bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions of metal-nucleotide complexes with aspartate carbamoyltransferase in the crystalline state.
Authors: Honzatko R B , Lipscomb W N
Published: May 2006

We report the results of crystallographic difference maps at 3.0-A resolution of complexes of metal-nucleoside triphosphates with aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli. The complexes Gd3+-ATP, Al3+-ATP, and Gd3+-CTP bind to the allosteric effector domain of the enzyme in nearly the same orientation as the metal-free nucleotides. The result is consistent with kinetic observations of nearly identical allosteric efficacy of ATP and CTP and their complexes with cations. The effector Gd3+-GTP, however, binds in a distinctly different conformation and location than does 8-bromoguanosine 5'-triphosphate, reported in a separate investigation [Honzatko, R. B. & Lipscomb, W.N. (1982) J. Mol. Biol. 160, 265-286]. The difference in the binding modes of Gd3+-GTP and the bromo derivative suggests a possible mechanism for the relief of allosteric inhibition of GTP due to metal cations. We observe no binding of metal-nucleoside triphosphates in the region of the phosphate crevice of aspartate carbamoyltransferase, consistent with the reduced ability of metal nucleotides to compete with carbamoyl phosphate for the active site. However, a single Gd3+ ion binds in the region of the active site as evidenced by strong density. The binding of cations near the active site probably causes the inhibition of catalysis observed in kinetics experiments reported earlier [Honzatko, R.B., Lauritzen, A.M. & Lipscomb, W.N. (1981) Proc. Natl. Acad. Sci. USA 78, 898-902].
X-ray structure of a dinucleoside monophosphate A2'p5'C that contains a 2'-5' link found in (2'-5')oligo(A)s induced by interferons: single-stranded helical conformation of 2'-5'-linked oligonucleotides.
Authors: Parthasarathy R , Malik M , Fridey S M
Published: May 2006

In order to understand why DNA and RNA have the 3'-5' and not the 2'-5' link and to delineate the stereochemistry of the 2'-5' phosphodiester links, we crystallized and carried out a very accurate x-ray diffraction analysis of A2 p5'C, an analog of A2' p5'A. Contrary to numerous reports in the literature that conclude that the tendency for 2'-5' nucleotides to stack intramolecularly is stronger than for 3'-5' counterparts, we find hardly any intramolecular base stacking for this molecule but find an intramolecular "stacking" of the ribose oxygen-4' of cytidine on top of the adenine ring. Although A2' p5'C shows the standard conformational features usually found for 3'-5' nucleotides, the overall stereochemistry of 2'-5' nucleotides is quite different because the 2' link orients the backbone inwards to the bases unlike the 3' and 5' links that orient it away from the bases. With the conformational features found for A2' p5'C, it is possible to build a very compact right-handed single-stranded helix but not a double helix. Such a preference for single-stranded helices may be the reason for the absence of 2'-5' bonds in DNA and RNA even though the 2'-5' bonds are formed more readily then 3'-5' bonds.
Monoclonal antibodies to the human insulin receptor block insulin binding and inhibit insulin action.
Authors: Roth R A , Cassell D J , Wong K Y , Maddux B A , Goldfine I D
Published: May 2006

Antibodies to the insulin receptor were prepared in BALB/c mice by immunization with IM-9 human lymphocytes, a cell type that has a large number of plasma membrane insulin receptors. The spleens of these mice were then removed, and their lymphocytes were fused to a mouse myeloma cell line, FO cells. After screening over 1,200 resulting hybrids, one stable hybrid was obtained that produced IgG1 antibodies directed towards the insulin receptor. This antibody blocked 125I-labeled insulin binding to its receptor by more than 90% in three human tissues: IM-9 cultured lymphocytes, freshly isolated adipocytes, and placenta membranes. In contrast, the antibody did not inhibit insulin binding to rat adipocytes and rat liver plasma membranes, suggesting that the antibody was species specific. In IM-9 cells, which had their proteins prelabeled with [35S]methionine, the antibody precipitated two polypeptides with molecular weights of 135,000 and 95,000; these molecular weights are identical to those previously identified as the alpha and beta subunits of the insulin receptor. The monoclonal antibody inhibited the actions of insulin on both human adipocytes and fibroblasts, suggesting that the antibody was an antagonist of insulin action. The present studies suggest, therefore, that monoclonal antibodies to the insulin receptor may provide new insights into the structure of the insulin receptor and its interaction with insulin.
Mutagenesis of bacteriophage T7 in vitro by incorporation of O6-methylguanine during DNA synthesis.
Authors: Dodson L A , Foote R S , Mitra S , Masker W E
Published: May 2006

