bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletion of the steroid-binding domain of the human androgen receptor gene in one family with complete androgen insensitivity syndrome: evidence for further genetic heterogeneity in this syndrome.
Authors: Brown T R , Lubahn D B , Wilson E M , Joseph D R , French F S , Migeon C J
Published: May 2006

The cloning of a cDNA for the human androgen receptor gene has resulted in the availability of cDNA probes that span various parts of the gene, including the entire steroid-binding domain and part of the DNA-binding domain, as well as part of the 5' region of the gene. The radiolabeled probes were used to screen for androgen receptor mutations on Southern blots prepared by restriction endonuclease digestion of genomic DNA from human subjects with complete androgen insensitivity syndrome (AIS). In this investigation, we considered only patients presenting complete AIS and with the androgen receptor (-) form as the most probable subjects to show a gene deletion. One subject from each of six unrelated families with the receptor (-) form of complete AIS and 10 normal subjects (6 females and 4 males) were studied. In the 10 normal subjects and in 5 of the 6 patients, identical DNA restriction fragment patterns were observed with EcoRI and BamHI. In one affected individual, a partial deletion of the androgen receptor gene involving the steroid-binding domain was detected. Analysis of other members of this family confirmed the apparent gene deletion. Our data provide direct proof that complete AIS in some families can result from a deletion of the androgen receptor structural gene. However, other families do not demonstrate such a deletion, suggesting that point mutations (or small, undetectable deletions) may also result in the receptor (-) form of complete AIS, adding further to the genetic heterogeneity of this syndrome.
T helper cell subsets require the expression of distinct costimulatory signals by antigen-presenting cells.
Authors: Weaver C T , Hawrylowicz C M , Unanue E R
Published: May 2006

We examined the ability of macrophages and B cells to function as antigen-presenting cells (APCs) for murine TH1 and TH2 cloned T helper cell lines. Antigen presented by concanavalin A-elicited peritoneal macrophages or resting splenic B cells stimulated antigen-dependent proliferation of both T helper subsets. Paraformaldehyde fixation of the APCs following different conditions of activation indicated differential requirements for costimulatory signals by TH1 and TH2 cells. TH2 proliferative responses were strictly dependent on APC expression of IL-1. TH1 proliferation was dependent on APC expression of a non-IL-1 costimulatory signal present on freshly isolated macrophages and on splenic B cells activated with anti-immunoglobulin plus interferon gamma.
Doxorubicin conjugates of monoclonal antibodies to hepatoma-associated antigens.
Authors: Shouval D , Adler R , Wands J R , Hurwitz E , Isselbacher K J , Sela M
Published: May 2006

A panel of six murine monoclonal antibodies against hepatocellular carcinoma-associated antigens, reactive with PLC/PRF/5 human hepatoma cells, was conjugated to Adriamycin (doxorubicin) via a dextran bridge. This library of antibodies includes three monoclonal antibodies against hepatitis B virus surface antigen, one anti-alpha-fetoprotein, and two other IgG2a antibodies against PLC/PRF/5 hepatoma-associated antigens. The use of dextran for conjugation of Adriamycin to antibodies enabled a 5- to 10-fold amplification of the number of drug molecules linked to antibody. Conjugation of Adriamycin to dextran caused an occasional reduction in the pharmacologic activity of dextran-Adriamycin in [3H]thymidine incorporation assays in hepatoma cells as compared to nonconjugated Adriamycin. This loss of anticellular activity was partially compensated for by conjugation of specific antibodies to the dextran-Adriamycin conjugate. Conjugated compounds completely retained their binding activity to purified hepatitis B virus surface antigen and alpha-fetoprotein fixed to a solid matrix as compared to binding of homologous nonconjugated antibodies. However, some reduction of the binding activity to intact hepatoma cells was observed in three of six conjugates. Binding activity to hepatoma cells and, as a consequence, suppression of tumor cell DNA synthesis by the various conjugates was enhanced as compared to the same effect in treated colorectal carcinoma cells that do not express the relevant hepatoma-associated proteins. Furthermore, two conjugates containing nonspecific antibodies did not bind to hepatoma cells and caused minimal suppression of DNA synthesis. These results suggest that this panel of monoclonal antibody-dextran-Adriamycin conjugates was effective in suppression of PLC/PRF/5 cell growth in vitro.
Hatchling turtles survive freezing during winter hibernation.
Authors: Storey K B , Storey J M , Brooks S P , Churchill T A , Brooks R J
Published: May 2006

Hatchlings of the painted turtle (Chrysemys picta marginata) are unique as the only reptile and highest vertebrate life form known to tolerate the natural freezing of extracellular body fluids during winter hibernation. Turtles survived frequent exposures to temperatures as low as -6 degrees C to -8 degrees C in their shallow terrestrial nests over the 1987-1988 winter. Hatchlings collected in April 1988 had a mean supercooling point of -3.28 +/- 0.24 degrees C and survived 24 hr of freezing at -4 degrees C with 53.4% +/- 1.98% of total body water as ice. Recovery appeared complete after 20 hr of thawing at 3 degrees C. However, freezing at -10.9 degrees C, resulting in 67% ice, was lethal. A survey of possible cryoprotectants revealed a 2- to 3-fold increase in glucose content of liver and blood and a 3-fold increase in blood glycerol in response to freezing. Although quantitatively low, these responses by spring turtles strongly indicate that these may be the winter-active cryoprotectants. The total amino acid pool of blood also increased 2.25-fold in freezing-exposed turtles, and taurine accounted for 52% of the increase. Most organs accumulated high concentrations of lactate during freezing, a response to the ischemic state imposed by extracellular freezing. Changes in glycogen phosphorylase activity and levels of glucose 6-phosphate and fructose 2,6-bisphosphate were also consistent with a dependence on anaerobic glycolysis during freezing. Studies of the molecular mechanisms of natural freeze tolerance in these turtles may identify protective strategies that can be used in mammalian organ cryopreservation technology.
A signal peptide encoded within the precore region of hepatitis B virus directs the secretion of a heterogeneous population of e antigens in Xenopus oocytes.
Authors: Standring D N , Ou J H , Masiarz F R , Rutter W J
Published: May 2006

