in the faithful replication of complex chromosomes of higher eukaryotes is the proper control of both the number and timing of the multiple initiations of replication on single chromosomes. When replication patterns are disrupted by any of a variety of agents, overreplication of DNA can occur. We propose a model that accounts for the generation of a wide variety of chromosomal aberrations-rearrangements, resulting from the various ways in which the overreplicated strands can undergo recombination. We also discuss certain implications of the generation of chromosomal alterations in higher eukaryotes as they may relate to cancer chemotherapy, cancer progression, aging, and rapid speciation-evolution.
Two distinct T-cell receptor alpha-chain transcripts in a rabbit T-cell line: implications for allelic exclusion in T cells.
Authors: Marche P N , Kindt T J
Published: May 2006

Information relevant to allelic exclusion in T cells has been obtained by a study of cDNA clones corresponding to alpha-chain genes of the T-cell receptor in the rabbit T-cell line RL-5. One clone contains a variable-joining-constant (VJC) sequence encoding a complete alpha chain of the T-cell receptor. A second has an identical constant region and includes a distinct variable-joining (VJ) sequence. However, a single-base deletion in the variable region places the remainder of the second transcript out-of-phase and appears to be the product of a rearrangement involving a variable region of the T-cell receptor alpha-chain pseudogene. Presence of two variable-joining-constant (VJC) transcripts in the same cell line indicates that alpha-chain gene rearrangement is not affected by transcription of a complete alpha-chain mRNA and suggests that steps after mRNA synthesis are involved in the allelic exclusion process for alpha-chain genes. Comparison of rabbit alpha-chain sequences with those of man and mouse revealed interspecies conservation in constant and variable regions. Genomic Southern blot analyses using a rabbit constant region of the T-cell receptor alpha-chain probe revealed the presence of a single constant region gene. Hybridization with variable region probes defined two distinct multigenic subfamilies. Homology between certain rabbit and murine variable regions of the T-cell receptor alpha-chain sequences suggests that the existence of subfamilies predated divergence of these species.
Synaptogenesis of cultured striatal neurons in serum-free medium: a morphological and biochemical study.
Authors: Weiss S , Pin J P , Sebben M , Kemp D E , Sladeczek F , Gabrion J , Bockaert J
Published: May 2006

Striatal neurons were cultured from the fetal mouse brain and maintained in serum-free medium for 14-21 days in vitro (DIV). Pretreatment of the culture dishes successively with a polycation followed by fetal calf serum resulted in rapid neuron attachment and neurite proliferation. After 9-10 DIV, electron microscope observations revealed the presence of vesicles in axon terminals forming mature synapses with axons and perikarya of adjacent neurons and in varicosities along extended axons. Synapsin I, a synaptic vesicle-specific protein, was present only in neuronal perikarya after 3 DIV, in perikarya and in varicosities along extended axons after 6 DIV, and in varicosities and contact points between axon terminals and adjacent axons or perikarya after 11-14 DIV. Neurotransmitter-stimulated intracellular formation of cAMP decreased markedly during neuronal differentiation. Inositol phosphate formation in response to neurotransmitters, however, increased significantly throughout the period of striatal neuronal development. K+ (56 mM) depolarization resulted in a 2-fold increase in endogenous gamma-aminobutyric acid (GABA) release from striatal neurons, 50% of which was Ca2+-dependent, between 3 and 11 DIV. Between 11 and 14 DIV, subsequent to synapse formation (as revealed by electron microscope observations), GABA release evoked by 56 mM K+ increased up to 5-fold, 75% of which was Ca2+-dependent. It appears that the complete differentiation of striatal neurons in serum-free medium may provide a suitable model for the study of the physiological and regulatory mechanisms involved in nerve cell development.
Use of specific antibodies to quantitate the guanine nucleotide-binding protein Go in brain.
Authors: Gierschik P , Milligan G , Pines M , Goldsmith P , Codina J , Klee W , Spiegel A
Published: May 2006

