bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of a chronic myelogenous leukemia-like syndrome in mice with v-abl and BCR/ABL.
Authors: Kelliher M A , McLaughlin J , Witte O N , Rosenberg N
Published: June 2006

The v-abl gene in Abelson virus induces pre-B-cell lymphoma in mice while the BCR/ABL oncogene is associated with chronic myelogenous leukemia and some cases of acute lymphocytic leukemia in humans. Understanding the mechanisms by which these oncogenes affect various cell types has been hampered by a paucity of experimental systems that reproduce the range of biological effects associated with them. We have developed an experimental system in which murine hematopoietic stem cell populations are infected with either v-abl or BCR/ABL retroviruses and are used to reconstitute lethally irradiated mice. Irrespective of the form of activated abl, greater than 90% of the animals reconstituted with such cells develop tumors. About 50% of them develop a myeloproliferative syndrome that shares several features with the chronic phase of chronic myelogenous leukemia; the remaining animals succumb to pre-B-cell lymphomas. The myeloproliferative syndrome is characterized by large numbers of clonally derived, infected myeloid cells. This model will allow study of the mechanism by which activated abl genes affect hematopoietic precursors in chronic myelogenous leukemia. Furthermore, our results demonstrate that introduction of an activated abl gene into the appropriate target cell, not the structure of the gene, is the major determinant in myeloid cell specificity.
Xenopus laevis alpha and beta thyroid hormone receptors.
Authors: Yaoita Y , Shi Y B , Brown D D
Published: June 2006

The Xenopus laevis genome encodes two genes for the alpha (TR alpha) and two genes for the beta (TR beta) thyroid hormone receptors. The two TR alpha genes closely resemble their rat, human, and chicken counterparts. No alternatively spliced TR alpha cDNA clones were found in the 5' untranslated region (5' UTR). In contrast, complex alternative splicing of TR beta mRNA occurs within the 5' UTR as well as possible alternative transcriptional start sites. As many as eight exons encoding mainly the 5' UTR are alternatively spliced, giving rise to at least two amino termini for each of the two TR beta proteins. The 5' UTR of transcripts from both TR alpha and TR beta genes contain multiple AUG sequences with short open reading frames suggesting translational control mechanisms might play a role in expression of TR genes.
Self-reactive T cells can escape clonal deletion in T-cell receptor V beta 8.1 transgenic mice.
Authors: Yui K , Komori S , Katsumata M , Siegel R M , Greene M I
Published: June 2006

To study the mechanisms of tolerance in detail, we have constructed transgenic mice expressing a V beta 8.1-D beta 2-J beta 2.3-C beta 2 T-cell receptor (TCR) gene. Since expression of V beta 8.1 is known to correlate with reactivity of CD4+CD8- T cells to minor lymphocyte-stimulating locus 1a (Mls-1a), we expected to induce tolerance in most CD4+CD8- T cells in V beta 8.1 transgenic mice of the Mls-1a allele. In one line of Mls-1b V beta 8.1 transgenic mice, the V beta 8.1 TCR was expressed on greater than 98% of mature T cells and their response to Mls-1a was highly enriched. In Mls-1a V beta 8.1 transgenic mice, CD4+CD8- T cells in these mice were severely reduced among both peripheral T cells and thymocytes. However, the deletion of these cells was not complete, and most of the residual CD4+CD8- mature T cells still expressed normal densities of V beta 8.1 TCR. The residual CD4+CD8- T cells did not respond to Mls-1a but were still able to proliferate in response to other stimuli via the TCR. Interestingly, CD4+CD8- V beta 8.1+ T-cell clones isolated from Mls-1a V beta 8.1 transgenic mice could respond to Mls-1a. We suggest that these types of T cells escape clonal deletion in the thymus.
mRNA coding for oxytocin is present in axons of the hypothalamo-neurohypophysial tract.
Authors: Jirikowski G F , Sanna P P , Bloom F E
Published: June 2006

Neuronal mRNA is thought to be restricted to perikaryal and dendritic compartments containing rough endoplasmic reticulum. We have used both in situ hybridization and DNA polymerase chain reaction methods to determine the precise intracellular distribution of oxytocin mRNA. Using light- and electron-microscopic detection of in situ hybridization with 5'-bromo-2'-deoxyuridine-labeled oligonucleotide probes, we found oxytocin mRNA in axons and Herring bodies in the lateral and ventral hypothalamus, the median eminence, and the posterior lobe of the pituitary in postpartum lactating rats. Southern blot analysis of the amplification products confirmed the presence of oxytocin mRNA in all three tissue samples. The present findings indicate that oxytocin mRNA can be transported axonally. Such transport could reflect an adventitious compartmentalization or a functional storage in Herring bodies for subsequent secretion.
Visna virus encodes a post-transcriptional regulator of viral structural gene expression.
Authors: Tiley L S , Brown P H , Le S Y , Maizel J V , Clements J E , Cullen B R
Published: June 2006

