bluuebunny/crossref_metadata_2025 huggingface dataset:
Retrovirus-mediated transduction of adult hepatocytes.
Authors: Wilson J M , Jefferson D M , Chowdhury J R , Novikoff P M , Johnston D E , Mulligan R C
Published: May 2006

Retrovirus-mediated gene transfer was used to develop a method for introducing genes into primary cultures of adult rat hepatocytes. Subconfluent monolayers of hepatocytes, cultured in hormonally defined media on different matrix substrata, were infected with helper-free stocks of a replication-defective retrovirus that constitutively expresses high levels of beta-galactosidase. Retrovirus-mediated transduction was measured by two methods: (i) an in situ cytochemical stain that specifically detects the expression of viral expressed beta-galactosidase, and (ii) Southern blot analysis, which measures the relative copy number of integrated provirus. Maximal transduction efficiency of approximately equal to 25% was achieved when the cells were infected after 3 days in culture; matrix had little effect on transduction efficiency. Enzyme cytochemical (catalase and glucose 6-phosphatase) and peroxidase immunocytochemical (asialoglycoprotein and UDP-glucuronosyltransferase) analyses of the cultures indicated that greater than 95% of cells were hepatocytes. The demonstration of hepatocyte-specific organelles in cells expressing the viral-directed beta-galactosidase provided unambiguous evidence for the transduction of hepatocytes. These methods should be useful in the development of liver-directed somatic gene therapy and in the study of liver-specific gene regulation.
Sequence analysis of the MYC oncogene involved in the t(8;14)(q24;q11) chromosome translocation in a human leukemia T-cell line indicates that putative regulatory regions are not altered.
Authors: Finver S N , Nishikura K , Finger L R , Haluska F G , Finan J , Nowell P C , Croce C M
Published: May 2006

We have cloned the translocation-associated and homologous normal MYC alleles from SKW-3, a leukemia T-cell line with the t(8;14)(q24;q11) translocation, and determined the sequence of the MYC oncogene first exon and flanking 5' putative regulatory regions. S1 nuclease protection experiments utilizing a MYC first exon probe demonstrated transcriptional deregulation of the MYC gene associated with the T-cell receptor alpha locus on the 8q+ chromosome of SKW-3 cells. Nucleotide sequence analysis of the translocation-associated (8q+) MYC allele identified a single base substitution within the upstream flanking region; the homologous nontranslocated allele contained an additional substitution and a two-base deletion. None of the deletions or substitutions localized to putative 5' regulatory regions. The MYC first exon sequence was germ line in both alleles. These results demonstrate that alterations within the putative 5' MYC regulatory regions are not necessarily involved in MYC deregulation in T-cell leukemias, and they show that juxtaposition of the T-cell receptor alpha locus to a germ-line MYC oncogene results in MYC deregulation.
Nonrandom X chromosome inactivation in B cells from carriers of X chromosome-linked severe combined immunodeficiency.
Authors: Conley M E , Lavoie A , Briggs C , Brown P , Guerra C , Puck J M
Published: May 2006

X chromosome-linked severe combined immunodeficiency (XSCID) is characterized by markedly reduced numbers of T cells, the absence of proliferative responses to mitogens, and hypogammaglobulinemia but normal or elevated numbers of B cells. To determine if the failure of the B cells to produce immunoglobulin might be due to expression of the XSCID gene defect in B-lineage cells as well as T cells, we analyzed patterns of X chromosome inactivation in B cells from nine obligate carriers of this disorder. A series of somatic cell hybrids that selectively retained the active X chromosome was produced from Epstein-Barr virus-stimulated B cells from each woman. To distinguish between the two X chromosomes, the hybrids from each woman were analyzed using an X-linked restriction fragment length polymorphism for which the woman in question was heterozygous. In all obligate carriers of XSCID, the B-cell hybrids demonstrated preferential use of a single X chromosome, the nonmutant X, as the active X. To determine if the small number of B-cell hybrids that contained the mutant X were derived from an immature subset of B cells, lymphocytes from three carriers were separated into surface IgM positive and surface IgM negative B cells prior to exposure to Epstein-Barr virus and production of B-cell hybrids. The results demonstrated normal random X chromosome inactivation in B-cell hybrids derived from the less mature surface IgM positive B cells. In contrast, the pattern of X chromosome inactivation in the surface IgM negative B cells, which had undergone further replication and differentiation, was significantly nonrandom in all three experiments [logarithm of odds (lod) score greater than 3.0]. These results suggest that the XSCID gene product has a direct effect on B cells as well as T cells and is required during B-cell maturation.
Transgenic rabbits with lymphocytic leukemia induced by the c-myc oncogene fused with the immunoglobulin heavy chain enhancer.
Authors: Knight K L , Spieker-Polet H , Kazdin D S , Oi V T
Published: May 2006

