bluuebunny/crossref_metadata_2025 huggingface dataset:
Endonucleolytic degradation of puf mRNA in Rhodobacter capsulatus is influenced by oxygen.
Authors: Klug G
Published: May 2006

The formation of pigment-protein complexes in facultatively photosynthetic bacteria is regulated by the oxygen tension in the culture. It is shown that the degradation of some mRNA species encoding components of the photosynthetic apparatus is affected by oxygen. The puf mRNA segment, encoding the pigment-binding proteins of the reaction center, and the 0.5-kb puc mRNA species, encoding pigment-binding proteins of the light-harvesting LHII antenna complex of Rhodobacter capsulatus were degraded more rapidly under high oxygen tension than under low oxygen tension. Studies on strains having deletions or insertions in the puf operon indicate that rate-limiting endonucleolytic cleavage in the reaction center coding region of the polycistronic puf mRNA was influenced by growth conditions. However, other mRNA segments, for which exonucleolytic degradation was postulated to be rate-limiting, decayed with the same rate under either high or low oxygen tension. Likewise, the degradation of the puhA mRNA, the cycA mRNA, and the cytfbc mRNA was found to be independent of the oxygen tension in the culture. The data strongly suggest that specific mRNA sequences or structures are responsible for the observed oxygen effect on mRNA stability.
Nerve growth factor corrects developmental impairments of basal forebrain cholinergic neurons in the trisomy 16 mouse.
Authors: Corsi P , Coyle J T
Published: May 2006

The trisomy 16 (Ts16) mouse, which shares genetic and phenotypic homologies with Down syndrome, exhibits impaired development of the basal forebrain cholinergic system. Basal forebrains obtained from Ts16 and euploid littermate fetuses at 15 days of gestation were dissociated and cultured in completely defined medium, with cholinergic neurons identified by choline acetyltransferase (ChAT) immunoreactivity. The Ts16 cultures exhibited fewer ChAT-immunoreactive neurons, which were smaller and emitted shorter, smoother, and more simplified neurites than those from euploid littermates. Whereas the addition of beta-nerve growth factor (100 ng/ml) augmented the specific activity of ChAT and neuritic extension for both Ts16 and euploid cholinergic neurons, only Ts16 cultures exhibited an increase in the number and size of ChAT-immunoreactive neurons. Furthermore, Ts16 ChAT-immunoreactive neurites formed varicosities only in the presence of beta-nerve growth factor.
Cloning and tissue-specific expression of five voltage-gated potassium channel cDNAs expressed in rat heart.
Authors: Roberds S L , Tamkun M M
Published: May 2006

Five distinct K+ channel cDNA molecules (RK1 to RK5) were cloned from either rat heart or rat aorta cDNA libraries. Four of the channels, RK1 to RK4, are similar or identical to Shaker-like K+ channels previously identified in rat brain cDNA libraries. Major differences among RK1 to RK4 exist in the amino- and carboxyl-terminal regions and in amino acids representing potential extracellular sequence between the S1 and S2 hydrophobic domains. RK5 encodes a unique channel of 490 amino acids having six hydrophobic domains but only five basic residues in the putative voltage-sensing domain. Unlike RK1 to RK4, RK5 is a rat homologue of the Drosophila Shal family of K+ channels, which have not been previously described in mammals. Although RK5 mRNA is present in cardiac atrium and ventricle, it is most abundant in brain. RK1, RK2, and RK3 transcripts are predominantly found in brain but are present also at lower levels in other tissues, such as heart and aorta. RK2 is absent from skeletal muscle whereas RK1 and RK3 are present in this tissue. RK4 mRNA is ubiquitous in electrically excitable tissue, being present at comparable levels in atrium, ventricle, aorta, brain, and skeletal muscle. The cloning of RK5 confirms the presence in mammals of all four Drosophila K+ channel families: Shaker, Shab, Shaw, and Shal.
Tunicamycin increases desensitization of acetylcholine receptors in cultured mouse muscle cells.
Authors: Giovannelli A , Grassi F , Eusebi F , Miledi R
Published: May 2006

Whole-cell currents activated by acetylcholine (AcCho) were recorded in C2 mouse myotubes before and after prolonged treatment with tunicamycin, an inhibitor of glycosylation. In control cells the AcCho-induced currents decayed slowly even in the continuous presence of AcCho. After 24 hr of treatment with tunicamycin AcCho still elicited currents, but their size was significantly reduced and their decay was greatly accelerated. The binding of 125I-labeled alpha-bungarotoxin, a specific and irreversible antagonist of muscle AcCho receptors, was greatly reduced after tunicamycin treatment, and an equivalent reduction was observed after a long-lasting application of the AcCho agonist carbachol. We suggest that, after inhibition of glycosylation by tunicamycin, AcCho receptors are expressed correctly on the plasma membrane but these receptors desensitize more rapidly and are less efficient in binding alpha-bungarotoxin.
Chromosome microdissection and cloning in human genome and genetic disease analysis.
Authors: Kao F T , Yu J W
Published: May 2006