Using synthetic hepatitis B virus (HBV) mRNAs, we have shown that expression of HBV core-antigen gene sequences in Xenopus oocytes leads to the stable accumulation of 21-kDa cytoplasmic core protein (P21). In contrast, expression of precore plus core sequences leads mainly to the secretion of a heterogeneous population of proteins ranging in size from 15 to 22 kDa that collectively display viral e antigen (HBeAg) activity. We demonstrate that the precore region contains a cleavable 19 amino acid signal peptide that targets the precore proteins to the secretory pathway. The initial product of translocation (P22) is further processed during migration through the secretory pathway, apparently by a series of cleavage events at the arginine-rich carboxyl terminus, to yield multiple proteins of 15-18 kDa (P15-P18) that are secreted along with some P22. Our results indicate that serum HBeAg is generated by a signal peptide-mediated secretion event dependent on precore sequences.
Amine cations promote concurrent conversion of prohistidine decarboxylase from Lactobacillus 30a to active enzyme and a modified proenzyme.
Authors: van Poelje P D , Snell E E
Published: May 2006

Activation of prohistidine decarboxylase (pi 6) from Lactobacillus 30a proceeds by an intramolecular, pH- and monovalent cation-dependent reaction in which its constituent pi chains are cleaved nonhydrolytically between Ser-81 and Ser-82 with loss of NH3 and conversion of Ser-82 to the pyruvoyl residue of active histidine decarboxylase (alpha beta)6. Amines with pKa values more than 7.0 substitute for K+ or NH4+ in the activation of prohistidine decarboxylase, but they also catalyze its inactivation in a competing reaction, pi 6----pi'6. Sequence analysis of the appropriate tryptic peptide from amine-inactivated prohistidine decarboxylase established that inactivation results from conversion of Ser-82 of the pi chain to an aminoacrylate residue. The inactivated proenzyme (pi'6) does not form histidine decarboxylase; this fact eliminates one of two postulated mechanisms of activation and, thus, favors activation by beta-elimination of the acyl group of an intermediate ester formed between Ser-81 and Ser-82. L-Histidine is bound by the proenzyme (Kd = 1.7 x 10(-4) M) and is an effective activator; one binding site is present per pi subunit. K+, NH4+, and Na+ competitively inhibit (Ki values = 2.8-4.4 x 10(-3) M) activation by histidine. The data suggest the presence of two classes of monovalent cation binding sites on prohistidine decarboxylase: one (near Ser-82) is readily saturable and one is unsaturable even by 2.4 M K+.
Properties of two 5'-coterminal RNAs transcribed part way and across the S component origin of DNA synthesis of the herpes simplex virus 1 genome.
Authors: Voss J H , Roizman B
Published: May 2006

In cells infected with herpes simplex virus 1 the domains of the two identical origins of viral DNA synthesis in the S component (Oris) are transcribed in two 5'-coterminal RNAs. OriSRNA1, synthesized early in infection and also in the absence of protein synthesis by infected cells, runs antisense to and terminates at the transcription initiation site for alpha genes 22 or 47. OriSRNA2, reported earlier, was detected in cells 14 hr after infection; it extends across the origin and is coterminal with the alpha 4 mRNA. Whereas the OriSRNA2 of herpes simplex virus 1 (HSV-1) contains an open reading frame, the corresponding herpes simplex virus 2 (HSV-2) transcript does not. However, in the two viruses, the transcription initiation sites are 260 (HSV-1) to 180 (HSV-2) nucleotides distant from the corresponding origins of DNA synthesis.
Induction of protooncogene c-jun by serum growth factors.
Authors: Ryder K , Nathans D
Published: May 2006

We have previously reported that one of the genes that is rapidly induced in mouse 3T3 cells by serum growth factors (jun-B) encodes a protein related to the onco-protein v-jun. By using jun-B as a probe, we have isolated a cDNA encoding a second member of the jun family (jun-A) that is the murine version of the protooncogene c-jun, which encodes the mammalian transcription factor AP-1. jun-B and jun-A (c-jun) have two highly conserved regions and two regions with little sequence similarity. Like jun-B, jun-A (c-jun) is rapidly activated by serum, platelet-derived growth factor, or fibroblast growth factor and is superinduced by serum in the presence of an inhibitor of protein synthesis. Both jun proteins are likely to play a role in regulating the genetic program induced by growth factors.
Orientation of carbon monoxide and structure-function relationship in carbonmonoxymyoglobin.
Authors: Ormos P , Braunstein D , Frauenfelder H , Hong M K , Lin S L , Sauke T B , Young R D
Published: May 2006