Transgenic rabbits with the rabbit c-myc oncogene fused with the rabbit immunoglobulin heavy chain enhancer region (E mu) DNA were developed by microinjecting pronuclei of single cell zygotes with the gene construct and implanting the microinjected eggs into pseudopregnant females. At age 17-20 days, 3 of 21 offspring born to seven females were found to have peripheral blood leukocyte counts of greater than 100,000 per mm3. Histology analyses showed extensive lymphocytic infiltration in the liver and kidney, indicating that these rabbits had a malignant lymphocytic leukemia. Genomic DNA analyses of thymus and peripheral blood lymphocytes revealed that the leukemic rabbits were transgenic and that both immunoglobulin heavy and kappa light chain genes were rearranged in the leukemic cells; thus, the leukemic cells are of B-cell lineage. One to four heavy and light chain gene rearrangements were observed, suggesting that the B-cell tumors were oligoclonal. Stable tissue culture cell lines from the bone marrow and peripheral blood of one of the transgenic rabbits have been developed. The development of B-cell leukemias in rabbits with the E mu-myc transgene contrasts with the development of B-cell lymphomas in mice carrying a similar transgene. The lymphomas in mice develop in adults and are monoclonal in origin. The leukemias in rabbits develop in juveniles, less than 3 weeks after birth, and appear oligoclonal in origin. The leukemias seem to develop in rabbit at a specific stage of development, and we suggest that a normal developmental signal may be involved in the oncogenesis.
Phospholipase C I and II brain isozymes: immunohistochemical localization in neuronal systems in rat brain.
Authors: Gerfen C R , Choi W C , Suh P G , Rhee S G
Published: May 2006

Two distinct inositol phospholipid-specific phospholipase C (PLC; phosphatidylcholine phosphatidohydrolase, EC 3.1.4.3) isozymes, PLC-I and PLC-II, have been purified and characterized from bovine brain. Monoclonal antibodies that distinguish between these isozymes are used in the present study to map isozyme distribution in the rat brain with immunohistochemical techniques. Both isozymes are localized in neurons, and, whereas PLC-II is rather ubiquitous--being expressed in most neurons, PLC-I is restricted in its distribution. The strongest immunoreactive labeling for PLC-I is in the neurons of the striatum, which provide inputs to the globus pallidus and substantia nigra, where terminals are also densely labeled. The neuronal targets of these terminals in the globus pallidus and substantia nigra do not express PLC-I immunoreactivity, but they do display PLC-II immunoreactivity. PLC-I immunoreactivity is also particularly well pronounced in the pyramidal cells of the hippocampus and, to a lesser extent, in the granule cells of the dentate gyrus. In the thalamus, PLC-I is localized to neurons in the reticular thalamic nucleus, in the medial subdivision of the mediodorsal thalamic nucleus, and in the anteromedial thalamic nucleus. Other areas displaying PLC-I immunoreactive neurons include the dorsal lateral septal nucleus and the basolateral amygdala. The expression of at least one or more forms of PLC in most neurons of the brain suggests that this enzyme may be part of a common system of signal transduction used universally by all neurons. However, the differential expression of PLC isozymes suggests further that certain neurotransmitter and receptor interactions may differ in the forms of the PLC enzyme used for signal transduction.
Electrical stimulation in vivo increases the expression of proenkephalin mRNA and decreases the expression of prodynorphin mRNA in rat hippocampal granule cells.
Authors: Morris B J , Feasey K J , ten Bruggencate G , Herz A , Höllt V
Published: May 2006

In situ hybridization histochemistry in combination with RNA blot techniques was used to study the regulation of opioid gene expression in rat hippocampus. By use of a prodynorphin cDNA probe, a strong hybridization signal was identified in the granule cell layer of the hippocampus. However, experiments using a proenkephalin cDNA probe revealed that the content of proenkephalin mRNA was considerably lower than that of prodynorphin mRNA. Following five brief trains of high-frequency electrical stimulation to the dentate gyrus, the proenkephalin mRNA content of the granule cells, measured 22 hr later, was substantially increased on the stimulated side. In contrast, levels of prodynorphin mRNA were markedly decreased ipsilateral to the stimulation site. These results were confirmed by RNA blot analysis of extracted mRNA. The decrease in prodynorphin mRNA content first became apparent between 4 and 7 hr after the end of stimulation. Distinct mechanisms, therefore, regulate the expression of proenkephalin mRNA and prodynorphin mRNA in rat hippocampus.
Detection of genes with a potential for suppressing the transformed phenotype associated with activated ras genes.
Authors: Noda M , Kitayama H , Matsuzaki T , Sugimoto Y , Okayama H , Bassin R H , Ikawa Y
Published: May 2006

Seven morphologically nontransformed (flat) revertants with reduced tumorigenicity in vivo have been isolated from populations of Kirsten sarcoma virus-transformed NIH 3T3 cells transfected with a cDNA expression library of normal human fibroblasts. Each revertant harbors 1-10 recombinant plasmids per cell and retains a rescuable transforming virus as well as high level expression of v-Ki-ras-specific RNA and the viral oncogene product, p21v-Ki-ras. Transformed phenotypes are suppressed in cell hybrids generated by fusing each revertant to v-Ki-ras-transformed NIH 3T3 cells. From two of the revertant lines, plasmids capable of giving rise to flat secondary transfectants have been recovered. Thus, in some, if not all, of the revertants, transfected cDNAs seem to be responsible for the suppression of specific transformed phenotypes.
Mercuric chloride induces autoantibodies against U3 small nuclear ribonucleoprotein in susceptible mice.
Authors: Reuter R , Tessars G , Vohr H W , Gleichmann E , Lührmann R
Published: May 2006