A procedure has been described for microdissection and microcloning of human chromosomal DNA sequences in which universal amplification of the dissected fragments by Mbo I linker adaptor and polymerase chain reaction is used. A very large library comprising 700,000 recombinant plasmid microclones from 30 dissected chromosomes of human chromosome 21 was constructed. Colony hybridization showed that 42% of the clones contained repetitive sequences and 58% contained single or low-copy sequences. The insert sizes generated by complete Mbo I cleavage ranged from 50 to 1100 base pairs with a mean of 416 base pairs. Southern blot analysis of microclones from the library confirmed their human origin and chromosome 21 specificity. Some of these clones have also been regionally mapped to specific sites of chromosome 21 by using a regional mapping panel of cell hybrids. This chromosome microtechnology can generate large numbers of microclones with unique sequences from defined chromosomal regions and can be used for processes such as (i) isolating corresponding yeast artificial chromosome clones with large inserts, (ii) screening various cDNA libraries for isolating expressed sequences, and (iii) constructing region-specific libraries of the entire human genome. The studies described here demonstrate the power of this technology for high-resolution genome analysis and explicate their use in an efficient search for disease-associated genes localized to specific chromosomal regions.
Affinity-purified CCAAT-box-binding protein (YEBP) functionally regulates expression of a human class II major histocompatibility complex gene and the herpes simplex virus thymidine kinase gene.
Authors: Zeleznik-Le N J , Azizkhan J C , Ting J P
Published: May 2006

Efficient major histocompatibility complex class II gene expression requires conserved protein-binding promoter elements, including X and Y elements. We affinity purified an HLA-DRA Y-element (CCAAT)-binding protein (YEBP) and used it to reconstitute Y-depleted HLA-DRA in vitro transcription. This directly demonstrates a positive functional role for YEBP in HLA-DRA transcription. The ability of YEBP to regulate divergent CCAAT elements was also assessed; YEBP was found to partially activate the thymidine kinase promoter. This functional analysis of YEBP shows that this protein plays an important role in the regulation of multiple genes.
Human cyclophilin B: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence.
Authors: Price E R , Zydowsky L D , Jin M J , Baker C H , McKeon F D , Walsh C T
Published: May 2006

We report the cloning and characterization of a cDNA encoding a second human cyclosporin A-binding protein (hCyPB). Homology analyses reveal that hCyPB is a member of the cyclophilin B (CyPB) family, which includes yeast CyPB, Drosophila nina A, and rat cyclophilin-like protein. This family is distinguished from the cyclophilin A (CyPA) family by the presence of endoplasmic reticulum (ER)-directed signal sequences. hCyPB has a hydrophobic leader sequence not found in hCyPA, and its first 25 amino acids are removed upon expression in Escherichia coli. Moreover, we show that hCyPB is a peptidyl-prolyl cis-trans isomerase which can be inhibited by cyclosporin A. These observations suggest that other members of the CyPB family will have similar enzymatic properties. Sequence comparisons of the CyPB proteins show a central, 165-amino acid peptidyl-prolyl isomerase and cyclosporin A-binding domain, flanked by variable N-terminal and C-terminal domains. These two variable regions may impart compartmental specificity and regulation to this family of cyclophilin proteins containing the conserved core domain. Northern blot analyses show that hCyPB mRNA is expressed in the Jurkat T-cell line, consistent with its possible target role in cyclosporin A-mediated immunosuppression.
Long-chain (sphingoid) bases inhibit multistage carcinogenesis in mouse C3H/10T1/2 cells treated with radiation and phorbol 12-myristate 13-acetate.
Authors: Borek C , Ong A , Stevens V L , Wang E , Merrill A H
Published: May 2006

Sphingosine and other long-chain (sphingoid) bases inhibit protein kinase C, the putative cellular receptor for the tumor promoter phorbol 12-myristate 13-acetate (PMA), and exert potent effects on diverse cell functions. We tested the ability of long-chain bases to modulate multistage carcinogenesis in mouse C3H/10T1/2 cells exposed to gamma-rays and PMA. Sphingosine and sphinganine completely blocked the enhancement of radiation-induced transformation by PMA (promotion) and partially suppressed transformation by radiation alone. N-Acetylsphingosine, a ceramide analog, did not inhibit transformation. Sphingosine was rapidly taken up by the cells and metabolized; hence, the long-chain bases were added daily to achieve prolonged inhibition. Long-chain bases inhibited protein kinase C activity in C3H/10T1/2 cells and suppressed the down-regulation of this enzyme by PMA. Our results establish that long-chain bases are highly effective inhibitors of carcinogenesis in this model. Our results also indicate that the suppressive effects may be mediated, in part, by inhibition of protein kinase C. The data suggest that sphingosine and other long-chain bases derived from complex sphingolipids may act as cancer-preventative agents.
The human immunodeficiency virus type 1 polyadenylylation signal: a 3' long terminal repeat element upstream of the AAUAAA necessary for efficient polyadenylylation.
Authors: Valsamakis A , Zeichner S , Carswell S , Alwine J C
Published: May 2006

Several polyadenylylation (PA) signals containing elements upstream of the AAUAAA have recently been characterized. Similar to PA elements found downstream of the AAUAAA, the upstream elements function to increase efficiency of AAUAAA use as a signal for cleavage and PA. Using deletion and linker scanning mutations we show that the PA signal of human immunodeficiency virus type 1 contains upstream elements transcribed from the U3 region of the 3' long terminal repeat. The element that has the greatest effect on PA site use lies 77 to 94 nucleotides upstream of the AAUAAA, between the TATA element and the transcriptional initiation site. Mutations in the adjacent region, between 59 and 76 nucleotides upstream of the AAUAAA, have a smaller effect on PA efficiency. Mutations in a region further upstream, between 141 and 176 nucleotides upstream of the AAUAAA, also affected PA modestly. Functional similarity between upstream elements was indicated by the ability of the human immunodeficiency virus upstream region to replace the upstream region of the simian virus 40 late PA signal. The sequence of the major upstream element of human immunodeficiency virus is uracil-rich, analogous to many defined downstream PA elements. This fact may imply that upstream and downstream elements have similar mechanisms of action.
Stable integration and expression in mouse cells of yeast artificial chromosomes harboring human genes.
Authors: Eliceiri B , Labella T , Hagino Y , Srivastava A , Schlessinger D , Pilia G , Palmieri G , D'Urso M
Published: May 2006