Fourier transform infrared spectroscopy of the CO stretch bands in carbonmonoxymyoglobin (MbCO) reveals three major bands implying that MbCO exists in three major substates, A0, A1, and A3. After photolysis at low temperatures the CO is in the heme pocket, and the resulting CO stretch bands represent the B substates. Photoselection experiments determine the orientation of CO in the A (bound) and B (photolyzed) substates: Small fractions of MbCO are photolyzed at 10 K with linearly polarized light at 540 nm. The resulting linear dichroism in the A and B IR bands yields the tilt angle between the heme normal and CO. The average angles are as follows: alpha (A0) = 15 degrees +/- 3 degrees; alpha (A1) = 28 degrees +/- 2 degrees, and alpha (A3) = 33 degrees +/- 4 degrees. The A bands are inhomogeneously broadened; the angle alpha shows a wavenumber dependence within the A bands. The wavenumber dependence is interpreted as a distribution of the tilt angle within the individually inhomogeneous A substates, thus providing a structural parameter to characterize the distribution of the conformational substates. The B substates exhibit no induced linear dichroism; in the photolyzed substates the ligand is randomly oriented with respect to the heme plane. The present results together with earlier data on static and kinetic properties of CO binding to Mb establish relations among spectroscopic, structural, energetic, and functional parameters.
Evolution of B-cell malignancy: pre-B-cell leukemia resulting from MYC activation in a B-cell neoplasm with a rearranged BCL2 gene.
Authors: Gauwerky C E , Haluska F G , Tsujimoto Y , Nowell P C , Croce C M
Published: May 2006

We have analyzed the molecular genetics of the breakpoints involved in the t(8;14) and t(14;18) translocations of an acute pre-B-cell leukemia from a patient with a history of follicular lymphoma. In this patient's leukemic cells, the breakpoint of the t(14;18) translocation occurred in the major breakpoint-cluster region of the BCL2 gene and became linked to the JH4 joining-region gene segment of the immunoglobulin heavy-chain locus on the 14q+ chromosome as previously observed in follicular lymphoma. An N region and heptamer and nonamer signal sequences indicated that this translocation occurred as a mistake in VH-DH-JH joining (where VH and DH are the variable and diversity segments). In the t(8;14) translocation, the breakpoint was located immediately 5' of the first exon of the MYC protooncogene, which was juxtaposed with the C gamma 2 constant gene segment of the second 14q+ chromosome. The finding of repeated sequences typical of switch regions suggested that this translocation occurred during heavy-chain isotype switching, resulting in progression to pre-B-cell leukemia with both the t(8;14) and the t(14;18) translocations. The terminal deoxynucleotidyltransferase-positive phenotype of the patient's leukemic cells further suggests that the pre-B-cell leukemia was derived from a pre-B cell carrying a t(14;18) translocation in the original follicular lymphoma. The polymerase chain reaction method was then used to identify cancer cells in the bone marrow of the patient.
Mutant gene phenotypes mediated by a Drosophila melanogaster retrotransposon require sequences homologous to mammalian enhancers.
Authors: Geyer P K , Green M M , Corces V G
Published: May 2006

We have analyzed the molecular structure of phenotypic revertants of gypsy-induced mutations to understand the molecular mechanisms by which this retrotransposon causes mutant phenotypes in Drosophila melanogaster. The independent partial revertants analyzed are caused by the insertion of different transposons into the same region of gypsy. One partial revertant of the yellow allele y2 arose as a consequence of the insertion of the jockey mobile element into gypsy sequences, whereas a second incomplete revertant is due to the insertion of the hobo transposon. In addition, a previously isolated partial revertant of the Hairy-wing allele Hw1 resulted from the integration of the BS transposable element into the same gypsy sequences. The region affected by the insertion of the three transposons contains 12 copies of a repeated motif that shows striking homology to mammalian transcriptional enhancers. Our results suggest that these sequences, which might be involved in the transcriptional control of the gypsy element, are also responsible for the induction of mutant phenotypes by this retrotransposon.
Cyclic AMP-dependent phosphorylation of a brain inositol trisphosphate receptor decreases its release of calcium.
Authors: Supattapone S , Danoff S K , Theibert A , Joseph S K , Steiner J , Snyder S H
Published: May 2006

We report the stoichiometric phosphorylation of an inositol 1,4,5-trisphosphate receptor-binding protein from rat brain by the cAMP-dependent protein kinase but not by protein kinase C or Ca2+/calmodulin-dependent protein kinase. This phosphorylation event does not markedly alter [3H]inositol 1,4,5-trisphosphate-binding characteristics. However, inositol 1,4,5-trisphosphate is only 10% as potent in releasing 45Ca2+ from phosphorylated, as compared with native, cerebellar microsomes. Phosphorylation of the inositol 1,4,5-trisphosphate-binding protein by the cAMP-dependent protein kinase may provide a biochemical substrate for second-messenger cross talk.
Beta gamma subunits of GTP-binding proteins inhibit muscarinic receptor stimulation of phospholipase C.
Authors: Moriarty T M , Gillo B , Carty D J , Premont R T , Landau E M , Iyengar R
Published: May 2006