Autoantibodies to nucleolar components are a common serological feature of patients suffering from scleroderma, a collagen vascular autoimmune disease. While animal models, which spontaneously develop abundant anti-nucleolar antibodies, have not yet been described, high titers of such antibodies may be induced by treating susceptible strains of mice with mercuric chloride. We have identified the nucleolar autoantigen against which the HgCl2-induced IgG autoantibodies from mice of strain B10.S are directed. It is a protein with an apparent molecular mass of 36 kDa and a pI value of approximately 8.6, which is associated with the nucleolar small nuclear RNA U3, and by these criteria must be identical with a polypeptide called fibrillarin. It is striking that scleroderma patients spontaneously produce autoantibodies against the same U3 ribonucleoprotein (RNP). The HgCl2-induced murine and the scleroderma-specific human anti-U3 RNP autoantibodies were indistinguishable in their reactivities toward fibrillarin. They further resemble each other insofar as both recognize epitopes on the 36-kDa protein, which have been highly conserved throughout evolution. Our results provide a basis to investigate at the molecular level whether similar immunoregulatory dysfunctions may lead to the preferential anti-U3 RNP autoantibody production in the animal model and in scleroderma patients.
Functional role of spines in the retinal horizontal cell network.
Authors: Winslow R L , Miller R F , Ogden T E
Published: May 2006

Compartmental models derived from serial electron-microscopic reconstructions of horizontal cell processes entering cone pedicles and rod spherules are used to show that these processes have the morphological and electrical characteristics of dendritic spines. Properties of these spines are incorporated into a distributed model of the horizontal cell network. Expressions relating the magnitude of conductance changes applied at the spine heads to hyperpolarization of cells within the network are derived. Model analyses show that spine properties play a critical role in determining network responses. Specifically, increasing spine stem resistance increases the network input resistance and space constant, hyperpolarizes the resting potential, decreases response to full-field light stimuli, and increases response to small light spots. Increasing spine-stem resistance also decouples potential at the spine head from potential at the cell body. This result suggests that the location of feedback neurotransmitter release sites (e.g., at the spine heads versus the cell body) may have a profound influence on properties of horizontal cell inhibition of cone response. Because of these important functional consequences, structurally realistic models of the horizontal cell network must incorporate spine properties.
Superconducting properties of copper oxide high-temperature superconductors
Authors: Chen Guanhua , Langlois Jean-Marc , Guo Yuejin , Goddard William A.
Published: May 2006

The equations for the magnon pairing theory of high-temperature copper-oxide-based superconductors are solved and used to calculate several properties, leading to results for specific heat and critical magnetic fields consistent with experimental results. In addition, the theory suggests an explanation of why there are two sets of transition temperatures (
T
c
≈ 90 K and
T
c
≈ 55 K) for the Y
1
Ba
2
Cu
3
O
6+x
class of superconductors. It also provides an explanation of why La
2-x
Sr
x
CuO
4
is a superconductor for only a small range of
x
(and suggests an experiment to independently test the theory). These results provide support for the magnon pairing theory of high-temperature superconductors. On the basis of the theory, some suggestions are made for improving these materials.
Characterization of human cardiac myosin heavy chain genes.
Authors: Yamauchi-Takihara K , Sole M J , Liew J , Ing D , Liew C C
Published: May 2006

We have isolated and analyzed the structure of the genes coding for the alpha and beta forms of the human cardiac myosin heavy chain (MYHC). Detailed analysis of four overlapping MYHC genomic clones shows that the alpha-MYHC and beta-MYHC genes constitute a total length of 51 kilobases and are tandemly linked. The beta-MYHC-encoding gene, predominantly expressed in the normal human ventricle and also in slow-twitch skeletal muscle, is located 4.5 kilobases upstream of the alpha-MYHC-encoding gene, which is predominantly expressed in normal human atrium. We have determined the nucleotide sequences of the beta form of the MYHC gene, which is 100% homologous to the cardiac MYHC cDNA clone (pHMC3). It is unlikely that the divergence of a few nucleotide sequences from the cardiac beta-MYHC cDNA clone (pHMC3) reported in a MYHC cDNA clone (pSMHCZ) from skeletal muscle is due to a splicing mechanism. This finding suggests that the same beta form of the cardiac MYHC gene is expressed in both ventricular and slow-twitch skeletal muscle. The promoter regions of both alpha- and beta-MYHC genes, as well as the first four coding regions in the respective genes, have also been sequenced. The sequences in the 5'-flanking region of the alpha- and beta-MYHC-encoding genes diverge extensively from one another, suggesting that expression of the alpha- and beta-MYHC genes is independently regulated.
S-phase induction and transformation of quiescent NIH 3T3 cells by microinjection of phospholipase C.
Authors: Smith M R , Ryu S H , Suh P G , Rhee S G , Kung H F
Published: May 2006

Two inositol phospholipid-specific phospholipase C (PLC) isozymes (PLC-I and -II) have been purified from bovine brain. When PLC-I or PLC-II was microinjected (100-700 micrograms/ml) into quiescent NIH 3T3 cells, a time- and dose-dependent induction of DNA synthesis occurred, as demonstrated by [3H]thymidine incorporation into nuclear DNA. In addition, approximately to 8 hr after PLC injection, NIH 3T3 fibroblasts appeared spindle-shaped, refractile, and highly vacuolated, displaying a morphology similar to transformed cells. The morphologic transformation was apparent for 26-30 hr after which the injected cells reverted back to a normal phenotype. Microinjected PLC at a high concentration (1 mg/ml) was cytotoxic, dissolving the cytoplasmic membrane and leaving behind cellular ghosts. PLC is a key regulatory enzyme involved in cellular membrane signal transduction. Introduction of exogenous PLC into NIH 3T3 cells by microinjection induced a growth and oncogenic potential, as demonstrated by the ability of microinjected PLC (approximately 10,000 molecules per cell) to override the cellular G0 block, inducing DNA synthesis and morphologic transformation of growth-arrested fibroblast cells.
Cloning and molecular characterization of the trithorax locus of Drosophila melanogaster.
Authors: Mozer B A , Dawid I B
Published: May 2006