We have developed a way to fit yeast artificial chromosomes (YACs) with markers that permit the selection of stably transformed mammalian cells, and have determined the fate and expression of such YACs containing the genes for human ribosomal RNA (rDNA) or glucose-6-phosphate dehydrogenase (G6PD). The YACs in the yeast cell are "retrofitted" with selectable markers by homologous recombination with the URA3 gene of one vector arm. The DNA fragment introduced contains a LYS2 marker selective in yeast and a thymidine kinase (TK) marker selective in TK-deficient cells, bracketed by portions of the URA3 sequence that disrupt the endogenous gene during the recombination event. Analyses of transformed L-M TK- mouse cells showed that YACs containing rDNA or G6PD were incorporated in essentially intact form into the mammalian cell DNA. For G6PD, a single copy of the transfected YAC was found in each of two transformants analyzed and was fully expressed, producing the expected human isozyme as well as the heterodimer composed of the human gene product and the endogenous mouse gene product.
The transcriptional regulator LevR of Bacillus subtilis has domains homologous to both sigma 54- and phosphotransferase system-dependent regulators.
Authors: Débarbouillé M , Martin-Verstraete I , Klier A , Rapoport G
Published: May 2006

The regulatory gene levR of the levanase operon of Bacillus subtilis was cloned and sequenced. It encodes a polypeptide of Mr 106,064 with two domains homologous to members of two families of bacterial activators. One domain in LevR is homologous with one region of bacterial regulators including SacT and SacY of B. subtilis and BglG from Escherichia coli. Another domain of LevR is homologous to one part of the central domain of NifA and NtrC, which control nitrogen assimilation in Gram-negative bacteria. The levanase promoter contains two regions almost identical to the -12, -24 consensus regions present in sigma 54-dependent promoters. The expression of the levanase operon in E. coli was strongly dependent on sigma 54. Taken together, these results suggest that the operon is expressed from a -12, -24 promoter regulated by a sigma 54-like-dependent system in B. subtilis.
Dual site model for the organogenic activity of angiogenin.
Authors: Hallahan T W , Shapiro R , Vallee B L
Published: May 2006

The residues that are indispensable for the ribonucleolytic activity of angiogenin are also known to be essential for its angiogenic activity. We now demonstrate that residues in another region of the protein, devoid of catalytic residues, are additionally required for angiogenesis. Endoproteinase Lys-C or a baby hamster kidney cell protease cleaves angiogenin at the peptide bond either between Lys-60 and Asn-61 or between Glu-67 and Asn-68, respectively. The two polypeptide fragments resulting from either cleavage remain linked by disulfide bonds. These two derivatives and des-(Asn61-Glu67)-angiogenin--in which both bonds are cleaved--retain their ribonucleolytic activities toward tRNA, 18S and 28S rRNA, and dinucleoside phosphates but are no longer angiogenic on the chicken embryo chorioallantoic membrane. Further, their capacity to elicit a second messenger response in endothelial cells is greatly decreased. Moreover, none of these three derivatives inhibit angiogenin-induced angiogenesis. This contrasts with two active site mutants of angiogenin. These results identify the residues from 60 to 68 as a region of angiogenin that is part of a cell-surface receptor binding site [see accompanying manuscript: Hu, G.-F., Chang, S.-I., Riordan, J.F. & Vallee, B.L. (1991) Proc. Natl. Acad. Sci. USA 88, 2227-2231] and serve as the basis for a dual site model of the organogenic activity of angiogenin.
Transport of lipoprotein lipase across endothelial cells.
Authors: Saxena U , Klein M G , Goldberg I J
Published: May 2006

Lipoprotein lipase (LPL), synthesized in muscle and fat, hydrolyzes plasma triglycerides primarily while bound to luminal endothelial cell surfaces. To obtain information about the movement of LPL from the basal to the luminal endothelial cell surface, we studied the transport of purified bovine milk LPL across bovine aortic endothelial cell monolayers. 125I-labeled LPL (125I-LPL) added to the basal surface of the monolayers was detected on the apical side of the cells in two compartments: (i) in the medium of the upper chamber, and (ii) bound to the apical cell surface. The amount of 125I-LPL on the cell surface, but not in the medium, reached saturation with time and LPL dose. Catalytically active LPL was transported to the apical surface but very little LPL activity appeared in the medium. Heparinase treatment of the basal cell surface and addition of dextran sulfate (0.15 microM) to the lower chamber decreased the amount of 125I-LPL appearing on the apical surface. Similarly, the presence of increasing molar ratios of oleic acid/bovine serum albumin at the basal surface decreased the transport of active LPL across the monolayer. Thus, a saturable transport system, which requires heparan sulfate proteoglycans and is inhibited by high concentrations of free fatty acids on the basal side of the cells, appears to exist for passage of enzymatically active LPL across endothelial cells. We postulate that regulation of LPL transport to the endothelial luminal surface modulates the physiologically active pool of LPL in vivo.
Normal and leukemic hematopoietic cells manifest differential sensitivity to inhibitory effects of c-myb antisense oligodeoxynucleotides: an in vitro study relevant to bone marrow purging.
Authors: Calabretta B , Sims R B , Valtieri M , Caracciolo D , Szczylik C , Venturelli D , Ratajczak M , Beran M , Gewirtz A M
Published: May 2006