This study examines the mechanism of guanine nucleotide-binding protein (G protein) coupling of receptors to phospholipase C. The Xenopus oocyte has a muscarinic receptor-activated Cl- current that is mediated by inositol 1,4,5-trisphosphate. Modulation of the muscarinic receptor-evoked Cl- current was examined under voltage clamp in oocytes injected with resolved G-protein subunits. The presence of an alpha subunit of G proteins in oocytes was shown by pertussis toxin-labeling of a 41-kDa band in oocyte membranes. The presence of the beta subunit of G proteins was demonstrated by immunoblotting experiments with an antiserum (U-49) that is specific for the beta subunit. Pertussis toxin treatment of oocytes resulted in the uncoupling of muscarinic receptors from activation of the Cl- current. Cells microinjected with 1.5 ng of human erythrocyte beta gamma-subunit complex or 1.0 ng of bovine brain beta gamma-subunit complex showed approximately a 95% reduction in the evoked Cl- current. Cells injected with equal volumes of protein storage vehicle showed no change in response. Cells injected with boiled beta gamma subunits, bovine serum albumin, or resolved alpha subunits also showed no reduction in response. Cells injected with various concentrations of beta gamma subunits showed a concentration dependence with half-maximal inhibition of the muscarinic activated Cl- current at about 10 nM. Cells injected with 1.0 ng of bovine brain beta gamma subunits could not respond to bath-applied agonist but could generate the Cl- current on intracellular injection of inositol 1,4,5-trisphosphate. These observations suggest that there is a G protein responsible for muscarinic receptor-mediated signal transduction through phospholipase C and that it is an alpha beta gamma heterotrimer. It appears that the mode of action of the G protein in the phospholipase C system may be similar to that of the hormone-activated adenylyl cyclase.
A nuclease that cuts specifically in the ribosome binding site of some T4 mRNAs.
Authors: Uzan M , Favre R , Brody E
Published: May 2006

We have identified a nucleolytic activity in Escherichia coli infected by bacteriophage T4 that introduces cuts in the ribosome binding site of at least two T4 mRNAs. Cutting takes place specifically in the GGAG sequences that are complementary to the 3' end of 16S rRNA (Shine-Dalgarno sequence). The nature of this nucleolytic cut has been investigated by reverse transcriptase mapping, anti-mRNA mapping, utilization of the vaccinia virus guanylyltransferase, and labeling by polynucleotide kinase. We have compared the sequences of target mRNAs with an mRNA of similar sequence but that is not a substrate for the nuclease. This allowed us to narrow down the possibilities for sequence elements that determine nuclease recognition. We hypothesize that this nuclease plays a physiological role in the inhibition of expression of a class of phage proteins.
Escherichia coli mutY gene product is required for specific A-G----C.G mismatch correction.
Authors: Au K G , Cabrera M , Miller J H , Modrich P
Published: May 2006

A-G mispairs are subject to correction by two distinct pathways in cell-free extracts of Escherichia coli [Su, S.-S., Lahue, R.S., Au, K.G. & Modrich, P. (1988) J. Biol. Chem. 263, 6829-6835; Lu, A.-L. & Chang, D.Y. (1988) Genetics 118, 593-600]. One is the mutHLS-dependent, methyl-directed pathway that recognizes a variety of mismatches and repairs the unmethylated strand of DNA heteroduplexes that are hemimethylated at d(GATC) sequences. The other pathway appears to be specific for A-G mispairs, yields C.G base pairs exclusively, and is independent of the presence of d(GATC) sites. Analyses of cell-free extracts prepared from E. coli mutY strains and isogenic parents have demonstrated that the mutY gene product is involved in the methyl-independent pathway, which converts A-G mispairs to C.G pairs. The specificity of this activity is consistent with the mutator phenotype associated with the mutY locus, which generates G.C----T.A transversions [Nghiem, Y., Cabrera, M., Cupples, C.G. & Miller, J.H. (1988) Proc. Natl. Acad. Sci. USA 85, 2709-2713]. We propose that the mutY product functions at a late stage of a pathway that excludes A-G mispairs during chromosome replication and that involves the function of the mutT gene product. This model suggests that the mutT function acts at an early stage of this pathway to exclude A-G mismatches where the adenine resides on the template DNA strand. A-G mispairs that persist after passage of the replication fork would contain guanine on the template strand and thus be processed to C.G base pairs by the mutY-dependent repair system.
Coexpression of granulocyte-macrophage colony-stimulating factor, gamma interferon, and interleukins 3 and 4 is random in murine alloreactive T-lymphocyte clones.
Authors: Kelso A , Gough N M
Published: May 2006

Lymphokine gene expression was examined in a panel of 116 short-term murine T-lymphocyte clones derived by single-cell micromanipulation from allogeneic mixed leukocyte cultures. About 30% of clonable T cells, including both CD4+ CD8- and CD4- CD8+ cells, could be expanded for assay at an average of 22 days after cloning. By RNA blot-hybridization analysis, most clones (85-96%) expressed detectable granulocyte-macrophage colony-stimulating factor, interleukin 3, and gamma interferon mRNAs, and 11% expressed interleukin 4 mRNA. Although no differences were noted between CD4+ and CD8+ clones in the combinations of lymphokines produced, CD4+ clones on average transcribed and secreted higher levels. When the frequencies of coexpression of any pair of lymphokine mRNAs were determined, all were found to correspond to the values predicted for random assortment of the individual frequencies. For example, among 13 interleukin 4-positive clones, 11 also transcribed gamma interferon, giving the frequency of double-positive clones expected for random association (9.6% versus 10.8%). Therefore, expression of the four lymphokine genes segregated independently among the clones and did not allow the division of T cells into subsets with distinct patterns of lymphokine synthesis.
Aminosugar derivatives as potential anti-human immunodeficiency virus agents.
Authors: Karpas A , Fleet G W , Dwek R A , Petursson S , Namgoong S K , Ramsden N G , Jacob G S , Rademacher T W
Published: May 2006