Visna virus is an ungulate lentivirus that is distantly related to the primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1). Replication of HIV-1 and of other complex primate retroviruses, including human T-cell leukemia virus type I (HTLV-I), requires the expression in trans of a virally encoded post-transcriptional activator of viral structural gene expression termed Rev (HIV-1) or Rex (HTLV-I). We demonstrate that the previously defined L open reading frame of visna virus encodes a protein, here termed Rev-V, that is required for the cytoplasmic expression of the incompletely spliced RNA that encodes the viral envelope protein. Transactivation by Rev-V was shown to require a cis-acting target sequence that coincides with a predicted RNA secondary structure located within the visna virus env gene. However, Rev-V was unable to function by using the structurally similar RNA target sequences previously defined for Rev or Rex and, therefore, displays a distinct sequence specificity. Remarkably, substitution of this visna virus target sequence in place of the HIV-1 Rev response element permitted the Rev-V protein to efficiently rescue the expression of HIV-1 structural proteins, including Gag, from a Rev- proviral clone. These results suggest that the post-transcriptional regulation of viral structural gene expression may be a characteristic feature of complex retroviruses.
DNA amplification-restricted transcription-translation: rapid analysis of rhesus rotavirus neutralization sites.
Authors: Mackow E R , Yamanaka M Y , Dang M N , Greenberg H B
Published: June 2006

DNA amplification-restricted transcription-translation (DARTT), is based on DNA amplification by the polymerase chain reaction (PCR) and uses PCR to truncate protein-encoding DNA while adding transcriptional and translational initiation signals to the segment. The amplified DNA segments are transcribed into RNA and translated into protein in vitro and the synthesized proteins are used to define functional sites. DARTT was applied to rhesus rotavirus gene segment 4 cDNA in order to create a series of carboxyl-terminal truncations and new amino termini in the encoded VP4 capsid protein. The truncated VP4 polypeptides were tested for reaction with 11 VP4-specific neutralizing monoclonal antibodies to identify the minimum polypeptides required for antibody recognition. Monoclonal antibodies 2G4, M2, and M7, which neutralize a number of serologically distinct rotaviruses, required amino acids 247-474 of VP4 for binding. DARTT is potentially applicable to the identification of discontinuous epitopes and functional domains on a variety of proteins.
Characterization and expression of the complementary DNA encoding rat histidine decarboxylase.
Authors: Joseph D R , Sullivan P M , Wang Y M , Kozak C , Fenstermacher D A , Behrendsen M E , Zahnow C A
Published: June 2006

Histamine is a neurotransmitter in the central nervous system and an important modulator of gastric acid secretion, vasomotor control, inflammation, and allergic reactions. In biological systems the formation of histamine from its precursor histidine is catalyzed by the enzyme L-histidine decarboxylase (HDC; L-histidine carboxy-lyase, EC 4.1.1.22). We have cloned HDC-encoding cDNA from a fetal rat liver cDNA library (phage lambda gt11) have deduced the amino acid sequence from the nucleotide sequence. The clone was proven to be HDC cDNA by expression of active recombinant enzyme in COS cells and by chromosomal mapping. The cDNA encodes a protein of Mr 73,450 (655 amino acid residues). The discrepancy between this molecular weight and the size of the purified fetal liver protein subunits [Taguchi, Y., Watanabe, T., Kubota, H., Hayashi, H. & Wada, H. (1984) J. Biol. Chem. 259, 5214-5221] (Mr = 54,000) suggests that HDC may be posttranslationally processed. The 469 amino acid residues from the amino-terminal portion of the protein share 50% identity with rat and Drosophila L-dopa decarboxylases and much less homology with other characterized amino acid decarboxylases.
Isolation of a new herpesvirus from human CD4+ T cells.
Authors: Frenkel N , Schirmer E C , Wyatt L S , Katsafanas G , Roffman E , Danovich R M , June C H
Published: June 2006

A new human herpesvirus has been isolated from CD4+ T cells purified from peripheral blood mononuclear cells of a healthy individual (RK), following incubation of the cells under conditions promoting T-cell activation. The virus could not be recovered from nonactivated cells. Cultures of lymphocytes infected with the RK virus exhibited a cytopathic effect, and electron microscopic analyses revealed a characteristic herpesvirus structure. RK virus DNA did not hybridize with large probes derived from herpes simplex virus, Epstein-Barr virus, varicella-zoster virus, and human cytomegalovirus. The genetic relatedness of the RK virus to the recently identified T-lymphotropic human herpesvirus 6 (HHV-6) was investigated by restriction enzyme analyses using 21 different enzymes and by blot hybridization analyses using 11 probes derived from two strains of HHV-6 (Z29 and U1102). Whereas the two HHV-6 strains exhibited only limited restriction enzyme polymorphism, cleavage of the RK virus DNA yielded distinct patterns. Of the 11 HHV-6 DNA probes tested, only 6 cross-hybridized with DNA fragments derived from the RK virus. Taken together, the maximal homology amounted to 31 kilobases of the 75 kilobases tested. We conclude that the RK virus is distinct from previously characterized human herpesviruses. We propose to designate it as the prototype of a new herpesvirus, the seventh human herpesvirus identified to date.
Site and strand specificity of UVB mutagenesis in the SUP4-o gene of yeast.
Authors: Armstrong J D , Kunz B A
Published: June 2006