The c-myb protooncogene is preferentially expressed in hematopoietic cells, and its encoded protein, Myb, is required for hematopoietic cell proliferation. To analyze the relative Myb dependence of normal and leukemic human hematopoietic progenitor cells, normal bone marrow cells, several types of leukemic blast cells, and 1:1 mixtures of normal and leukemic cells were cultured in the presence of c-myb sense or antisense oligodeoxynucleotides; cell viability and cloning efficiency were then assessed. c-myb sense oligomers had negligible effects on normal and leukemic cells. In contrast, c-myb antisense oligomers strongly inhibited or completely abolished clonogenic growth of a T-cell leukemia line, 78% (18 of 23) of primary acute myelogenous leukemia cases examined, and 4 of 5 primary chronic myelogenous leukemia (CML) cases in blast crisis. In three of the latter patients, polymerase chain reaction analysis of a 1:1 mixture of c-myb antisense-treated normal and CML cells revealed a complete absence of bcr-abl expression, suggesting that the CML clonogenic units had been completely eliminated from the cultures. At antisense doses that inhibited leukemic cell growth, normal hematopoietic progenitor cells survived. Thus, normal and leukemic hematopoietic cells show differential sensitivity to the toxic effects of c-myb antisense DNA. Perturbation of c-myb function with antisense oligodeoxynucleotides might eventually form the basis for a molecular approach to leukemia therapy, perhaps most immediately as ex vivo bone marrow purging agents.
DNA polymerization in the absence of exonucleolytic proofreading: in vivo and in vitro studies.
Authors: Reha-Krantz L J , Stocki S , Nonay R L , Dimayuga E , Goodrich L D , Konigsberg W H , Spicer E K
Published: May 2006

Classical genetic selection was combined with site-directed mutagenesis to study bacteriophage T4 DNA polymerase 3'----5' exonuclease activity. A mutant DNA polymerase with very little (less than or equal to 1%) 3'----5' exonuclease activity was generated. In vivo, the 3'----5' exonuclease-deficient DNA polymerase produced the highest level of spontaneous mutation observed in T4, 500- to 1800-fold above that of wild type. The large reduction in 3'----5' exonuclease activity appears to be due to two amino acid substitutions: Glu-191 to Ala and Asp-324 to Gly. Protein sequence similarities have been observed between sequences in the Escherichia coli DNA polymerase I 3'----5' exonuclease domain and conserved sequences in eukaryotic, viral, and phage DNA polymerases. It has been proposed that the conserved sequences contain metal ion binding ligands that are required for 3'----5' exonuclease activity; however, we find that some proposed T4 DNA polymerase metal binding residues are not essential for 3'----5' exonuclease activity. Thus, our T4 DNA polymerase studies do not support the hypothesis by Bernad et al. [Bernad, A., Blanco, L., Lazaro, J.M., Martin, G. & Salas, M. (1989) Cell 59, 219-228] that many DNA polymerases, including T4 DNA polymerase, share an extensively conserved 3'----5' exonuclease motif. Therefore, extrapolation from E. coli DNA polymerase I sequence and structure to other DNA polymerases for which there is no structural information may not be valid.
Quox-1, a quail homeobox gene expressed in the embryonic central nervous system, including the forebrain.
Authors: Xue Z G , Gehring W J , Le Douarin N M
Published: May 2006

This paper reports the cloning and sequencing of a quail homeobox-containing gene, Quox-1, and its expression pattern in embryos from 3 to 6 days (E3 to E6) of development as determined by in situ hybridization. The opening reading frame in cDNA clone g11 corresponds to a predicted protein of 242 amino acids. Quox-1 protein displays high sequence similarity to the Antennapedia family, especially to the mouse homeodomain-containing protein Hox-1.1 (100% identity in the homeobox region, 77% at the 5' end beyond the homeobox). However, the carboxyl-terminal domain of the postulated protein has no significant homology with other known homeoproteins, including Hox-1.1. In situ hybridization experiments showed that Quox-1 is widely expressed in the developing central nervous system including the entire brain and the spinal cord. Outside the central nervous system, transcription of Quox-1 was mainly detected in the endoderm-derived epithelium of esophagus, trachea, and other digestive organs, as well as in the sensory epithelium of the olfactory region and perichondrium of the vertebrae. Thus, Quox-1 transcripts have a remarkably wide distribution that, unlike the other vertebrate homeobox genes examined to date, encompasses the rostral part of the developing nervous system, including the forebrain.
A constitutively active holoenzyme form of the cAMP-dependent protein kinase.
Authors: Wang Y H , Scott J D , McKnight G S , Krebs E G
Published: May 2006