An in vitro system in which bacteriophage T7 DNA is replicated and efficiently packaged into procapsids to form viable phage has been used to examine mutagenesis. The fidelity of replication was assayed both by measuring reversion of an amber mutation in an essential gene and by generation of temperature-sensitive mutants among the phage produced in vitro. Under standard reaction conditions, the fidelity of DNA replication is about equal to that normally found in vivo. However, when O6-methyldeoxyguanosine triphosphate is included in the reaction, O6-methylguanine is incorporated into newly synthesized DNA and the mutation frequencies increase 10- to 70-fold over the control. These experiments demonstrate in vitro mutagenesis with the T7 DNA replication-packaging system and provide more direct evidence for the premutagenic role of O6-methylguanine.
Identification of a human neuroectodermal tumor antigen (OFA-I-2) as ganglioside GD2.
Authors: Cahan L D , Irie R F , Singh R , Cassidenti A , Paulson J C
Published: May 2006

Two monospecific human antibodies (anti-OFA-I-1 and anti-OFA-I-2) produced in vitro by lymphoblast cell lines originating from melanoma patients have been shown previously to recognize cell surface antigens (OFA-I-1 and OFA-I-2) on human tumors and fetal brain: OFA-I-1 is expressed on a variety of human tumors, while OFA-I-2 has been detected only on tumors of neuroectodermal origin. Evidence presented in this report suggests that the two antigens expressed by a cultured human melanoma cell line (M14) are chemically distinct and that OFA-I-2 is a cell surface glycolipid, ganglioside GD2: GalNAc beta 1 leads to 4 NeuAc alpha 2 leads to 8NeuAc alpha 2 leads to 3 Gal beta 1 leads to 4Glc-ceramide.
Cloning the double-stranded RNA genes of reovirus: sequence of the cloned S2 gene.
Authors: Cashdollar L W , Esparza J , Hudson G R , Chmelo R , Lee P W , Joklik W K
Published: May 2006

The genes of the Dearing strain of reovirus serotype 3, which consist of double-stranded RNA, have been cloned into pBR322 by tailing both strands of each gene with poly(A), transcribing them with reverse transcriptase, self-hybridizing the cognate plus and minus cDNA strands, incubating them with Escherichia coli DNA polymerase I to ensure that they are complete, and cloning the double-stranded cDNA molecules by standard procedures. The sequence of the cloned S2 gene has been determined. The sequence at the termini are exactly the same as those at the ends of the native double-stranded RNA gene. The gene is 1,329 nucleotides long and possesses a single long open reading frame that starts at the first initiation codon (residue 19) and extends for 331 codons, sufficient to encode a protein of the same size as the known S2 gene product, protein sigma 2, a major reovirus core component (Mr, 38,000). A second open reading frame of 85 codons, in a different phase, starts close to where the first ends. The protein translated from this reading frame is unknown.
Na+/H+ antiport in Swiss 3T3 cells: mitogenic stimulation leads to cytoplasmic alkalinization.
Authors: Schuldiner S , Rozengurt E
Published: May 2006

Addition of Na+ to Na+-depleted Swiss 3T3 cells causes a rapid and dramatic increase in intracellular pH, as monitored by uptake of the weak acid 5,5-dimethyloxazolidine-2,4-dione. The effect of Na+ is concentration dependent (half-maximal effect at 38 mM); this cation can be replaced by Li+ but not by K+ or the choline ion. Amiloride prevents the Na+-induced increase in intracellular pH and also blocks the entry of Na+ into 3T3 cells; the half-maximal concentrations of amiloride for inhibiting the two processes are similar (40 microM). Increase in extracellular pH caused an increase in the initial rate of Na+ influx that was of sufficient magnitude to stimulate the activity of the Na+/K+ pump in quiescent 3T3 cells. Taken together, these findings suggest the presence of a functional Na+/H+ antiport in Swiss 3T3 cells. Addition of the potent mitogenic combination platelet-derived growth factor, vasopressin, and insulin to quiescent Swiss 3T3 cells increased the intracellular pH from 7.21 +/- 0.07 to 7.36 +/- 0.09 in 10 independent experiments (P less than 0.001). This combination of growth factors also stimulated Na+ entry and ouabain-sensitive Rb+ uptake. The data support the hypothesis that early changes in ion fluxes play a role in signaling mitogenesis in 3T3 cells.
Chromosomal localization of human leukocyte, fibroblast, and immune interferon genes by means of
in situ
hybridization
Authors: Trent Jeffrey M. , Olson Sharon , Lawn Richard M.
Published: May 2006