Recent data suggest that aminosugar derivatives which inhibit glycoprotein processing have potential anti-human immunodeficiency virus (HIV) activity. These inhibitory effects may be due to disruption of cell fusion and subsequent cell-cell transmission of the acquired immunodeficiency syndrome (AIDS) virus. Free virus particles able to bind CD4-positive cells are still produced in the presence of these compounds with only partial reduction of infectivity. We now report a method to score in parallel both the degree of antiviral activity and the effect on cell division of aminosugar derivatives. We find that (i) the compounds 1,4-dideoxy-1,4-imino-L-arabinitol and N-(5-carboxymethyl-1-pentyl)-1,5-imino-L-fucitol partially inhibit the cytopathic effect (giant cell formation, etc.) of HIV and yield of infectious virus; (ii) the compounds N-methyldeoxynojirimycin and N-ethyldeoxynojirimycin reduce the yield of infectious HIV by an order of four and three logarithms, respectively; and (iii) one compound, N-butyldeoxynojirimycin, of the 47 compounds previously screened reduces infectious viral particles by a logarithmic order greater than five at noncytotoxic concentrations. In addition, long-term growth of infected cells in the presence of N-butyldeoxynojirimycin gradually decreases the proportion of infected cells, leading to eventual elimination of HIV from culture. This result suggests that replication is associated with cytolysis. The ability to break the cycle of replication and reinfection has important implications in the chemotherapy of AIDS.
Purification and characterization of a bovine acetyl low density lipoprotein receptor.
Authors: Kodama T , Reddy P , Kishimoto C , Krieger M
Published: May 2006

The acetyl low density lipoprotein (LDL) receptor is expressed on macrophages and some endothelial cells and mediates macrophage-foam cell formation in culture. A 220-kDa acetyl LDL binding protein was partially purified from bovine liver membranes and was used to make a specific monoclonal antibody. The 220-kDa protein immunoprecipitated by this antibody retained binding activity, and the antibody was used to detect this protein in cells lining bovine liver sinusoids and on the surface of cultured bovine alveolar macrophages. In the human monocytic cell line THP-1, the expression of both acetyl LDL receptor activity and a 220-kDa acetyl LDL binding protein were dramatically induced in parallel after differentiation to a macrophage-like state induced by phorbol ester. The ligand specificity, tissue and cell-type specificity, and coinduction data indicated that this 220-kDa cell-surface binding protein is probably a receptor that mediates acetyl LDL endocytosis. The 220-kDa protein, which was purified 238,000-fold from bovine lung membranes to near homogeneity using monoclonal antibody affinity chromatography, is a trimer of 77-kDa subunits that contain asparagine-linked carbohydrate chains.
Analysis of RAS oncogene mutations in human lymphoid malignancies.
Authors: Neri A , Knowles D M , Greco A , McCormick F , Dalla-Favera R
Published: May 2006

We investigated the frequency of mutations activating RAS oncogenes in human lymphoid malignancies, including B- and T-cell-derived acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. By the polymerase chain reaction/oligonucleotide hybridization method, DNA from 178 cases was analyzed for activating mutations involving codons 12 and 61 of the HRAS, KRAS and NRAS genes and codon 13 of the NRAS gene. Mutations involving codons 12 or 13 of the NRAS gene were detected in 6 of 33 cases of acute lymphoblastic leukemia (6/33, 18%), whereas no mutations were found in non-Hodgkin lymphoma or chronic lymphocytic leukemia. Direct nucleotide sequence analysis of polymerase chain reaction products showed that the mutations involved a G----A transition in five of the six cases of acute lymphocytic leukemia. In four cases the mutations seemed to occur in only a fraction of the neoplastic cells, and one case displayed two distinct NRAS mutations, most likely present in two distinct cell populations. These results indicate the following: (i) RAS oncogenes are not found in all types of human malignancies, (ii) significant differences in the frequency of RAS mutations can be found among subtypes of neoplasms derived from the same tissue, (iii) in lymphoid neoplasms the NRAS mutation correlates with the most undifferentiated acute lymphocytic leukemia phenotype, and (iv) NRAS mutations present in only a fraction of malignant cells may result from either the selective loss or the acquisition of mutated alleles during tumor development.
Stable DNA unwinding, not "breathing," accounts for single-strand-specific nuclease hypersensitivity of specific A+T-rich sequences.
Authors: Kowalski D , Natale D A , Eddy M J
Published: May 2006

A long A+T-rich sequence in supercoiled pBR322 DNA is hypersensitive to single-strand-specific nucleases at 37 degrees C but not at reduced temperature. The basis for the nuclease hypersensitivity is stable DNA unwinding as revealed by (i) the same temperature dependence for hypersensitivity and for stable unwinding of plasmid topoisomers after two-dimensional gel electrophoresis, (ii) preferential nuclease digestion of stably unwound topoisomers, and (iii) quantitative nicking of stably unwound topoisomers in the A+T-rich region. Nuclease hypersensitivity of A+T-rich sequences is hierarchical, and either deletion of the primary site or a sufficient increase in the free energy of supercoiling leads to enhanced nicking at an alternative A+T-rich site. The hierarchy of nuclease hypersensitivity reflects a hierarchy in the free energy required for unwinding naturally occurring sequences in supercoiled DNA. This finding, along with the known hypersensitivity of replication origins and transcriptional regulatory regions, has important implications for using single-strand-specific nucleases in DNA structure-function studies.
Molecular analysis of the 60-kDa human Ro ribonucleoprotein.
Authors: Deutscher S L , Harley J B , Keene J D
Published: May 2006