The major function of the regulatory (R) subunit of the cAMP-dependent protein kinase is to bind tightly to the catalytic (C) subunit to form an inactive holoenzyme in the absence of cAMP. The hinge region of the R subunit resembles the substrate recognition site for the C subunit and is known to be involved in the R.C subunit interaction. Two arginine residues in this region, Arg-92 and Arg-93, are suggested to be essential for holoenzyme formation. In this study, a mutant in which Arg-92 and Arg-93 of type II regulatory subunit (RII) were replaced with alanine was constructed. Formation of the holoenzyme from mutant RII and C subunits was analyzed by gel-filtration and cation-exchange chromatography. Mutant RII in its cAMP-free form formed a stable holoenzyme with the C subunit, which dissociated in the presence of cAMP. Interestingly, the holoenzyme formed from mutant RII and C subunits retained full enzymatic activity even in the absence of cAMP. Although mutant RII could no longer be phosphorylated by the C subunit, the rate of [3H]cAMP release from mutant RII.cAMP was increased by addition of the C subunit, indicating that C-induced cAMP release is not the result of the interaction of the C subunit with the hinge region. These results demonstrate that Arg-92 and Arg-93 are not essential for holoenzyme formation but are critical for inhibiting kinase activity in the holoenzyme probably by occupying the substrate binding site. The results suggest that, in addition to the hinge region, a second site on the RII subunit may interact with the C subunit in a cAMP-dependent manner.
Distinct muscarinic receptors inhibit release of gamma-aminobutyric acid and excitatory amino acids in mammalian brain.
Authors: Sugita S , Uchimura N , Jiang Z G , North R A
Published: May 2006

Intracellular recordings were made from neurons of rat lateral amygdala, nucleus accumbens, and striatum in vitro. Synaptic potentials mediated by gamma-aminobutyric acid and by excitatory amino acids were isolated pharmacologically by using receptor antagonists, and their amplitudes were used as a measure of transmitter release. Muscarine and acetylcholine inhibited the release of both gamma-aminobutyric acid and excitatory amino acids, but measurements of the dissociation equilibrium constants for the antagonists pirenzepine, 11-(2-[(diethylamino)methyl]-1-piperidinyl)acetyl-5,11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepine-6-one, methoctramine, and hexahydrosiladifenidol indicated clearly that different muscarinic receptors were involved (M1 and probably M3, respectively). The differential localization of distinct muscarinic receptor subtypes on terminals releasing the major inhibitory and excitatory transmitters of the brain could be exploited therapeutically in some movement disorders and Alzheimer disease.
Deletion analysis of a phytochrome-regulated monocot rbcS promoter in a transient assay system.
Authors: Rolfe S A , Tobin E M
Published: May 2006

We have developed a transient gene expression assay system in the aquatic monocot Lemna gibba in which DNA was introduced into intact tissue by particle bombardment. Constructs based on the Lemna rbcS gene SSU5B, which is positively regulated by phytochrome in vivo, also showed phytochrome regulation in the transient assay system. Reporter gene expression increased 12-fold over dark levels in response to a single treatment with red light. This increase was not observed if far-red light was immediately followed by the red light. A 5' deletion analysis of the promoter defined a region from position -205 to position -83 relative to the start of transcription as necessary to observe the phytochrome response. This region contains the binding site for the light-induced binding activity (LRF-1) found in Lemna nuclear extracts. Upstream of position -205, we found evidence for the presence of at least two upstream activating sequences and a silencer.
Functional interdependence of the yeast SNF2, SNF5, and SNF6 proteins in transcriptional activation.
Authors: Laurent B C , Treitel M A , Carlson M
Published: May 2006

The SNF2, SNF5, and SNF6 genes of Saccharomyces cerevisiae are required for expression of a variety of differently regulated genes. Previous evidence implicated the SNF5 protein in transcriptional activation, and a DNA-bound LexA-SNF5 fusion protein was shown to activate expression of a nearby promoter. Here, we examine the functional relationship of the SNF2, SNF5, and SNF6 proteins. Activation by DNA-bound LexA-SNF5 fusion protein was greatly reduced in snf2 and snf6 mutants, indicating that activation by LexA-SNF5 requires SNF2 and SNF6 function. An spt6 mutation, which suppresses transcriptional defects caused by snf2, restored activation by LexA-SNF5 in a snf2 mutant. The SNF2 gene was sequenced and encodes a 194-kDa protein that is targeted to the nucleus. DNA-bound LexA-SNF2 fusion protein also activated transcription, dependent on SNF5 and SNF6. These findings suggest that SNF2, SNF5, and SNF6 function interdependently in transcriptional activation, possibly forming a heteromeric complex.
Simian varicella virus DNA in dorsal root ganglia.
Authors: Mahalingam R , Smith D , Wellish M , Wolf W , Dueland A N , Cohrs R , Soike K , Gilden D
Published: May 2006

Clinical, pathological, immunological, and virological evidence suggests that simian varicella virus (SVV) infection of primates is the counterpart of varicella-zoster virus infection of humans. To determine whether these two viruses share similarities in their properties during latency, we analyzed ganglia and brain of an African green monkey experimentally infected with SVV for the presence of viral nucleic acid using the polymerase chain reaction technique. We detected SVV DNA in dorsal root ganglia but not in brain of this monkey, which demonstrated no apparent clinical signs of SVV infection. Our results suggest that SVV becomes latent in monkey ganglia and that latency can develop in the absence of clinical varicella (chickenpox). These studies provide an animal model system to study varicella virus latency.
Interleukin 1 alpha inhibits prostaglandin E2 release to suppress pulsatile release of luteinizing hormone but not follicle-stimulating hormone.
Authors: Rettori V , Gimeno M F , Karara A , Gonzalez M C , McCann S M
Published: May 2006