The trithorax (trx) locus of Drosophila melanogaster affects segment determination primarily in the thoracic region. Mutant flies show transformations of the third and, to a lesser extent, first thoracic segment toward the second thoracic segment; abdominal transformations also occur. Prior genetic evidence suggested that these effects are based on interactions between trx and genes of the bithorax complex and Antennapedia complex. Further, interactions between the maternal effect locus female sterile homeotic (fsh) and trx have been observed. To aid in a molecular analysis of trx function, we have cloned the locus by a P-element transposon tagging approach. Five insertion mutations have been mapped within a region of about 10 kilobases; one of these mutations reverted coincident with the loss of the insertion. Transcription mapping suggests that two RNAs of about 12 and 15 kilobases are the major transcripts of the trx locus and that the transcription unit comprises a region of about 25 kilobases. Transcripts from the trx locus are distributed uniformly in early embryos, but at 14-16 hr after fertilization the ventral nerve cord contains a higher concentration of trx RNA than other regions of the embryo.
Regulation of nerve growth factor biosynthesis by beta-adrenergic receptor activation in astrocytoma cells: a potential role of c-Fos protein.
Authors: Mocchetti I , De Bernardi M A , Szekely A M , Alho H , Brooker G , Costa E
Published: May 2006

The chain of events that results in increased production of nerve growth factor (NGF) following beta-adrenergic receptor (BAR) stimulation has been investigated in the C6-2B rat astrocytoma cell line. Exposure of these cells to the BAR agonist isoproterenol elicits the following cascade of events: (i) increase of cAMP content; (ii) increase of c-Fos mRNA content; (iii) accumulation of c-Fos protein immunoreactivity in the nucleus; (iv) increase of NGF mRNA content. The increase in c-Fos mRNA and its translation product are early events (15 and 40 min, respectively) and precede the accumulation of NGF mRNA, which peaks at 3 hr. The increase in the two mRNAs appears interrelated because cycloheximide inhibits the accumulation of c-Fos protein and NGF mRNA elicited by isoproterenol. Moreover, the accumulation of nuclear c-Fos protein and NGF mRNA induced by BAR stimulation is reduced by 2-aminopurine, an inhibitor of c-Fos mRNA induction. These data suggest that, in C6-2B astrocytoma cells, the nuclear accumulation of c-Fos protein is required for the induction of NGF mRNA expression by BAR stimulation.
Corticosterone differentially regulates the expression of Gs alpha and Gi alpha messenger RNA and protein in rat cerebral cortex.
Authors: Saito N , Guitart X , Hayward M , Tallman J F , Duman R S , Nestler E J
Published: May 2006

The possibility that glucocorticoids regulate specific guanine nucleotide binding regulatory proteins (G proteins) was investigated in rat cerebral cortex. Corticosterone was administered to normal and bilaterally adrenalectomized rats, and hormone regulation of individual G-protein subunits was investigated in cerebral cortex in three ways: (i) immunoblot analysis of subunit protein, (ii) hybridization blot analysis of subunit mRNA, and (iii) ADP-ribosylation analysis of stimulatory G protein (Gs alpha) subunits. Chronic (7 days) corticosterone administration to normal rats increased levels of Gs alpha immunoreactivity, mRNA, and ADP-ribosylation but decreased levels of inhibitory G protein (Gi alpha) mRNA and tended to decrease levels of Gi alpha immunoreactivity. In contrast, levels of Go alpha and G beta immunoreactivity and mRNA were not influenced by corticosterone treatment. In adrenalectomized rats, corticosterone treatment produced a 25-50% increase in the levels of Gs alpha immunoreactivity, mRNA, and ADP-ribosylation, whereas the hormone produced a 20-35% decrease in the levels of Gi alpha immunoreactivity and mRNA. Adrenalectomy, without corticosterone replacement, produced the opposite effects on Gs alpha and Gi alpha compared to sham-operated controls, indicating that these G proteins are regulated by this class of steroid hormone under physiological conditions in vivo. The results indicate that specific G-protein subunits--namely, Gs alpha and Gi alpha--are under the coordinated control of glucocorticoids in rat brain and demonstrate that G proteins are physiological targets of glucocorticoids in vivo. Possible roles played by these G-protein responses in mediating the effects of glucocorticoids on brain function are discussed.
Activation of prophage P4 by the P2 Cox protein and the sites of action of the Cox protein on the two phage genomes.
Authors: Saha S , Haggård-Ljungquist E , Nordström K
Published: May 2006

Phage P2 induces the unrelated prophage P4. In this paper we show that this is due to the activation of the P4 late promoter PII by the P2 Cox protein. This is in contrast to the effects of Cox on P2, for which it is known from previous work that it acts as a repressor of the promoter Pc, which is responsible for expression of the immunity repressor C. The activator role of Cox was revealed by its effect on replication of P4 DNA and on the formation of chloramphenicol acetyltransferase when a promoterless cat gene was inserted downstream of the P4 PII promoter. DNase I protection studies revealed that the Cox protein binds to the repressor promoter Pc of phage P2 and to the promoter PII of phage P4. In the latter case the Cox protein binds upstream of the -35 region, in analogy to several other activators of promoters. A weak binding was found in the promoters Pe of phage P2 and Ple of phage P4. The Cox protein is a case of viral transactivation of the replication genes of one phage by a control protein of the other. However, the effects of the Cox protein are totally different in the two phages, repressive in one case and activating in the other.
Phospholipids containing polyunsaturated fatty acyl groups are mitogenic for normal mouse mammary epithelial cells in serum-free primary cell culture.
Authors: Imagawa W , Bandyopadhyay G K , Wallace D , Nandi S
Published: May 2006