Ro, or Sjogren syndrome type A (SS-A), antigen is the most prevalent of the human systemic autoimmune specificities and exists as an inabundant ribonucleoprotein complex (RNP) composed of a 60,649-Da protein, as defined here by cDNA cloning, and the human Y RNAs. The recombinant 60-kDa Ro protein and human Y1 RNA were reconstituted in vitro, and the binding was enhanced by divalent cations. A region of the Ro amino acid sequence revealed a resemblance to the RNP consensus motif found in most RNA-binding proteins. In addition, Ro contained a potential "zinc-binding finger" motif that was distinct from the RNP consensus region and that may participate in the interaction with human Y RNAs or with other proteins. The recombinant Ro fusion protein also proved useful for the detection of autoantibodies in the sera of patients with autoimmune disorders. Possible functions of the Ro RNPs and their relationship to RNA polymerase III transcription are discussed.
Structure and regulation of the rat 1,25-dihydroxyvitamin D3 receptor.
Authors: Burmester J K , Wiese R J , Maeda N , DeLuca H F
Published: May 2006

Complementary DNA clones encoding the entire rat 1,25-dihydroxyvitamin D3 receptor were isolated, and the total 423-amino acid sequence was deduced. The amino acid sequence of the rat receptor is similar but not identical to the reported human receptor sequence. The cysteine-rich DNA-binding domain is completely conserved and the steroid-binding domain is greater than 93% conserved between rat and human. The cDNA was used as a probe in blot analysis of polyadenylylated RNA to show that the 1,25-dihydroxyvitamin D3 receptor mRNA is a single 4.4-kilobase mRNA that is expressed in intestine and kidney, slightly expressed in heart, and not detectable in liver and spleen. The receptor mRNA concentration is markedly increased during development of the rat intestine between day 14 and day 21, in accord with previous results obtained by measurement of receptor concentration by ligand binding or immunoblotting.
Purification and reconstitution of the proton-translocating ATPase of Golgi-enriched membranes.
Authors: Young G P , Qiao J Z , Al-Awqati Q
Published: May 2006

Kidney cortex microsomes enriched in Golgi markers and probably also containing endosomes were isolated by cell fractionation and found to contain a proton-translocating ATPase that was inhibited by N-ethylmaleimide (NEM). This NEM-sensitive ATPase was solubilized with n-octyl glucoside and purified using anion-exchange sievorptive chromatography on sequential DEAE-Sephadex and QAE-Sephadex columns followed by a final hydroxyapatite HPLC column. The purified enzyme, with a specific activity of 4.4 mumol.mg-1.min-1 was completely inhibited by NEM. Addition of asolectin and removal of the detergent by dialysis resulted in reconstitution of NEM-sensitive electrogenic proton transport. This vacuolar ATPase is composed of five polypeptides with apparent molecular masses of 68, 58, 40, 37, and 16 kDa.
Maternal transmission of retroviral disease: transgenic mice as a rapid test system for evaluating perinatal and transplacental antiretroviral therapy.
Authors: Sharpe A H , Hunter J J , Ruprecht R M , Jaenisch R
Published: May 2006

Transgenic mouse strains carrying Moloney murine leukemia virus (Mo-MuLV) in the germ line were found to serve as rapid and quantitative models of in utero and perinatal retroviral infection for evaluating strategies of antiretroviral therapy. In these strains virus activation leads to virus expression, viral spread associated with the development of viremia, and subsequent T-cell leukemia/lymphoma. To test these transgenic strains for their usefulness in evaluating antiretroviral agents, the effect of the drug 3'-azido-3'-deoxythymidine (AZT) on the development of viremia and subsequent disease was examined. The assessment of mice for viremia at 1 month of age appeared to be the most useful assay because it was rapid and quantitative. AZT was most effective in preventing viremia in transgenic strains that activate Mo-MuLV after birth and had more variable effects in strains that activate prior to birth. However, in six of seven of the strains examined, AZT led to a marked improvement in survival and reduced the onset of T-cell leukemia/lymphoma. These results provide evidence for effective antiretroviral therapy during gestation and in the perinatal period and are of potential significance for the management of the maternal transmission of human retroviruses.
Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor.
Authors: de Nucci G , Thomas R , D'Orleans-Juste P , Antunes E , Walder C , Warner T D , Vane J R
Published: May 2006

Endothelin releases prostacyclin and thromboxane A2 from guinea pig or rat isolated lungs and endothelium-derived relaxing factor in the perfused mesentery of the rat. Endothelin is also substantially removed by the pulmonary circulation of the rat in vitro and in vivo and by guinea pig lungs in vitro. In the rat, the effects of endothelin on the blood pressure vary from pressor (in pithed rats) to purely depressor in anesthetized rats where the resting blood pressure is high. It therefore has the characteristics of a local pressor hormone, rather than a circulating one.
Galanin antagonizes acetylcholine on a memory task in basal forebrain-lesioned rats.
Authors: Mastropaolo J , Nadi N S , Ostrowski N L , Crawley J N
Published: May 2006