Interleukin 1 alpha (IL-1 alpha), a powerful endogenous pyrogen released from monocytes and macrophages by bacterial endotoxin, stimulates corticotropin, prolactin, and somatotropin release and inhibits thyrotropin release by hypothalamic action. We injected recombinant human IL-1 alpha into the third cerebral ventricle, to study its effect on the pulsatile release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in conscious, freely moving, ovariectomized rats. Intraventricular injection of 0.25 pmol of IL-1 alpha caused an almost immediate reduction of plasma LH concentration; this decrease was statistically significant 20 min after injection and occurred through a highly significant reduction in the number of LH pulses, with no effect on pulse amplitude. In contrast, there was no change in pulse frequency but a small significant elevation in amplitude of FSH pulses. Intraventricular injection of the diluent had no effect on gonadotropin release. The results provide further evidence for separate hypothalamic control mechanisms for FSH and LH release. To determine the mechanism of the suppression of LH release, mediobasal hypothalamic fragments were incubated in vitro with IL-1 alpha (10 pM) and the release of LH-releasing hormone (LHRH) and prostaglandin E2 into the medium was measured by RIA in the presence or absence of norepinephrine (50 microM). IL-1 alpha reduced basal LHRH release and blocked LHRH release induced by norepinephrine. It had no effect on the basal release of prostaglandin E2; however, it completely inhibited the release of PGE2 evoked by norepinephrine. To evaluate the possibility that IL-1 alpha might also interfere with the epoxygenase pathway of arachidonic acid metabolism, epoxyeicosatrienoic acids were also measured. IL-1 alpha had no effect on the content of epoxyeicosatrienoic acids in the hypothalamic fragments as measured by gas chromatography and mass spectrometry. In conclusion, IL-1 alpha suppresses LH but not FSH release by an almost complete cessation of pulsatile release of LH in the castrated rat. The mechanism of this effect appears to be by inhibition of prostaglandin E2-mediated release of LHRH.
Clonal analysis of hematopoietic stem-cell differentiation in vivo.
Authors: Smith L G , Weissman I L , Heimfeld S
Published: May 2006

Previous work has shown that the 0.02-0.05% of adult mouse bone marrow cells that bear the cell surface phenotype Thy-1loLin-Sca-1+ are enriched 1000- to 2000-fold for hematopoietic stem-cell activity in a variety of assays. When 50-100 cells of this phenotype are injected into an irradiated animal, they can permanently repopulate the entire hematopoietic system. In the present study, limiting-dilution and single-cell experiments were used to address the question of how individual Thy-1loLin-Sca-1+ stem cells contribute to repopulation of the hematopoietic system following irradiation. We calculated that 1 of 13 Thy-1loLin-Sca-1+ cells formed a clone comprising greater than 1% of peripheral white blood cells 3-7 weeks after injection. The majority of these clones included both lymphoid and myeloid lineages. Approximately one-third of the clones continued to produce new blood cells for 9 weeks or more, but the remainder disappeared earlier, including many that were multilineage. Thus, while the majority of Thy-1loLin-Sca-1+ bone marrow cells whose progeny are detected in the in vivo repopulation assay are pluripotential, only a subset undergo long-term self-renewal in vivo. Repopulation appears to be oligoclonal when limiting numbers of Thy-1loLin-Sca-1+ cells are injected. However, the number of clones contributing to hematopoiesis increases in proportion to the number of Thy-1loLin-Sca-1+ cells injected, bringing into question the notion that steady-state hematopoiesis in normal individuals is oligoclonal.
A single gene encodes membrane-bound and free forms of GP-2, the major glycoprotein in pancreatic secretory (zymogen) granule membranes.
Authors: Fukuoka S , Freedman S D , Scheele G A
Published: May 2006

GP-2, a 75-kDa glycoprotein, was isolated from dog pancreatic zymogen granule membranes (ZGMs). In a carbohydrate-shift strategy, N-terminal and internal peptide sequences were obtained on glycosylated and deglycosylated forms of GP-2, respectively, by gas-phase sequencing. Sets of mixed oligonucleotides and the polymerase chain reaction were used to obtain a double-stranded cDNA probe, which was used to isolate overlapping cDNA clones from a dog pancreatic cDNA library. The sequence of these clones revealed an open reading frame that encodes a protein of 509 amino acids, eight N-linked oligosaccharide attachment sites, and an N-terminal signal sequence absent from the mature form of GP-2 associated with ZGMs. The C terminus shows a 20-residue hydrophobic transmembrane domain preceded by a decapeptide containing potential phosphatidylinositol-glycan attachment sites. GP-2 completely released from ZGMs by exogenous phospholipase C showed similar immunochemical properties and electrophoretic mobilities compared to the form associated with ZGMs. A similar form of GP-2 was released from zymogen granules permeabilized with saponin and incubated in the absence of added phospholipase C. Kinetic analysis of GP-2 release at 0 degrees C and 37 degrees C suggested the presence of a granule enzyme responsible for endogenous release of GP-2 to granule contents and into the apical medium. The data indicate that GP-2 is a phosphatidylinositol-glycan-linked membrane protein released from the membrane of mature zymogen granules by an enzymatic mechanism. The cDNA structure presented here thus encodes both membrane-bound and free forms of GP-2.
Structural similarities in glutaminyl- and methionyl-tRNA synthetases suggest a common overall orientation of tRNA binding.
Authors: Perona J J , Rould M A , Steitz T A , Risler J L , Zelwer C , Brunie S
Published: May 2006