DNA sequencing was used to characterize 208 mutations induced in the SUP4-o tRNA gene of the yeast Saccharomyces cerevisiae by UVB (285-320 nm) radiation. The results were compared to those for an analysis of 211 SUP4-o mutations induced by 254-nm UVC light. In each case, greater than 90% of the mutations were single base-pair changes but G.C----A.T transitions predominated and accounted for more of the mutations induced by UVB than UVC. Double substitutions, single base-pair deletions, and more complex events were also recovered. However, UVB induced 3-fold more tandem substitutions than UVC and nontandem double events were detected only after irradiation with UVC. Virtually all induced substitutions occurred at sites where the pyrimidine of the base pair was part of a dipyrimidine sequence. Although the site specificities were consistent with roles for cyclobutane dimers and pyrimidine-pyrimidone(6-4) lesions in mutation induction, preliminary photoreactivation data implicated cyclobutane dimers as the major form of premutational DNA damage for both agents. Intriguingly, there was a preference for both UVB- and UVC-induced mutations to occur at sites where the dipyrimidine was on the transcribed strand.
An antifreeze glycopeptide gene from the antarctic cod Notothenia coriiceps neglecta encodes a polyprotein of high peptide copy number.
Authors: Hsiao K C , Cheng C H , Fernandes I E , Detrich H W , DeVries A L
Published: June 2006

The antarctic fish Notothenia coriiceps neglecta synthesizes eight antifreeze glycopeptides (AFGP 1-8; Mr 2600-34,000) to avoid freezing in its ice-laden freezing habitat. We report here the sequence of one of its AFGP genes. The structural gene contains 46 tandemly repeated segments, each encoding one AFGP peptide plus a 3-amino acid spacer. Most of the repeats (44/46) code for peptides of AFGP 8; the remaining 2 code for peptides of AFGP 7. At least 2 of the 3 amino acids in the spacers could act as substrate for chymotrypsin-like proteases. The nucleotide sequence between the translation initiation codon (ATG) and the first AFGP-coding segment is G + T-rich and encodes a presumptive 37-residue signal peptide of unusual sequence. Primer extension establishes the transcription start site at nucleotide 43 upstream from ATG. CAAT and TATA boxes begin at nucleotides 53 and 49, respectively, upstream from the transcription start site. The polyadenylylation signal, AATAAA, is located approximately 240 nucleotides downstream from the termination codon. A mRNA (approximately 3 kilobases) was found that matches the size of this AFGP gene. Thus, this AFGP gene encodes a secreted, high-copy-number polyprotein that is processed posttranslationally to produce active AFGPs.
RETRACTED: Sodium channels in the cytoplasm of Schwann cells.
Authors: Ritchie J M , Black J A , Waxman S G , Angelides K J
Published: June 2006

Immunoblotting, ultrastructural immunocytochemistry, and tritiated saxitoxin ([3H]STX) binding experiments were used to study sodium channel localization in Schwann cells. Polyclonal antibody 7493, which is directed against purified sodium channels from rat brain, specifically recognizes a 260-kDa protein corresponding to the alpha subunit of the sodium channel in immunoblots of crude glycoproteins from rat sciatic nerve. Electron microscopic localization of sodium channel immunoreactivity within adult rat sciatic nerves reveals heavy staining of the axon membrane at the node of Ranvier, in contrast to the internodal axon membrane, which does not stain. Schwann cells including perinodal processes also exhibit antibody 7493 immunoreactivity, localized within both the cytoplasm and the plasmalemma of the Schwann cell. To examine further the possibility that sodium channels are localized within Schwann cell cytoplasm, [3H]STX binding was studied in cultured rabbit Schwann cells, both intact and after homogenization. Saturable binding of STX was significantly higher in homogenized Schwann cells (410 +/- 37 fmol/mg of protein) than in intact Schwann cells (214 +/- 21 fmol/mg of protein). Moreover, the equilibrium dissociation constant was higher for homogenized preparations (1.77 +/- 0.37 nM) than for intact Schwann cells (1.06 +/- 0.29 nM). These data suggest the presence of an intracellular pool of sodium channels or channel precursors in Schwann cells.
Cation-dependent mannose 6-phosphate receptor contains two internalization signals in its cytoplasmic domain.
Authors: Johnson K F , Chan W , Kornfeld S
Published: June 2006

The signals required for rapid internalization of the bovine cation-dependent mannose 6-phosphate receptor have been localized to two distinct regions of the 67-amino acid cytoplasmic domain. One signal includes phenylalanine 13 and phenylalanine 18, while the other involves tyrosine 45. The former signal is more potent than the latter, but both must be present for the maximal rate of receptor internalization. Each signal shares similarities with the known internalization signals of other recycling receptors.
Dynamics of the distribution of cyclic AMP-dependent protein kinase in living cells.
Authors: Meinkoth J L , Ji Y , Taylor S S , Feramisco J R
Published: June 2006

The intracellular distribution of regulatory molecules may provide a mechanism for controlling gene expression. The subcellular location of cAMP-dependent protein kinase was analyzed in living cells by microinjection of regulatory and catalytic subunits labeled with fluorescein. Following microinjection, type I holoenzyme was found in the cytoplasm and remained there for up to 4 hr. Upon dissociation of holoenzyme with 8-bromo-cAMP, free catalytic subunit appeared in the nucleus while regulatory subunit remained in the cytoplasm. Similarly, purified catalytic subunit was transported to the nucleus in the absence of elevated intracellular cAMP following its introduction into the cytoplasm. Translocation to the nucleus was apparent within 10 min and persisted for at least 2 hr. In contrast, purified regulatory subunit, like holoenzyme, was maintained in the cytoplasm. These results suggest that one function of the type I regulatory subunit is to serve as a cytoplasmic anchor, sequestering the catalytic subunit in the cytoplasm until holoenzyme dissociates in response to increased cAMP.
Neuropathological changes in transgenic mice carrying copies of a transcriptionally activated Mos protooncogene.
Authors: Propst F , Rosenberg M P , Cork L C , Kovatch R M , Rauch S , Westphal H , Khillan J , Schulz N T , Neumann P E , Newmann P E
Published: June 2006