Galanin coexists with acetylcholine in medial septal neurons projecting to the ventral hippocampus, a projection thought to modulate memory functions. Neurochemical lesions of the nucleus basalis-medial septal area in rats impaired choice accuracy on a delayed alternation t-maze task. Acetylcholine (7.5 or 10 micrograms intraventricularly or 1 micrograms micro-injected into the ventral hippocampus) significantly improved performance in the lesioned rats. Atropine (5 mg/kg intraperitoneally or 10 micrograms intraventricularly), but not mecamylamine (3 mg/kg intraperitoneally or 20 micrograms intraventricularly), blocked this action of acetylcholine, suggesting involvement of a muscarinic receptor. Galanin (100-500 ng intraventricularly or 200 ng into the ventral hippocampus) attenuated the ability of acetylcholine to reverse the deficit in working memory in the lesioned rats. The antagonistic interaction between galanin and acetylcholine suggests that endogenous galanin may inhibit cholinergic function in memory processes, particularly in pathologies such as Alzheimer disease that involve degeneration of basal forebrain neurons.
Expression of neurotransmitter transport from rat brain mRNA in Xenopus laevis oocytes.
Authors: Blakely R D , Robinson M B , Amara S G
Published: May 2006

To permit a molecular characterization of neurotransmitter transporter proteins, we have studied uptake activities induced in Xenopus laevis oocytes after injection of adult rat forebrain, cerebellum, brainstem, and spinal cord poly(A)+ RNA. L-Glutamate uptake could be observed as early as 24 hr after injection, was linearly related to the quantity of mRNA injected, and could be induced after injection of as little as 1 ng of cerebellar mRNA. Transport of radiolabeled L-glutamate, gamma-aminobutyric acid, glycine, dopamine, serotonin, and choline could be measured in single microinjected oocytes with a regional profile consistent with the anatomical distribution of particular neurotransmitter synthesizing soma. Forebrain L-glutamate and dopamine uptake, as well as cerebellar L-glutamate transport, were found to be Na+-dependent. Cerebellar mRNA-induced L-glutamate transport was both time and temperature-dependent, was saturable by substrate, suggesting a single activity with an apparent transport Km of 14.2 microM and a Vmax of 15.2 pmol/hr per oocyte, and was sensitive to inhibitors of brain L-glutamate transport. Thus, the oocyte L-glutamate transport induced by injection of adult rat cerebellar mRNA appears essentially identical to the high-affinity, Na+-dependent L-glutamate uptake found in brain slices and nerve terminals. Experiments with size-fractionated cerebellar mRNA reveal single, comigrating peaks for cerebellar L-glutamate and gamma-aminobutyric acid transport, with peak activity obtained in fractions of approximately 2.7 kilobases, suggesting the presence of single or similarly sized mRNAs encoding each of these activities.
Protein tyrosine phosphorylation in synaptic vesicles.
Authors: Pang D T , Wang J K , Valtorta F , Benfenati F , Greengard P
Published: May 2006

Protein tyrosine phosphorylation in purified synaptic vesicles from rat forebrain has been studied in the presence of Mn2+ and orthovanadate. High levels of endogenous protein tyrosine phosphorylation were observed. Four major phosphoproteins, with apparent molecular masses of 105, 94, 38, and 30 kDa, were shown to contain phosphotyrosine. The 38-kDa phosphoprotein was identified as synaptophysin (p38), a well-characterized integral membrane protein of synaptic vesicles. The three other phosphotyrosine-containing proteins distributed in the same manner as synaptophysin in all subcellular fractions. Like synaptophysin, the two high molecular weight phosphotyrosine proteins (105 and 94 kDa) were found to be glycoproteins by lectin chromatography. Tyrosine phosphorylation of synaptophysin was an intravesicular reaction and reached 50% of maximal level within 3 min. Triton X-100, a nonionic detergent, inhibited tyrosine phosphorylation of endogenous protein substrates but not the phosphorylation of an exogenous substrate, poly(Glu80,-Tyr20). Tyrosine phosphorylation of synaptophysin was also demonstrated in synaptosomes, indicating that tyrosine phosphorylation of synaptic vesicle proteins occurs in intact nerve terminals.
Differential expression of DNA topoisomerases I and II during the eukaryotic cell cycle.
Authors: Heck M M , Hittelman W N , Earnshaw W C
Published: May 2006

We have utilized antibody probes to examine the expression of DNA topoisomerases I and II and chromosome scaffold protein Sc-2 in normal and transformed cells. Neither topoisomerase I nor Sc-2 shows significant fluctuations in content or stability across the cell cycle. In contrast, topoisomerase II undergoes significant cell cycle-dependent alterations in both amount and stability. As cells progress from mitosis into G1, much of the topoisomerase II is degraded. During the first 2 hr of G1, the half life of topoisomerase II is decreased from that measured in asynchronous cell populations by a factor of 7. This suggests that the chromosome condensation/decondensation cycle is coupled to a parallel cycle of synthesis and degradation of topoisomerase II. In control experiments, we also found that the half-life of topoisomerase II is shorter in normal cells than in transformed cells by a factor of 4. Since the number of copies of topoisomerase II per cell is also lower in normal cells, this suggests that control of topoisomerase II stability is altered upon transformation. The stability of topoisomerase I and Sc-2 does not differ significantly between normal and transformed cells.
Directional mutation pressure and transfer RNA in choice of the third nucleotide of synonymous two-codon sets.
Authors: Osawa S , Ohama T , Yamao F , Muto A , Jukes T H , Ozeki H , Umesono K
Published: May 2006