Detailed comparisons between the structures of the tRNA-bound Escherichia coli glutaminyl-tRNA (Gln-tRNA) synthetase [L-glutamine:tRNA(Gln) ligase (AMP-forming), EC 6.1.1.18] and recently refined E. coli methionyl-tRNA (Met-tRNA) synthetase [L-methionine:tRNA(Met) ligase (AMP-forming), EC 6.1.1.10] reveal significant similarities beyond the anticipated correspondence of their respective dinucleotide-fold domains. One similarity comprises a 23-amino acid alpha-helix-turn-beta-strand motif found in each enzyme within a domain that is inserted between the two halves of the dinucleotide binding fold. A second correspondence, which consists of two alpha-helices connected by a large loop and beta-strand, is located in the Gln-tRNA synthetase within a region that binds the inside corner of the "L"-shaped tRNA molecule. This structural motif contains a long alpha-helix, which extends along the entire length of the D and anticodon stems of the complexed tRNA. We suggest that the positioning of this helix relative to the dinucleotide fold plays a critical role in ensuring the proper global orientation of tRNA(Gln) on the surface of the enzyme. The structural correspondences suggest a similar overall orientation of binding of tRNA(Met) and tRNA(Gln) to their respective synthetases.
Lesions of the hypothalamus and pituitary inhibit volume-expansion-induced release of atrial natriuretic peptide.
Authors: Antunes-Rodrigues J , Ramalho M J , Reis L C , Menani J V , Turrin M Q , Gutkowska J , McCann S M
Published: May 2006

Expansion of the blood volume causes a release of atrial natriuretic peptide (ANP) that is believed to be important in induction of the subsequent natriuresis and diuresis which, in turn, acts to reduce the increase in blood volume. Since stimulation of the anteroventral portion of the third cerebral ventricle (AV3V) induced a rapid elevation of plasma ANP, whereas lesions of the AV3V were followed by a marked decline in plasma concentration of the peptide, we hypothesized that release of ANP from the brain ANP neuronal system might be important to the control of plasma ANP. The perikarya of the ANP-containing neurons are densely distributed in the AV3V and their axons project to the median eminence and neural lobe. To test the hypothesis that these neurons are involved in volume-expansion-induced ANP release, by using electrolysis we destroyed the AV3V, the site of the perikarya, in male rats. Other lesions were made in the median eminence and posterior pituitary, sites of termination of the axons of these neurons, and also hypophysectomy was performed in other animals. In conscious freely moving animals, volume expansion and stimulation of postulated sodium receptors in the hypothalamus were induced by injection of hypertonic NaCl solution [0.5 or 0.3 M NaCl; 2 ml/100 g (body weight)]. Volume expansion alone was induced with the same volume of an isotonic solution (NaCl or glucose). In the sham-operated rats, volume expansion with hypertonic or isotonic solutions caused equivalent rapid increases in plasma ANP that peaked at 5 min and returned nearly to control values by 15 min. Lesions caused a decrease in the initial levels of plasma ANP on comparison with values from the sham-operated rats, and each type of lesion induced a highly significant suppression of the response to volume expansion on testing 1-5 days after lesions were made. Because a common denominator of the lesions was elimination of the brain ANP neuronal system, these results suggest that the brain ANP plays an important role in the mediation of the release of ANP that occurs after volume expansion. Since the content of ANP in this system is much less than that in the atria, there must be a remarkable increase in synthesis and release of brain ANP associated with this stimulus. It is also possible that blockade of volume-expansion-induced release of other neurohypophyseal hormones, such as endothelin, may block release of ANP from atrial myocytes. It is probable that volume expansion detected by stretch of atrial and carotid-aortic baroreceptors causes afferent input to the brain ANP system, thereby causing increased release of the peptide from the median eminence and neural lobe. Our results emphasize the importance of brain ANP to the control of ANP release to the blood.
Differential effect of cysteine-to-serine substitutions in metallothionein on cadmium resistance.
Authors: Chernaik M L , Huang P C
Published: May 2006

A set of mutant coding sequences for Chinese hamster metallothionein (MT) 2 in which codons for individual cysteines were replaced by serine codons was cloned into a yeast expression system. MT gene expression was placed under control of a constitutive promoter on a multicopy Escherichia coli-yeast shuttle vector. MTs were expressed in a metal-sensitive host that lacks the endogenous MT gene. The expressed MTs conferred increased metal resistance to the yeast host. A sensitive assay for cadmium resistance was developed in which population doubling times were monitored in rich liquid medium supplemented with a sublethal dose of CdCl2. Measurements on mutants with single cysteine replacements at 12 positions revealed two mutant classes. One class (Cys----Ser at position 5, 13, 19, or 33) did not affect the detoxification capacity of MT. A second class (Cys----Ser at position 7, 15, 26, 29, 44, 48, 50, or 60) conferred to the host markedly less resistance to cadmium. Bridging cysteines were more critical to cadmium resistance. All five bridging cysteine mutants studied (at positions 7, 15, 44, 50, and 60) conferred lower cadmium resistance. In contrast, mutation of four out of seven terminal cysteines (at position 5, 13, 19, or 33) was shown to be inconsequential. Mutations tend to be more detrimental in the alpha domain than in the beta domain in conveying cadmium resistance, suggesting that the contribution of individual cysteine to the detoxification function of MT is site specific.
Selective abstraction of 2H from C-5' of thymidylate in an oligodeoxynucleotide by the radical center at C-6 of the diradical species of neocarzinostatin: chemical evidence for the structure of the activated drug-DNA complex.
Authors: Meschwitz S M , Goldberg I H
Published: May 2006