Independent transgenic mouse lines carrying the mouse Mos protooncogene linked to a retroviral transcriptional control sequence display behavioral abnormalities including circling, head tilting, and head bobbing. This dominant phenotype shows various degrees of penetrance in different transgenic founder animals and lines. Neuronal and axonal degeneration, gliosis, and inflammatory infiltrates are found in all transgenic mouse lines in which behavioral traits are present. Recordings of auditory-evoked potentials in mice of one of these lines demonstrate that transgenic mice are deaf; in these mice spiral ganglia degenerate and most of the cochlear hair cells are absent. By using an S1 nuclease protection assay, we have detected RNA expression of the transgene in all tissues examined and, in particular, at high levels in brain. In situ hybridization experiments show that Mos expression can be detected in specific areas of the central nervous system. Lesions are present in areas with demonstrable overexpression of Mos.
Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication.
Authors: Guatelli J C , Whitfield K M , Kwoh D Y , Barringer K J , Richman D D , Gingeras T R
Published: June 2006

A target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNA-dependent RNA polymerase. By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target. Product accumulation is exponential with respect to time, indicating that newly synthesized cDNAs and RNAs function as templates for a continuous series of transcription and reverse transcription reactions. Ten million-fold amplification occurs after a 1- to 2-hr incubation, with an initial rate of amplification of 10-fold every 2.5 min. This self-sustained sequence replication system is useful for the detection and nucleotide sequence analysis of rare RNAs and DNAs. The analogy to aspects of retroviral replication is discussed.
Genomic subtraction for cloning DNA corresponding to deletion mutations.
Authors: Straus D , Ausubel F M
Published: June 2006

We have developed a technique, called genomic subtraction, for isolating the DNA that is absent in deletion mutants. The method removes from wild-type DNA the sequences that are present in both the wild-type and the deletion mutant genomes. The DNA that corresponds to the deleted region remains. Enrichment for the deleted sequences is achieved by allowing a mixture of denatured wild-type and biotinylated mutant DNA to reassociate. After reassociation, the biotinylated sequences are removed by binding to avidin-coated beads. This subtraction process is then repeated several times. In each cycle we hybridize the unbound wild-type DNA from the previous round with fresh biotinylated deletion mutant DNA. The unbound DNA from the final cycle is ligated to adaptors and amplified by using one strand of the adaptor as a primer in the polymerase chain reaction. The amplified sequences can then be used to probe a genomic library. We applied genomic subtraction to a yeast strain that has a 5-kilobase deletion, corresponding to 1/4000th of the genome. In the experiment reported here, three rounds of subtraction were sufficient to accurately identify genomic clones containing sequences that are missing in the deletion mutant. We discuss the limitations and some potential applications of the method.
Structure and regulation of the gene for dGTP triphosphohydrolase from Escherichia coli.
Authors: Wurgler S M , Richardson C C
Published: June 2006

Escherichia coli encodes an enzyme, deoxyguanosine triphosphate triphosphohydrolase (dGTPase, EC 3.1.5.1), that cleaves dGTP into deoxyguanosine and tripolyphosphate. An E. coli mutant, optA1, contains a 50-fold increased level of dGTPase and cannot support the growth of phage T7 defective in [corrected] gene 1.2, whose product is an inhibitor of dGTPase. The optA1 mutation maps to 3.6 min on the E. coli chromosome and is closely linked to dapD. We have isolated the gene encoding dGTPase (dgt) from wild-type E. coli and determined its nucleotide sequence. The dgt gene lies immediately upstream of htrA and 6 kilobases from dapD, in the same region as the optA1 mutation. The dgt structural gene is 1515 base pairs, encoding a protein of 59,315 daltons, in agreement with the size and N-terminal amino acid sequence of the purified protein. An E. coli strain containing a null allele has no detectable phenotype when grown at 30-42 degrees C in rich medium. A transition of C to T in a potential promoter of dgt is required for expression of the optA1 phenotype.
crnA encodes a nitrate transporter in Aspergillus nidulans.
Authors: Unkles S E , Hawker K L , Grieve C , Campbell E I , Montague P , Kinghorn J R
Published: June 2006

The nucleotide sequence of the Aspergillus nidulans crnA gene for the transport of the anion nitrate has been determined. The crnA gene specifies a predicted polypeptide of 483 amino acids (molecular weight 51,769). A hydropathy plot suggests that this polypeptide has 10 membrane-spanning helices with an extensive hydrophilic region between helices six and seven. No striking homology was observed between the crnA protein and other reported membrane proteins of either prokaryotic or eukaryotic organisms, indicating that the crnA transporter may represent another class of membrane protein. Northern blotting results with wild-type cells show that (i) control of crnA expression is subject to nitrate (and nitrite) induction as well as nitrogen metabolite repression and (ii) regulation of the crnA gene is exerted at the level of mRNA accumulation, most likely at transcription, in response to the nitrogen source in the growth medium. Furthermore, similar studies with mutants of nirA and areA control genes and the niaD nitrate reductase structural gene show that crnA expression is mediated by the products of nirA (nitrate induction control gene), areA (nitrogen metabolite repression control gene), and niaD (involved in autoregulation of nitrate reductase).
Sorting-out of acceptor-donor relationships in the transglutaminase-catalyzed cross-linking of crystallins by the enzyme-directed labeling of potential sites.
Authors: Lorand L , Parameswaran K N , Velasco P T
Published: June 2006