Epithelial cells obtained by collagenase digestion of mammary glands from virgin BALB/c mice were cultured in collagen gels in serum-free basal medium containing insulin (10 micrograms/ml), to which lipids or growth factors were added. Synthetic phospholipids were added as liposomes. Dilinoleoyl phosphatidic acid or phosphatidylserine or epidermal growth factor stimulated multifold growth. The optimum mitogenic effect of the phospholipids was dependent upon the presence of a polyunsaturated fatty acid esterified to the sn-2 position of the glycerol moiety. Dilinoleoyl phosphatidylcholine also stimulated growth but was generally less stimulatory than phosphatidylserine or phosphatidic acid, and phosphatidylethanolamine did not stimulate growth. Studies using phospholipids radiolabeled in either the sn-2 fatty acyl group or the glycerol backbone showed that the relative effect of phospholipids on growth did not correlate directly with the extent of their incorporation into cellular lipid, indicating that phospholipid turnover was the more important determinant for mitogenesis. Analysis of phosphatidic acid-stimulated growth suggested that both cAMP-dependent and cAMP-independent pathways were involved. Thus, mitogenic phospholipids stimulate proliferation by activating (directly or indirectly) multiple growth-regulatory pathways in mammary epithelial cells.
Retroviral protease-like gene in the vaccinia virus genome.
Authors: Slabaugh M B , Roseman N A
Published: May 2006

The retroviral protease-encoding region, PR, situated between the gag and pol genes, underwent gene duplication in the lineage now represented by simian retrovirus type 1; the sequence of the duplicated segment has diverged considerably from the present PR sequence [Power, M.D., Marx, P.A., Bryant, M.L., Gardner, M.B., Barr, P.J. & Luciw, P.A. (1986) Science 231, 1567-1572]. The PR-like duplicated gene segment was at some point translocated to a new site within the pol gene of a lentivirus (subsequent to the divergence of human immunodeficiency virus type 1), where it has been maintained. We have identified in the vaccinia virus genome a sequence that is homologous to the PR-like duplicated gene segment of both types of retrovirus in an open reading frame whose product is predicted to be a 16.2-kDa protein. The vaccinia PR-like gene is located in the HindIII F fragment, and its product displays 31-34% amino acid identity to the two types of retroviral duplicated protease sequences over a region encompassing 125 amino acid residues. Sequences flanking the vaccinia gene showed no significant homology at either the DNA or amino acid level to the retroviruses. Nuclease S1 and primer extension analyses determined that the vaccinia gene is transcribed early in infection.
Clustering of breakpoints on chromosome 10 in acute T-cell leukemias with the t(10;14) chromosome translocation.
Authors: Kagan J , Finger L R , Letofsky J , Finan J , Nowell P C , Croce C M
Published: May 2006

The T-cell receptor (TCR) alpha/delta chain locus on chromosome 14q11 is nonrandomly involved in translocations and inversions in human T-cell neoplasms. We have analyzed three acute T-lymphoblastic leukemia samples carrying a t(10;14)(q24;q11) chromosome translocation by means of somatic cell hybrids and molecular cloning. In all cases studied the translocation splits the TCR delta chain locus. Somatic cell hybrids containing the human 10q+ chromosome resulting from the translocation retain the human terminal deoxynucleotidyltransferase gene mapped at 10q23-q24 and the diversity and joining, D delta 2-J delta 1, regions of the TCR delta chain, but not the V alpha region (variable region of the TCR alpha chain), demonstrating that the split occurred within the V alpha-D delta 2 region. Molecular cloning of the breakpoint junctions revealed that the TCR delta chain sequences involved are made from the D delta 2 segment. The chromosome breakpoints are clustered within a region of approximately 263 base pairs of chromosome 10. The results suggest that the translocation of the TCR delta chain locus to a locus on 10q, which we have designated TCL3, results in deregulation of this putative oncogene, leading to acute T-cell leukemia.
Turnover of Ia-peptide complexes is facilitated in viable antigen-presenting cells: biosynthetic turnover of Ia vs. peptide exchange.
Authors: Harding C V , Roof R W , Unanue E R
Published: May 2006

Macrophages and B cells process antigens to produce antigenic peptides that associate with class II major histocompatibility complex molecules (e.g., Ia molecules); these Ia-peptide complexes are recognized by CD4+ T lymphocytes. Processing of the antigen hen egg white lysozyme was inhibited by cycloheximide in peritoneal exudate cells (PECs, largely macrophages), but not in TA3 B-lymphoma cells. The uptake and metabolism of hen egg white lysozyme was largely intact in cycloheximide-treated PECs, implicating a blockade in other steps in the formation of Ia-peptide complexes. Turnover of Ia-peptide complexes was markedly enhanced in viable antigen-presenting cells (TA3 and PEC) as compared to such complexes studied on fixed cells or in isolated preparations of Ia and peptide. In B cells the half-life of Ia-peptide complexes was much shorter than the half-life of the Ia molecules, implying turnover of Ia-peptide complexes by dissociatin and peptide exchange. In PECs, the dissociation of Ia-peptide complexes was more limited; the enhanced Ia-peptide turnover in viable PECs reflected in part biosynthetic turnover of Ia molecules. Specific mechanisms may exist in TA3 cells to facilitate exchange of peptides bound to Ia, allowing recycling of Ia to present another antigenic peptide; such Ia recycling would explain the ability of these cells to process and present antigen in the absence of Ia synthesis.
Unexpected heterogeneity of BCR-ABL fusion mRNA detected by polymerase chain reaction in Philadelphia chromosome-positive acute lymphoblastic leukemia.
Authors: Hooberman A L , Carrino J J , Leibowitz D , Rowley J D , Le Beau M M , Arlin Z A , Westbrook C A
Published: May 2006