Use has been made of the mechanism of DNA deoxyribose damage by the ene-diyne-containing chromophore of the antitumor antibiotic neocarzinostatin to provide chemical evidence for the structure of the activated drug-DNA complex. Radical centers at C-2 and C-6 of the diradical form of the glutathione-activated chromophore abstract hydrogen atoms from C-1' of the C residue and C-5' of the T residue in AGC.GCT to generate a bistranded lesion consisting of an abasic site at C and a strand break at T. This laboratory has proposed a molecular model for the drug-DNA interaction in which the naphthoate moiety of the chromophore intercalates between A.T and G.C, placing the diradical core in the minor groove, so that the radical centers at C-6 and C-2 are close to C-5' of T and C-1' of C, respectively. To determine which radical center abstracts one of the hydrogen atoms from C-5', the self-complementary oligodeoxynucleotide GCAGCGCTGC was synthesized with 2H at both 5' positions of the T residue and treated with glutathione-activated chromophore. Sequencing-gel electrophoresis showed that drug attack was limited to the T and C residues and that abstraction of 2H from C-5' exhibited a small isotope selection effect of 1.25. 1H NMR spectroscopic examination of the reacted chromophore, isolated by HPLC, indicated that 2H was selectively abstracted by C-6, providing experimental corroboration of the model and further elucidating the chemical mechanism. Since direct strand breakage at the T residue exceeds (44% more) abasic site formation at the C residue, other models of drug-DNA interaction leading to only single-strand breaks are also considered.
Induction of anti-phospholipid syndrome in naive mice with mouse lupus monoclonal and human polyclonal anti-cardiolipin antibodies.
Authors: Blank M , Cohen J , Toder V , Shoenfeld Y
Published: May 2006

The primary anti-phospholipid syndrome is characterized by recurrent venous and arterial thromboembolic phenomena, recurrent fetal loss, thrombocytopenia, and serological evidence of anti-cardiolipin (aCL) antibodies or/and the presence of lupus anticoagulant (prolonged activated partial thromboplastin time). The exact role of aCL antibodies in pathogenesis is not clear and the mechanism by which the antibodies may induce the various manifestations is unknown. In the current study we evaluated the effect of passive transfer of aCL antibodies (to the tail vein of naive mice) on fecundity, fetal loss (fetal resorption), and the weight of embryos and placentae. Two types of aCL antibodies were employed: (i) mouse monoclonal aCL antibodies derived from a BALB/c mouse in which experimental systemic lupus erythematosus was induced by a pathogenic idiotype (idiotype 16/6) of anti-DNA antibodies and (ii) polyclonal IgG and IgM aCL antibodies derived from serum of a patient with primary anti-phospholipid syndrome. After infusion of either antibody (10 micrograms per mouse) we could demonstrate lower fecundity rate, increased resorption index of embryos (equivalent to recurrent fetal loss), lower number of embryos per pregnancy, and lower mean weights of embryos and placentae in comparison to mice infused with appropriate control immunoglobulins. We conclude that the aCL antibodies may have direct effects on fecundity and on the outcome of pregnancy.
Sequence of a cysteine-rich galactose-specific lectin of Entamoeba histolytica.
Authors: Mann B J , Torian B E , Vedvick T S , Petri W A
Published: May 2006

Entamoeba histolytica trophozoites adhere to human colonic mucins and epithelial cells by a cell surface galactose-specific lectin. This lectin, which is composed of two subunits linked by disulfide bonds, has been shown to be a protective antigen in an animal model of amebiasis. We have determined the sequence of the mature form of the 170-kDa heavy subunit from cDNA clones and PCR-amplified fragments. The heavy subunit sequence consisted of a putative extracellular domain containing 1209 amino acids with 16 potential sites for N-linked glycosylation, a 26-amino acid hydrophobic region, and a 41-amino acid cytoplasmic tail. The presence of N-linked oligosaccharides was confirmed by culturing amebae with tunicamycin, which resulted in a decrease in the heavy subunit molecular mass to 160 kDa and a loss of lectin activity. The extracellular domain was remarkable for an extensive cysteine-rich domain that shared identify with similar regions of several other cell surface proteins and appeared to confer protease resistance to the subunit.
Use of heteropolymeric monoclonal antibodies to attach antigens to the C3b receptor of human erythrocytes: a potential therapeutic treatment.
Authors: Taylor R P , Sutherland W M , Reist C J , Webb D J , Wright E L , Labuguen R H
Published: May 2006

We have prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine gamma globulin) to human RBCs under conditions that either allow or preclude complement activation. Quantitative analyses of this binding agree well with the number of complement receptors per RBC. In vitro "whole-blood" model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37 degrees C. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.
Coordinated regulation of apoptosis and cell proliferation by transforming growth factor beta 1 in cultured uterine epithelial cells.
Authors: Rotello R J , Lieberman R C , Purchio A F , Gerschenson L E
Published: May 2006

Cell and tissue growth is regulated through a complex interplay of stimulatory and inhibitory signals. We describe two biological actions of transforming growth factor beta 1 (TGF-beta 1) in primary cultures of rabbit uterine epithelial cells: (i) inhibition of cell proliferation and (ii) a concomitant increase in cells undergoing apoptosis (programmed cell death). It is proposed that proliferation and apoptosis together comprise normal cell growth regulation.
Early visual deprivation results in a degraded motor map in the optic tectum of barn owls.
Authors: du Lac S , Knudsen E I
Published: May 2006

The optic tectum contains a precise map of orienting movements: the size and direction of movements of the eyes, head, and/or body vary systematically with the locus of neural activation within the tectum. In adult animals, this motor map aligns closely with the tectal map of visual space. This study addressed the question of whether the motor map develops entirely independently of visual experience. We found that in barn owls (Tyto alba) raised without vision, although a tectal map of head movement develops, its topography and alignment with the map of visual (and auditory) space are abnormal. The results demonstrate that during early life vision is necessary either to maintain or to guide the development of a normal tectal motor map.