The dansyl-conjugated (Dns) peptides Dns-Pro-Gly-Gly-Gln-Gln-Ile-Val and Dns-Ala-Gln-Gln-Ile-Val, patterned on the N-terminal sequence of fibronectin, were synthesized and used for the transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13)-directed selective blocking of lens proteins that otherwise might participate in donating lysyl side chains in forming N epsilon-(gamma-glutamyl)-lysine cross-linked oligomers and polymers. Labeling profiles with these peptides could be readily visualized by fluorescence as well as by immunoblotting with anti-dansyl antibody. The labeling patterns in rabbit lens homogenates were quite different with the dansylated peptides than those obtained with dansylcadaverine. Use of such glutamine-containing dansylated peptides should clearly aid in identifying, isolating, and sequencing potential donor substrates of transglutaminases in many biological systems.
Inactivation of the Zfx gene on the mouse X chromosome.
Authors: Adler D A , Bressler S L , Chapman V M , Page D C , Disteche C M
Published: June 2006

ZFX, an X chromosome-linked gene encoding a zinc-finger protein, has previously been shown to escape X inactivation in humans. Here we report studies of the inactivation status of the homolog, Zfx, on the mouse X chromosome. We took advantage of both the preferential inactivation of the normal X chromosome in females carrying the T(X;16)16H translocation and the high degree of nucleotide sequence variation between the laboratory strain of mouse [corrected] and Mus spretus genomes. An EcoRV restriction fragment difference between laboratory strain of mouse [corrected] and M. spretus was detected after amplification of Zfx transcripts using the reverse transcription-polymerase chain reaction. Using this allelic variation, we assessed expression of the two Zfx genes in females carrying the T(X;16)16H translocation (from laboratory strain of mouse [corrected]) and an intact X chromosome (from M. spretus). Such females exhibit Zfx transcription from the active laboratory strain of mouse [corrected] chromosome but not from the inactive M. spretus chromosome. These results indicate that the mouse Zfx gene is subject to X inactivation.
RETRACTED: Splicing-defective mutants of the yeast mitochondrial COXI gene can be corrected by transformation with a hybrid maturase gene.
Authors: Anziano P Q , Butow R A
Published: June 2006

We have developed a recombinant vector, termed pMIT, for transient expression of genes delivered to yeast mitochondria by biolistic transformation. Using that vector, we introduced a hybrid RNA maturase (splicing) gene into mitochondria of rho 0 petite cells and showed the gene to be functional in crosses. The hybrid maturase is an in-frame fusion between the N-terminal half of the maturase encoded by intron 1 of the COXI (cytochrome oxidase) gene and the C-terminal half of a similar maturase encoded by COXI intron 2. pMIT transformants can provide a functional maturase in crosses to restore respiration and COXI polypeptide synthesis to a respiratory-deficient strain defective in the synthesis of a maturase encoded by COXI intron 1; the transformant will also restore respiration to two splicing-defective cis mutants of COXI introns 1 and 3. We detect a 68-kDa polypeptide comparable in abundance to other major mitochondrial translation products as a likely product of the hybrid maturase gene. Transformants containing an internal 218-amino acid deletion mutation of the hybrid maturase gene no longer express a functional maturase in crosses and produce the expected shortened polypeptide of approximately 40 kDa; however, those transformants still restore respiration to the COXI cis mutants. These studies show the utility of the pMIT transformation system for the expression and reverse genetic analysis of yeast mitochondrial genes.
The glucocorticoid hormone signal transduction pathway in mice homozygous for chromosomal deletions causing failure of cell type-specific inducible gene expression.
Authors: DeFranco D , Bali D , Torres R , DePinho R A , Erickson R P , Gluecksohn-Waelsch S
Published: June 2006

Wild-type newborn mice are characterized by the ability of certain liver-specific genes encoding various enzymes and mapping on different chromosomes to respond to glucocorticoid induction. Newborn mice homozygous for deletions at and around the albino locus on chromosome 7 fail to develop this competence for hormone-inducible gene expression even through they do show normal constitutive expression of the same genes. Studies of the glucocorticoid hormone signal transduction pathway reported here show identical expression of glucocorticoid receptor mRNA and protein in deletion homozygotes and normal littermates. Furthermore, the receptor interacts normally with the 90-kDa heat shock protein hsp90. Elevated glucocorticoid hormone levels in newborn deletion homozygotes, most likely resulting from their stressed condition, provide an explanation for the reduced binding activities of receptors reported previously. The elimination of receptors and hormones as direct targets of the chromosomal deletion effects suggests that the failure of inducible gene expression might reside in defective competence of the affected structural genes to respond to the hormonal stimulus.
Identification and characterization of a ouabain-like compound from human plasma.
Authors: Hamlyn J M , Blaustein M P , Bova S , DuCharme D W , Harris D W , Mandel F , Mathews W R , Ludens J H
Published: June 2006