Bacterial species have diverged into a series of families, some with high G + C content in their DNA, and other with high A + T content, resulting, respectively, from G.C- and A.T-directional mutation pressures. Such mutation pressure (G.C/A.T pressure) may be an important determinant for codon usage. It has also been suggested that tRNA acts as a selective constraint for determining codon usage. We have studied the relation between G.C/A.T pressure and tRNA constraints in determining choice of the third nucleotide of eight two-codon sets, using codon usage data obtained from protein genes in four bacterial species, Mycoplasma capricolum, Bacillus subtilis, Escherichia coli, and Micrococcus luteus, and in liverwort (Marchantia polymorpha) chloroplasts. The genomic G + C contents of these range from 25% to 74%. The results demonstrate that tRNA levels act additively to A.T and G.C pressure in affecting contents of A (pairing with *UNN anticodons, in which *U indicates a 2-thiouridine derivative) and C (pairing with GNN anticodons) or G (pairing with CNN anticodons), respectively, in third nucleotide positions of codons.
An all-purine precursor of nucleic acids.
Authors: Wächtershäuser G
Published: May 2006

The theory is proposed that the pyrimidines in extant nucleic acids are postenzymatic substitutes for their isoelectronic and isogeometric position 3-bonded purine analogs xanthine and isoguanine, which were sibling products in a preenzymatic de novo purine pathway.
Evidence that the modulator of the glucocorticoid-receptor complex is the endogenous molybdate factor.
Authors: Bodine P V , Litwack G
Published: May 2006

We have recently purified the modulator of the glucocorticoid-receptor complex from rat liver. Purified modulator inhibits glucocorticoid-receptor complex activation and stabilizes the steroid-binding ability of the unoccupied glucocorticoid receptor. Since these activities are shared by exogenous sodium molybdate, modulator appears to be the endogenous factor that sodium molybdate mimics. In this report, we present additional evidence for the mechanism of action of purified modulator. (i) Molybdate and modulator inhibit receptor activation as measured by DNA-cellulose binding, DEAE-cellulose chromatography, and Sepharose 4B gel filtration. (ii) The ability of molybdate and modulator to inhibit receptor activation and stabilize the unoccupied receptor appears to be additive. (iii) Scatchard analysis of heat-destabilized unoccupied receptors indicates that the number of steroid-binding sites is reduced during destabilization, whereas the steroid dissociation constant remains unchanged. Molybdate and modulator stabilize the receptor by maintaining the number of steroid-binding sites. (iv) Molybdate and modulator do not inhibit alkaline phosphatase-induced destabilization of the unoccupied receptor. However, alkaline phosphatase-induced destabilization is reversed by the addition of dithiothreitol in the presence, but not in the absence, of molybdate or modulator. These results suggest that the mechanism of action for modulator is identical to that of sodium molybdate, and we propose that modulator is the endogenous molybdate factor for the glucocorticoid receptor.
Activity-dependent accumulation of calcium in Purkinje cell dendritic spines.
Authors: Andrews S B , Leapman R D , Landis D M , Reese T S
Published: May 2006

The calcium content of synapses of parallel fibers on Purkinje cell dendritic spines was determined by electron probe x-ray microanalysis of freeze-dried cryosections from directly frozen slices of mouse cerebellar cortex. In fresh slices frozen within 20-30 sec of excision, calcium concentrations ranging from 0.8 to 18.6 mmol/kg of dry weight were measured in cisterns of smooth endoplasmic reticulum within Purkinje cell dendritic spines. The average calcium content of spine cisterns in rapidly excised slices (6.7 +/- 0.6 mmol/kg of dry weight +/- SEM) was higher than the average calcium content of spine cisterns in brain slices incubated without stimulation for 1-2 hr before direct freezing (2.5 +/- 0.4 mmol/kg of dry weight). Depolarization of incubated cerebellar slices by isotonic 55 mM KCl resulted in the accumulation within spine cisterns of very high amounts of calcium or isotonically substituted strontium, both derived from the extracellular fluid. These results suggest that one function of spine cisterns is to sequester free calcium that enters the spine through ligand-gated or voltage-gated channels during synaptic transmission.
Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs.
Authors: Travis G H , Sutcliffe J G
Published: May 2006

To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA.
5-Fluorodeoxyuridine as an alternative to the synthesis of mixed hybridization probes for the detection of specific gene sequences.
Authors: Habener J F , Vo C D , Le D B , Gryan G P , Ercolani L , Wang A H
Published: May 2006

Synthetic complementary oligonucleotides are useful hybridization probes for the detection of mRNAs and genes encoding proteins for which only a partial amino acid sequence is known. Usually this involves the synthesis of mixtures of oligonucleotides complementary to all possible bases in degenerate positions of codons. As an alternative we have prepared and characterized a series of unique oligonucleotides containing a pyrimidine analog, 5-fluorodeoxyuridine (F). Thermodynamic parameters and the melting temperatures of hybrid duplexes containing A.F and G.F base pairs showed that they are considerably more stable than duplexes containing A.T and G.T base pairs. The stability of a duplex decreased linearly with the number of mismatches introduced at positions at least a codon apart. A 5-fluorodeoxyuridine-substituted oligonucleotide cDNA detects rat liver pyruvate carboxylase mRNA on a RNA gel blot with a dissociation temperature only 10 degrees C below the measured melting temperature in solution. We suggest that the complexity of oligonucleotide cDNAs used for screening gene libraries can be reduced by the design of single hybridization probes containing the substituted bases--5-fluorodeoxyuridine to pair with adenosine or guanosine, guanosine to pair with cytidine or thymidine, and deoxyinosine to pair with adenosine or cytidine at positions of codon degeneracy--and still retain near-maximum stability of hybrid duplexes.