The Philadelphia (Ph1) chromosome results in a fusion of portions of the BCR gene from chromosome 22 and the ABL gene from chromosome 9, producing a chimeric BCR-ABL mRNA and protein. In lymphoblastic leukemias, there are two molecular subtypes of the Ph1 chromosome, one with a rearrangement of the breakpoint cluster region (bcr) of the BCR gene, producing the same 8.5-kilobase BCR-ABL fusion mRNA seen in chronic myelogenous leukemia (CML), and the other, without a bcr rearrangement, producing a 7.0-kilobase BCR-ABL fusion mRNA that is seen only in acute lymphoblastic leukemia (ALL). We studied the molecular subtype of the Ph1 chromosome in 11 cases of Ph1-positive ALL, including 2 with a previous diagnosis of CML, using a sensitive method to analyze the mRNA species based on the polymerase chain reaction (PCR). We observed unexpected heterogeneity in BCR-ABL mRNA in this population; in particular, 1 of 6 bcr-rearranged cases and 1 of 5 bcr-unrearranged cases contained none of the known fusion mRNA species, while 1 of the bcr-rearranged cases contained both. This latter case is particularly interesting because it suggests that the acquisition of an additional BCR-ABL fusion species may be a mechanism of disease progression. We conclude that the PCR gives additional information about the Ph1 chromosome gene products that cannot be obtained by genomic analysis, but that it cannot be used as the sole means of detection of this chromosomal abnormality in ALL because of the high incidence of false negative results.
Inhibition of K+ efflux and dehydration of sickle cells by [(dihydroindenyl)oxy]alkanoic acid: an inhibitor of the K+ Cl- cotransport system.
Authors: Vitoux D , Olivieri O , Garay R P , Cragoe E J , Galacteros F , Beuzard Y
Published: May 2006

[(Dihydroindenyl)oxy]alkanoic acid (DIOA) was recently introduced as a potent inhibitor of the K+Cl- cotransport system without side effects on other cation transport systems [Garay, R. P., Nazaret, C., Hannaert, P.A. & Cragoe, E. J., Jr. (1988) Mol. Pharmacol. 33, 696-701]. In sickle cells, an abnormal activation of this K+Cl- cotransport system was proposed to be involved in cell K+ loss and dehydration. We found that DIOA inhibited the abnormal sickle cell K+ loss and specifically reduced sickle cell density upon stimulation of the net outward K+Cl- cotransport--i.e., low pH, hypoosmolarity, and activation by N-ethylmaleimide. DIOA opens another therapeutic approach to sickle cell disease by inhibiting cell dehydration, which favors HbS polymerization and reduces erythrocyte deformability.
Blue-light regulation of transcription for nuclear genes in pea
Authors: Marrs Kathleen A. , Kaufman Lon S.
Published: May 2006

The expression of many nuclear genes in plants is light regulated. Previous investigations have shown that the excitation of phytochrome can affect transcription rate and steady-state RNA levels for several of these genes. No direct demonstration of the effects of blue light has been reported. We have identified several nuclear genes in pea whose transcription rate and steady-state RNA levels are affected by a single pulse of blue light. Pea seedlings, grown in continuous red light to saturate any phytochrome response, were treated with a single pulse (10
3
μmol·m
-2
) of blue light 6 days after planting. The blue-light treatment resulted in an increase in the steady-state RNA level and rate of transcription for the
Cab
(chlorophyll a/b binding protein) gene family and a decrease in the steady-state RNA level and the rate of transcription for the gene families corresponding to cDNA clones pEA25 and pEA207. Changes in the rate of transcription for
Cab
and pEA207 are apparent within 3 hr of the blue-light treatment.
Failure of glucose to elicit a normal secretory response in fetal pancreatic beta cells results from glucose insensitivity of the ATP-regulated K+ channels.
Authors: Rorsman P , Arkhammar P , Bokvist K , Hellerström C , Nilsson T , Welsh M , Welsh N , Berggren P O
Published: May 2006

Fetal pancreatic beta cells demonstrate a deficient insulin release in response to glucose, but the underlying mechanism at the cellular level is unknown. By using beta cells from 21-day fetal rats we made an attempt to clarify the mechanism(s) behind this reduced glucose response. In addition to measuring insulin release, glucose metabolism, and cellular ATP content, ATP-regulated K+ channels (G channels) and voltage-activated Ca2+ currents were investigated with the patch-clamp technique. It was thus demonstrated that the ATP-regulated K+ channels in fetal beta cells were not sensitive to glucose but otherwise had similar characteristics as those of adult beta cells. Also, the characteristics of the voltage-activated Ca2+ currents were similar in adult and fetal beta cells. However, as judged from measurements of both glucose oxidation and glucose utilization, glucose metabolism was impaired in fetal beta cells. In addition, there was no increase in the ATP content, even when the cells were stimulated for 30 min. It is therefore concluded that the attenuated glucose-induced insulin release in fetal pancreatic beta cells is due to an immature glucose metabolism resulting in impaired regulation of the ATP-sensitive K+ channels. These findings may be relevant to the understanding of the deficient stimulus-secretion coupling associated with non-insulin-dependent diabetes.
Archaebacterial DNA-dependent RNA polymerases testify to the evolution of the eukaryotic nuclear genome.
Authors: Pühler G , Leffers H , Gropp F , Palm P , Klenk H P , Lottspeich F , Garrett R A , Zillig W
Published: May 2006