The plasma membrane sodium-potassium pumps that regulate intracellular sodium in most animal cells have specific, high-affinity receptors for the digitalis glycosides and their aglycones. This has fostered speculation that there is an endogenous ligand. We have purified and structurally identified by mass spectroscopy an endogenous substance from human plasma that binds with high affinity to this receptor and that is indistinguishable from the cardenolide ouabain. This human ouabain-like compound (OLC) displaces [3H]ouabain from its receptor, inhibits Na,K-ATPase and ouabain-sensitive 86Rb+ uptake, and has cardiotonic actions quantitatively similar to commercial ouabain. Immunoreactive OLC was detected in the plasma of many mammals, and high concentrations were found in the adrenals. The circulating OLC may modulate intracellular Na+ and affect numerous Na+ gradient-dependent processes including intracellular Ca2+ and pH homeostasis in many tissues. Furthermore, altered circulating levels of OLC may be associated with the pathogenesis of certain forms of hypertension.
Resistance of primary isolates of human immunodeficiency virus type 1 to soluble CD4 is independent of CD4-rgp120 binding affinity.
Authors: Ashkenazi A , Smith D H , Marsters S A , Riddle L , Gregory T J , Ho D D , Capon D J
Published: June 2006

The infection of human cells by laboratory strains of human immunodeficiency virus type 1 (HIV-1) can be blocked readily in vitro by recombinant soluble CD4 and CD4-immunoglobulin hybrid molecules. In contrast, infection by primary isolates of HIV-1 is much less sensitive to blocking in vitro by soluble CD4-based molecules. To investigate the molecular basis for this difference between HIV-1 strains, we isolated the gp120-encoding genes from several CD4-resistant and CD4-sensitive HIV-1 strains and characterized the CD4-binding properties of their recombinant gp120 (rgp120) products. Extensive amino acid sequence variation was found between the gp120 genes of CD4-resistant and CD4-sensitive HIV-1 isolates. However, the CD4-binding affinities of rgp120 from strains with markedly different CD4 sensitivities were essentially the same, and only small differences were observed in the kinetics of CD4 binding. These results suggest that the lower sensitivity of primary HIV-1 isolates to neutralization by CD4-based molecules is not due to lower binding affinity between soluble CD4 and free gp120.
Conformational switching at cytochrome a during steady-state turnover of cytochrome c oxidase.
Authors: Copeland R A
Published: June 2006

As an electron transfer-driven proton pump, cytochrome c oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.1) must alternate between two conformations in each valence state of the redox element associated with ion translocation. Using second derivative absorption spectroscopy, the conformation of the cytochrome a cofactor has been investigated during steady-state turnover of this enzyme. Resting cytochrome c oxidase displays a transition for ferric cytochrome a at 430 nm. During aerobic steady-state turnover, this band is replaced by a ferrous cytochrome a transition at 450 nm. When anaerobicity is achieved, the transition occurs at 444 nm. The 450-nm-absorbing species is thus the dominant form during turnover, suggesting that conformational transitions of cytochrome a direct electron transfer during catalysis and may direct as well proton translocation in the last step of the respiratory electron transfer chain.
RETRACTED: Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy.
Authors: Rockman H A , Ross R S , Harris A N , Knowlton K U , Steinhelper M E , Field L J , Ross J , Chien K R
Published: June 2006

To study the mechanisms that activate expression of the atrial natriuretic factor (ANF) gene during pressure-induced hypertrophy, we have developed and characterized an in vivo murine model of myocardial cell hypertrophy. We employed microsurgical techniques to produce a stable 35- to 45-mmHg pressure gradient across the thoracic aorta of the mouse that is associated with rapid and transient expression of an immediate-early gene program (c-fos/c-jun/junB/Egr-1/nur-77), an increase in heart weight/body weight ratio, and up-regulation of the endogenous ANF gene. These responses that are identical to those in cultured cell and other in vivo models of hypertrophy. To determine whether tissue-specific and inducible expression of the ANF gene can be segregated, we used a transgenic mouse line in which 500 base pairs of the human ANF promoter region directs atrial-specific expression of the simian virus 40 large tumor antigen (T antigen), with no detectable expression in the ventricles. Thoracic aortic banding of these mice led to a 20-fold increase in the endogenous ANF mRNA in the ventricle but no detectable expression of the T-antigen marker gene. This result provides evidence that atrial-specific and inducible expression of the ANF gene can be segregated, suggesting that a distinct set of regulatory cis sequences may mediate the up-regulation of the ANF gene during in vivo pressure overload hypertrophy. This murine model demonstrates the utility of microsurgical techniques to study in vivo cardiac physiology in transgenic mice and should allow the application of genetic approaches to identify the mechanisms that activate ventricular expression of the ANF gene during in vivo hypertrophy.
The polymeric immunoglobulin receptor (secretory component) mediates transport of immune complexes across epithelial cells: a local defense function for IgA.
Authors: Kaetzel C S , Robinson J K , Chintalacharuvu K R , Vaerman J P , Lamm M E
Published: June 2006