We immunized rabbits with purified guanine nucleotide-binding proteins (G proteins) from bovine brain and obtained an antiserum, RV3, that reacts specifically with the alpha subunit (39 kDa) of a G protein of unknown function, termed Go, as well as with the beta subunit (35 kDa) common to all G proteins. RV3 showed no crossreactivity with the alpha subunits of the stimulatory (Gs) or inhibitory (Gi) G proteins associated with adenylate cyclase, nor with that of the rod outer segment G protein, transducin. Immunoblots with crude and affinity-purified antiserum showed that RV3 specifically recognizes the Go alpha subunit and the beta subunit in crude brain membranes. Using RV3, we found approximately equal amounts of Go in brain membranes from frog, chicken, rat, cow, and man. Quantitative immunoblotting gave Go alpha subunit/ beta subunit ratios approximately equal to 1 in cerebral cortex, raising the possibility that free Go alpha subunit (unassociated with beta subunit) may exist in brain. The concentration of Go alpha subunit in cortex is about 5 times that of Gi alpha subunit. The results show that Go is an immunochemically distinct, highly conserved protein distributed throughout the brain, with particularly high concentrations in forebrain.
Cayley-Menger coordinates.
Authors: Sippl M J , Scheraga H A
Published: May 2006

A major obstacle in applying distance geometry techniques is the analytical complexity of the Cayley-Menger determinants that are used to characterize euclidian spaces in terms of distances between points. In this paper we show that, with the aid of a theorem of Jacobi, the complex Cayley-Menger determinants can be replaced by simpler determinants, and we derive the concept of Cayley-Menger coordinates, a coordinate system in terms of which each point of En is characterized by n + 1 distances to n + 1 points of a reference. We also show that this coordinate system provides a natural norm for the incomplete embedding problem. This paper provides the tools to treat the problem of filling out an incomplete distance matrix so that our previous procedure can then be used to embed the corresponding structure in a three-dimensional space.
Harvey ras genes transform without mutant codons, apparently activated by truncation of a 5' exon (exon -1).
Authors: Cichutek K , Duesberg P H
Published: May 2006

The hypothesis is tested that the ras gene of Harvey sarcoma virus (Ha-SV) and the proto-ras DNAs from certain tumor cells derive transforming function from specific codons in which they differ from normal proto-ras genes. Molecularly cloned Harvey proviral vectors carrying viral ras, normal rat proto-ras, and recombinant ras genes in which the virus-specific ras codons 12 and 59 were replaced by proto-ras equivalents each transformed aneuploid mouse 3T3 cells after latent periods that ranged from 4 to 10 days. Viruses with or without virus-specific ras codons all transformed diploid rat cells in 3-5 days equally well. However, in the absence of virus replication, mutant codons were beneficial for transforming function. Deletion of non-ras regions of Ha-SV did not affect transforming function. We conclude that specific ras codons are not necessary for transforming function. Comparisons of the ras sequences of Ha-SV, BALB SV, and Rasheed SV with sequences of proto-ras genes from rat and man revealed an upstream proto-ras exon, termed exon -1. The 3' end of this exon is present in all three viruses and in a ras pseudogene of the rat. Since ras genes transform without mutation and since exon -1 is truncated in viral ras genes and all transforming proto-ras DNAs of the Harvey and the Kirsten ras family, we propose that ras genes are activated by truncation of exon -1 either via viral transduction or artificially via cloning and transfection. The proposal implies that untruncated proto-ras genes with point mutations may not be cellular cancer genes.
Partial sequencing of human adult, human fetal, and bovine intestinal alkaline phosphatases: comparison with the human placental and liver isozymes.
Authors: Hua J C , Berger J , Pan Y C , Hulmes J D , Udenfriend S
Published: May 2006

Purification, molecular weights, amino acid compositions, and partial sequencing of intestinal alkaline phosphatases (EC 3.1.3.1) from human adult, human fetal, and bovine sources is reported. Additional sequence information is presented for the bovine liver isozyme. Comparisons are made of the partial primary structures of intestinal alkaline phosphatases with those of the isozymes from liver and placenta. Homologies among these isozymes provide structural data to corroborate some concepts of the etiology of these isozymes and refute others.
Cloning of a portion of the chromosomal gene for human erythrocyte alpha-spectrin by using a synthetic gene fragment.
Authors: Linnenbach A J , Speicher D W , Marchesi V T , Forget B G
Published: May 2006

A region of minimal codon degeneracy was selected from the amino acid sequence of the amino-terminal alpha I domain of human erythrocyte spectrin to design a 90-base-pair DNA probe for the screening of a human genomic library. Five complementary oligonucleotides were assembled to form a full-length double-stranded DNA, which was then cloned in an M13 phage vector to generate hybridization probes. Under stringent conditions, a single hybridizing clone was isolated from a total human genomic library. Partial DNA sequence analysis established the 16.8-kilobase-pair isolate as erythrocyte alpha-spectrin by correlation to a known sequence of 131 amino acids. The spectrin 106 amino acid repeat segment is encoded by multiple exons separated by introns of various sizes. Of the 3074 base pairs of DNA sequenced thus far, 12.8% code for amino acids.
Primary structure and cDNA cloning of human fibroblast collagenase inhibitor.
Authors: Carmichael D F , Sommer A , Thompson R C , Anderson D C , Smith C G , Welgus H G , Stricklin G P
Published: May 2006