Genes for DNA-dependent RNA polymerase components B, A, and C from the archaebacterium Sulfolobus acidocaldarius and for components B", B', A, and C from the archaebacterium Halobacterium halobium were cloned and sequenced. They are organized in gene clusters in the order above, which corresponds to the order of the homologous rpoB and rpoC genes in the corresponding operon of the Escherichia coli genome. Derived amino acid sequences of archaebacterial components A and C were aligned with each other and with the sequences of corresponding (largest) subunits from the archaebacterium Methanobacterium thermoautotrophicum, with sequences of various eukaryotic nuclear RNA polymerases I, II, and III, and with the sequence of the beta' component from E. coli polymerase. The archaebacterial genes for component A are homologous to about the first two-thirds of genes for the eukaryotic component A and the eubacterial component beta', and the archaebacterial genes for component C are homologous to the last third of the genes for the eukaryotic component A and the eubacterial component beta'. Unrooted phylogenetic dendrograms derived from both distance matrix and parsimony analyses show the archaebacteria are a coherent group closely related to the eukaryotic nuclear RNA polymerase II and/or III lineages. The eukaryotic polymerase I lineage appears to arise separately from a bifurcation with the eubacterial beta' component lineage.
Targeted homologous recombination at the endogenous adenine phosphoribosyltransferase locus in Chinese hamster cells.
Authors: Adair G M , Nairn R S , Wilson J H , Seidman M M , Brotherman K A , MacKinnon C , Scheerer J B
Published: May 2006

We have developed a system that permits analysis of targeted homologous recombination at an endogenous, chromosomal gene locus in cultured mammalian cells. Using a hemizygous, adenine phosphoribosyltransferase (APRT)-deficient, Chinese hamster ovary (CHO) cell mutant as a transfection recipient, we have demonstrated correction of a nonrevertible deletion mutation by targeted homologous recombination. Transfection with a plasmid carrying a fragment of the APRT gene yielded APRT+ recombinants at a frequency of approximately 4.1 x 10(-7). The ratio of targeted recombination to nontargeted integrations of plasmid sequences was approximately 1:4000. Analysis of 31 independent APRT+ recombinants revealed conversions of the endogenous APRT gene, targeted integration at the APRT locus, and a third class of events in which the plasmid donor APRT fragment was converted to a full-length, functional gene.
Juxtaposition of domains homologous to protein kinases and histidyl-tRNA synthetases in GCN2 protein suggests a mechanism for coupling GCN4 expression to amino acid availability.
Authors: Wek R C , Jackson B M , Hinnebusch A G
Published: May 2006

The GCN2 protein of Saccharomyces cerevisiae stimulates the expression of amino acid biosynthetic genes under conditions of amino acid starvation by derepressing GCN4, a transcriptional activator of these genes. GCN2 contains sequences homologous to the catalytic domain of protein kinases. We show here that substitution of a highly conserved lysine in the presumed ATP-binding site of this domain impairs the derepression of histidine biosynthetic genes under GCN4 control. This result supports the idea that protein kinase activity is required for GCN2 positive regulatory function. Determination of the nucleotide sequence of the entire GCN2 complementation unit, and measurement of the molecular weight of GCN2 protein expressed in vivo, indicate that GCN2 is a Mr approximately 180,000 protein and contains a Mr approximately 60,000 segment homologous to histidyl-tRNA synthetases (HisRSs) juxtaposed to the protein kinase domain. Several two-codon insertion mutations in the HisRS-related coding sequences inactivate GCN2 regulatory function. Based on these results, we propose that the GCN2 HisRS domain responds to the presence of uncharged tRNA by activating the adjacent protein kinase moiety, thus providing a means of coupling GCN2-mediated derepression of GCN4 expression to the availability of amino acids.
Two murine natural polyreactive autoantibodies are encoded by nonmutated germ-line genes.
Authors: Baccala R , Quang T V , Gilbert M , Ternynck T , Avrameas S
Published: May 2006

Two monoclonal IgM natural autoantibodies (E7 and D23) obtained from the fusion of normal, nonimmunized, BALB/c mouse spleen cells and nonsecreting myeloma cells were selected on the basis of their polyreactivity with auto- and xenoantigens and chemical haptens. Nucleotide sequence analysis of the variable and constant regions of the heavy and light chains showed the following. (i) The antibodies arise from different genetic elements with very low or no homology--E7 from a heavy-chain variable region (VH) of family 36-60 and kappa light-chain variable region (V kappa) from a group 19--whereas D23 derives from a VH of family Q52 and V kappa derives from group 8. (ii) E7 and D23 are probably of germ-line origin, as suggested by high homology with VH genes from the unrearranged genome. Compared with the germ-line VH 1210.7 gene, E7 has a single nucleotide difference leading to a silent mutation at position 15, whereas D23 seems to be encoded by germ-line VH 101 with one nucleotide difference causing replacement of Ser-84 by Ala. (iii) The genetic V kappa and VH elements for E7 and D23 also give rise to different responses to phenyloxazolone, dinitrophenyl, 5-(dimethylamino)naphthalene-1-sulfonyl, arsonate, phosphocholine, and influenza virus hemagglutinin. Antibodies from normal and autoimmune mice with rheumatoid factor-like activity are also homologous to E7 and D23. These results indicate that polyreactive autoantibodies are encoded by germ-line genes and that, starting with the preimmune poly- and autoreactive repertoire, mutated forms of antibodies recognizing exogenous antigens can be obtained and selected.
Complete nucleotide sequence of primitive vertebrate immunoglobulin light chain genes.
Authors: Shamblott M J , Litman G W
Published: May 2006