The polymeric immunoglobulin receptor (pIgR) on mucosal epithelial cells binds dimeric IgA (dIgA) on the basolateral surface and mediates transport of dIgA to the apical surface. Using Madin-Darby canine kidney epithelial cells stably transfected with pIgR cDNA, we found that soluble immune complexes (ICs) of 125I-labeled rat monoclonal antidinitrophenyl (DNP) dIgA (125I-dIgA) and DNP/biotin-bovine serum albumin were transported from the basolateral to the apical surface and then released. Monomeric IgA ICs were not transported, consistent with the specificity of pIgR for polymeric immunoglobulins. Essentially all the 125I-dIgA in apical culture supernatants was streptavidin precipitable, indicating that dIgA remained bound to antigen during transcytosis. While both dIgA and dIgA ICs bound pIgR with equal affinity (Kd approximately 8 nM), the number of high-affinity binding sites per cell was 2- to 3-fold greater for dIgA than for dIgA ICs. The extent of endocytosis of dIgA and dIgA ICs was correlated with the number of high-affinity binding sites. SDS/PAGE analysis of intracellular dIgA and dIgA ICs demonstrated that in both cases IgA remained undegraded during transport. The results suggest that the pathways of epithelial transcytosis of free dIgA and dIgA ICs are the same. Given the high population density of mucosal IgA plasma cells and the enormous surface area of pIgR-expressing mucosal epithelium, it is likely that significant local transcytosis of IgA ICs occurs in vivo. Such a process would allow direct elimination of IgA ICs at the mucosal sites where they are likely to form, thus providing an important defense function for IgA.
cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase.
Authors: Finocchiaro G , Taroni F , Rocchi M , Martin A L , Colombo I , Tarelli G T , DiDonato S
Published: June 2006

We have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase (CPTase; palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21), an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH2-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH2-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hamster somatic cell hybrids.
Isolation of a cellular protein that binds to the human immunodeficiency virus Tat protein and can potentiate transactivation of the viral promoter.
Authors: Desai K , Loewenstein P M , Green M
Published: June 2006

The human immunodeficiency virus type 1 Tat protein is a powerful transactivator of the viral long terminal repeat (LTR). We have identified a cellular protein that strongly binds to Tat and can complement Tat transactivation in rodent cells. The cellular protein of about 36 kDa was isolated from extracts of human cells by Tat peptide-affinity chromatography and can form a complex with Tat in vitro. Tat transactivation is inefficient in rodent cells microinjected or transfected with the reporter plasmid pHIV-LTRCAT plus the Tat-expressing plasmid pCV-1. Remarkably, coinjection of purified 36-kDa protein with pHIV-LTRCAT plus pCV-1 stimulated Tat transactivation 2.7- to 4.9-fold. Taken together, our findings suggest that the 36-kDa protein may be a transcription factor or modulator that is important for efficient Tat transactivation.
Brain and heart sodium channel subtype mRNA expression in rat cerebral cortex.
Authors: Yarowsky P J , Krueger B K , Olson C E , Clevinger E C , Koos R D
Published: June 2006

The expression of mRNAs coding for the alpha subunit of rat brain and rat heart sodium channels has been studied in adult and neonatal rat cerebral cortex using the reverse transcription-polymerase chain reaction (RT-PCR). Rat brain sodium channel subtype I, II, IIA, and III sequences were simultaneously amplified in the same PCR using a single oligonucleotide primer pair matched to all four subtype sequences. Identification of each subtype-specific product was inferred from the appearance of unique fragments when the product was digested with specific restriction enzymes. By using this RT-PCR method, products arising from mRNAs for all four brain sodium channel subtypes were identified in RNA extracted from adult rat cerebral cortex. The predominant component was type IIA with lesser levels of types I, II, and III. In contrast, the type II and IIA sequences were the predominant RT-PCR products in neonatal rat cortex, with slightly lower levels of type III and undetectable levels of type I. Thus, from neonate to adult, type II mRNA levels decrease relative to type IIA levels. Using a similar approach, we detected mRNA coding for the rat heart sodium channel in neonatal and adult rat cerebral cortex and in adult rat heart. These results reveal that mRNAs coding for the heart sodium channel and all four previously sequenced rat brain sodium channel subtypes are expressed in cerebral cortex and that type II and IIA channels may be differentially regulated during development.
Augmentation of synthesis of plasminogen activator inhibitor type 1 by insulin and insulin-like growth factor type I: implications for vascular disease in hyperinsulinemic states.
Authors: Schneider D J , Sobel B E
Published: June 2006

Accelerated atherosclerosis accompanying diabetes mellitus, obesity, and some types of hypertension has been associated with hyperinsulinemia, augmented plasma plasminogen activator inhibitor type 1 (PAI-1), or both. We hypothesized that insulin and insulin-like growth factor type I (IGF-I) can influence synthesis of PAI-1, thereby potentially attenuating fibrinolysis. In HepG2 cells used as a model system, concentrations of insulin and IGF-I consistent with those seen in plasma independently stimulated PAI-1 synthesis. Accumulation of PAI-1 protein in conditioned medium over 24 hr was stimulated more with insulin alone than with the combination. Synergistic increases were evident, however, in the accumulation of PAI-1 protein over 48 hr with a concomitant increase in PAI-1 mRNA. A 10- to 20-fold increase in IGF binding protein I mRNA was seen 16-48 hr after exposure of the HepG2 cells to insulin and IGF-I, an increase abolished by cycloheximide. The results obtained are consistent with the hypothesis that hyperinsulinemia coupled with physiologic concentrations of IGF-I may attenuate fibrinolytic activity in vivo, thereby contributing to accelerated atherosclerosis.
Aminoguanidine treatment inhibits the development of experimental diabetic retinopathy.
Authors: Hammes H P , Martin S , Federlin K , Geisen K , Brownlee M
Published: June 2006