We report the primary structure and cDNA cloning of human fibroblast collagenase inhibitor, a glycoprotein that appears to play a central role in modulating the activity of a number of metalloendoproteases of connective tissue origin including collagenase, gelatinase, and proteoglycanase. Secreted human fibroblast collagenase inhibitor was purified and subjected to automated Edman degradation. The secreted protein consists of 184 amino acid residues; it contains two sites of N-linked oligosaccharide linkage and six disulfide bonds. Synthetic oligonucleotide probes based on selected amino acid sequences of the inhibitor were used to screen a lambda gt10 cDNA library from a human fibroblast line. Two overlapping cDNA clones were characterized to determine the complete coding and noncoding sequences of the specific mRNA. The amino acid sequence deduced from the nucleotide sequence agrees with that determined by protein sequencing. One clone appears to contain the complete 5' end and, in addition, the cDNA sequence predicts a 23-amino acid leader peptide. The other clone represents the 3' end of the mature message and includes a short poly(A)+ tract. This 3' sequence is remarkably similar to a reported cDNA encoding part of the protein derived from mouse fibroblast poly(A)+ RNA. However, this inhibitor has no substantial homology with previously sequenced protease inhibitors.
Rapid activation by fungal elicitor of genes encoding “pathogenesis-related” proteins in cultured parsley cells
Authors: Somssich Imre E. , Schmelzer Elmon , Bollmann Joachim , Hahlbrock Klaus
Published: May 2006

Administration of a cell-wall preparation from the fungus
Phytophthora megasperma
f. sp.
glycinea
, which acts as an elicitor of phytoalexin production in cell suspension cultures of parsley (
Petroselinum crispum
), also results in a rapid and dramatic increase in the relative amounts of mRNAs coding for a number of small proteins having low isoelectric points. According to various operational criteria, the translation products are classified as “pathogenesis-related” (PR) proteins. Here we report that the cDNA inserts of two pBR322-derived plasmids, pcPR1 and pcPR2, are homologous to mRNAs coding for one (PR1) and three (PR2) of these proteins in hybrid-selected
in vitro
translation experiments. Nuclear run-off transcription studies show that activation of the corresponding genes is extremely rapid; we observed a 4-fold increase in the transcription rate of the PR1 gene within 5 min and a 3-fold increase for the PR2 gene within 20 min following elicitation. Subsequent increases in the amounts of PR1 and PR2 mRNAs indicate that regulation of PR protein synthesis occurs at the transcriptional level.
Developmental regulation of a gene that encodes a cysteine-rich intestinal protein and maps near the murine immunoglobulin heavy chain locus.
Authors: Birkenmeier E H , Gordon J I
Published: May 2006

Mouse and rat small intestinal cDNA libraries were screened for recombinants derived from mRNAs whose concentration changed during the transition from suckling to weaning. cDNAs transcribed from a 570-nucleotide-long mRNA were isolated. Dot blot hybridization analyses of RNA recovered at various stages of rat gastrointestinal ontogeny indicated that the concentration of this mRNA begins to increase during the mid-suckling period, reaching a peak during weaning. There is considerable variation in the relative amount of this mRNA in adult tissues, with highest levels encountered in the rat small intestine and colon. Its concentration in duodenum, jejunum, and ileum is approximately the same. It is more concentrated in villi than in crypts. The rat mRNA encodes a 77 amino acid, 8.55-kDa polypeptide that has seven cysteine residues. This cysteine-rich intestinal protein (named CRIP) has two internal repeated sequence blocks. Computer-assisted comparisons of CRIP to proteins of known function disclosed that it is homologous to certain ferredoxins. Southern blot analyses revealed that sequences homologous to the rat gene are present in sea squirt, fish, bird, and human DNA, indicating that this gene is highly conserved and that related proteins may be present in many if not all vertebrates. Recombinant inbred mouse strains were utilized to show that the CRIP gene is closely linked to the immunoglobulin heavy chain constant region locus, Igh-c, on chromosome 12. CRIP mRNA is a molecular marker for the suckling-to-weaning transition of rodent intestinal development. The cloned cDNA may be a useful probe for identifying factors that regulate intestinal development during this period.