Antibody to Heterodontus francisci (horned shark) immunoglobulin light chain was used to screen a spleen cDNA expression library, and recombinant clones encoding light chain genes were isolated. The complete sequences of the mature coding regions of two light chain genes in this phylogenetically distant vertebrate have been determined and are reported here. Comparisons of the sequences are consistent with the presence of mammalian-like framework and complementarity-determining regions. The predicted amino acid sequences of the genes are more related to mammalian lambda than to kappa light chains. The nucleotide sequences of the genes are most related to mammalian T-cell antigen receptor beta chain. Heterodontus light chain genes may reflect characteristics of the common ancestor of immunoglobulin and T-cell antigen receptors before its evolutionary diversification.
Coamplification of human acetylcholinesterase and butyrylcholinesterase genes in blood cells: correlation with various leukemias and abnormal megakaryocytopoiesis.
Authors: Lapidot-Lifson Y , Prody C A , Ginzberg D , Meytes D , Zakut H , Soreq H
Published: May 2006

To study the yet unknown role of the ubiquitous family of cholinesterases (ChoEases) in developing blood cells, the recently isolated cDNAs encoding human acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) and butyrylcholinesterase (BtChoEase; cholinesterase; acylcholine acylhydrolase, EC 3.1.1.8) were used in blot hybridization with peripheral blood DNA from various leukemic patients. Hybridization signals (10- to 200-fold intensified) and modified restriction patterns were observed with both cDNA probes in 4 of the 16 leukemia DNA preparations examined. These reflected the amplification of the corresponding AcChoEase and BtChoEase genes (ACHE and CHE) and alteration in their structure. Parallel analysis of 30 control samples revealed nonpolymorphic, much weaker hybridization signals for each of the probes. In view of previous reports on the effect of acetylcholine analogs and ChoEase inhibitors in the induction of megakaryocytopoiesis and production of platelets in the mouse, we further searched for such phenomena in nonleukemic patients with platelet production disorders. Amplifications of both ACHE and CHE genes were found in 2 of the 4 patients so far examined. Pronounced coamplification of these two related but distinct genes in correlation with pathological production of blood cells suggests a functional role for members of the ChoEase family in megakaryocytopoiesis and raises the question whether the coamplification of these genes could be causally involved in the etiology of hemocytopoietic disorders.
Axonal arborizations of a magnocellular basal nucleus input and their relation to the neurons in the thalamic reticular nucleus of rats.
Authors: Asanuma C
Published: May 2006

A dense axonal plexus, arising in a portion of the magnocellular basal nucleus, was identified in the thalamic reticular nucleus in adult rats. The details of these axonal arbors as well as their relation to the neurons of the reticular nucleus were investigated by using Phaseolus vulgaris leucoagglutinin injections into the basal nucleus and intracellular injections of Lucifer yellow into reticular nucleus neurons. Axons arising in the caudal basal nucleus at the medial margin of the globus pallidus do not enter the dorsal thalamus but are confined to the reticular nucleus, where they arborize widely and densely. Neurons in the reticular nucleus are large, with sparsely spined and beaded dendrites, which radiate within the plane of the nucleus. Bouton-like swellings along basal nucleus axons are often found apposed to the somata of reticular nucleus neurons, although many are also apposed to dendrites. These morphological observations suggest a second potentially significant route, in addition to its well-known direct cortical projection, through which the magnocellular basal nucleus could influence cortical function: it may, by strategically modulating the excitability of reticular nucleus neurons, alter the general state of the thalamus and hence affect the initial transmission of information to the cortex.
Thymidine kinase synthesis is repressed in nonreplicating muscle cells by a translational mechanism that does not affect the polysomal distribution of thymidine kinase mRNA.
Authors: Gross M K , Merrill G F
Published: May 2006

The molecular basis for replication-dependent expression of thymidine kinase (TK) activity (EC 2.7.1.21) was investigated in mouse skeletal muscle cells transformed with multiple copies of the chicken TK gene. When shifted to mitogen-depleted medium, proliferating myoblasts irreversibly withdraw from the cell cycle and commit to terminal differentiation. Early after commitment, postreplicative myocytes maintain nearly proliferative levels of TK mRNA but have greatly reduced levels of TK activity. Metabolic labeling studies with [35S]methionine indicated that the decrease in TK activity was associated with a 10-fold reduction in the rate of TK protein synthesis. Commitment had little effect on the stability or catalytic efficiency of TK protein. The decrease in TK synthetic rate in the continued presence of TK mRNA indicated that translation of TK mRNA was repressed in committed cells. The distribution of TK mRNA between ribonucleoprotein particles and polysomes was determined. In both proliferative cells and committed cells, TK mRNA levels were maximal in polysomes containing five to seven ribosomes. Thus, the synthesis of TK protein in nonreplicating muscle cells was inhibited by a translational mechanism that did not alter the average number of ribosomes engaged by TK mRNA.