Retinal capillary closure induced by hyperglycemia is the principal pathophysiologic abnormality underlying diabetic retinopathy, but the mechanisms by which this induction occurs are not clear. Treatment of diabetic rats for 26 weeks with aminoguanidine, an inhibitor of advanced glycosylation product formation, prevented a 2.6-fold accumulation of these products at branching sites of precapillary arterioles where abnormal periodic acid/Schiff reagent-positive deposits also occurred. Aminoguanidine treatment completely prevented abnormal endothelial cell proliferation and significantly diminished pericyte dropout. After 75 weeks, untreated diabetic animals developed an 18.6-fold increase in the number of acellular capillaries and formed capillary microaneurysms, characteristic pathologic features of background diabetic retinopathy. In contrast, aminoguanidine-treated diabetic animals had only a 3.6-fold increase in acellular capillaries and no microaneurysms. These findings indicate that advanced glycosylation product accumulation contributes to the development of diabetic retinopathy and suggest that aminoguanidine may have future therapeutic use in this disorder.
Cardiac fibroblasts are predisposed to convert into myocyte phenotype: specific effect of transforming growth factor beta.
Authors: Eghbali M , Tomek R , Woods C , Bhambi B
Published: June 2006

Cardiac fibroblasts are mainly responsible for the synthesis of major extracellular matrix proteins in the heart, including fibrillar collagen types I and III and fibronectin. In this report we show that these cells, when stimulated by transforming growth factor beta 1 (TGF-beta 1), acquire certain myocyte-specific properties. Cultured cardiac fibroblasts from adult rabbit heart were treated with TGF-beta 1 (10-15 ng/ml) for different periods of time. Northern hybridization analysis of total RNA showed that cells treated with TGF-beta 1 for 24 hr expressed mRNA corresponding to sarcomeric actin mRNA. Immunofluorescence staining and light microscopy showed that cultured cardiac fibroblasts treated with TGF-beta 1 became stained with a monoclonal antibody to muscle-specific actin. After treatment of quiescent cells with TGF-beta 1, cell proliferation (as measured by [3H]thymidine incorporation) was moderately increased (1.5-fold, P less than 0.001). NIH 3T3 cells and human skin fibroblasts, treated with TGF-beta 1, did not express sarcomeric actin mRNA. Treatment of cardiac fibroblasts with the mitogenic agent phorbol 12-myristate 13-acetate or with norepinephrine, angiotensin II, or interleukin 1 beta did not induce myocyte-specific actin mRNA. Cultured cardiac fibroblasts at the subconfluent stage, when exposed to TGF-beta 1 in the presence of 10% fetal bovine serum, gave rise to a second generation of slowly growing cells that expressed muscle-specific actin filaments. Our findings demonstrate that cardiac fibroblasts can be made to differentiate into cells that display many characteristics of cardiac myocytes. TGF-beta 1 seems to be a specific inducer of such conversion.
Expression of the bacterioopsin gene in Halobacterium halobium using a multicopy plasmid.
Authors: Krebs M P , Hauss T , Heyn M P , RajBhandary U L , Khorana H G
Published: June 2006

Bacteriorhodopsin (bR) was expressed in Halobacterium halobium by using a multicopy plasmid containing the bop gene. The plasmid contains pGRB1, a 1.8-kilobase-pair plasmid; a 70-base-pair fragment from ISH11, a recently characterized insertion sequence; and a 1.6-kilobase-pair fragment carrying the bop gene from H. halobium S9. When transformed with this plasmid, a bop- insertion mutant of H. halobium yielded purple (Pum+) colonies. The insertion at the chromosomal bop locus remained intact in transformed cells, indicating that the plasmid bop gene was responsible for the Pum+ phenotype. bR was induced in early stationary phase in both wild-type and transformed cells. The final level of bR in transformed cells was 25-40% of that in wild type. The lower level of expression was presumably due to plasmid instability. Purple membrane purified from transformed strains had absorption and visible CD properties similar to wild type and contained bR in a hexagonal lattice with the same unit-cell dimension as wild type. The structure of bR from wild-type and transformed strains was identical at a resolution of 7.2 A. When reconstituted into vesicles, the purple membrane from wild-type and transformed strains showed similar light-dependent proton-pumping activity.
Expression of v-src in a murine T-cell hybridoma results in constitutive T-cell receptor phosphorylation and interleukin 2 production.
Authors: O'Shea J J , Ashwell J D , Bailey T L , Cross S L , Samelson L E , Klausner R D
Published: June 2006

Ligand binding to the T-cell antigen receptor results in phosphatidylinositol hydrolysis and the resultant activation of protein kinase C, as well as the activation of a receptor-coupled protein-tyrosine kinase. As a model for tyrosine kinase activation in T cells, we used retroviral gene transfer to express the v-src oncogene in an antigen-specific murine T-cell hybridoma. Clones that expressed v-src mRNA demonstrated constitutive tyrosine phosphorylation of several cellular substrates, including the zeta chain of the T-cell receptor, and constitutive interleukin 2 production. Thus, expression of a constitutively active protein-tyrosine kinase such as pp60v-src appears to be sufficient to induce the expression of at least one gene critical to the process of T-cell activation.