Recombinant plasmids containing cDNA inserts from human leukocyte interferon (IFN-α), fibroblast interferon (IFN-β), or immune interferon (INF-γ) genes were radiolabeled and hybridized
in situ
to human metaphase chromosome preparations. The results localized the human IFN-α and IFN-β genes to the short arm of chromosome 9(p21→pter) and localized the IFN-γ gene to the long arm of chromosome 12(q24.1).
Radioimmunoassay for leukotriene B
4
Authors: Lewis Robert A. , Mencia-Huerta Jean-Michel , Soberman Roy J. , Hoover Dennis , Marfat Anthony , Corey E. J. , Austen K. Frank
Published: May 2006

A rabbit immunized with leukotriene B
4
[LTB
4
; (5
S
,12
R
)-6, 14-
cis
-8, 10-
trans
-icosatetraenoic acid] coupled to bovine serum albumin via the 12-oxy function of the lipid produced antibodies having an average association constant (
K
a
) for [14,15-
3
H]LTB
4
of 3.2 × 10
9
M
-1
at 37°C and in a concentration of 0.37 μg/ml of the immune plasma. When 10 μl of anti-LTB
4
and 3.9 nCi of [14,15-
3
H]LTB
4
(28 Ci/mmol; 1 Ci = 3.7 × 10
10
becquerels) were incubated in a volume of 250 μl, 50% inhibition of radioligand binding was achieved with 0.31 ng of LTB
4
and with 1.95 ng of (5
S
,12
S
)-6-
trans
-8-
cis
-LTB
4
. The sulfidopeptide leukotrienes, LTC
4
and LTD
4
, displaced the radioligand from this antibody with less than 1/100th the activity of LTB
4
, and the diastereoisomers of 6-
trans
-LTB
4
, 5-L-hydroxy-6-
trans
-8,11,14-
cis
-icosatetraenoic acid (5-HETE), and three prostaglandins were minimally effective. The specificity of this radioimmunoassay was further shown by assessment of the immunoreactive products generated from calcium ionophore (A23187)-activated rat serosal mast cells and human neutrophils after reversed-phase HPLC. Resolution of the supernatants from each cell type yielded a single immunoreactive peak that coeluted with synthetic LTB
4
and quantitatively correlated with the physical measurement by integrated A
269
in that peak; UV-absorbing peaks eluting at other retention times were not immunoreactive. The immunoreactive LTB
4
generated averaged 4.6 ng per 10
6
rat mast cells and resolution of the supernatants by reversed-phase HPLC without a prior extraction step gave a recovery of 54%, validating the direct applicability of this sensitive and specific assay for LTB
4
, a highly potent chemotactic factor, to unfractionated biologic fluids.
Simple rapid method for the synthesis of radioactively labeled cDNA hybridization probes utilizing bacteriophage M13mp7.
Authors: Ricca G A , Taylor J M , Kalinyak J E
Published: May 2006

Double-stranded cDNA sequences for rat alpha 1-acid glycoprotein and rat glutathione S-transferase mRNAs were inserted into the Pst I site of bacteriophage M13mp7 and used to develop a new method for preparing specific cDNA hybridization probes directly from cloned template DNA. A palindrome sequence surrounding the Pst I site in the vector DNA permitted single-stranded DNA isolated from the recombinant phage to fold back, thus forming a stable hybrid bounded on the ends by a large loop of M13mp7 single-stranded DNA and a small loop of inserted foreign DNA. A primer corresponding to an internal sequence of the foreign DNA was hybridized, then Escherichia coli DNA polymerase I was used to synthesize a 32P-labeled complementary DNA copy of the cloned inserted DNA. The single-stranded cDNA reaction product was easily isolated by subsequent sedimentation through alkaline sucrose gradients. Gel electrophoresis of the labeled cDNA product, after denaturation with glyoxal, indicated a single discrete band with an electrophoretic mobility corresponding to the length of the inserted DNA sequence. About 95% of the cDNA product formed S1 nuclease-resistant hybrids in hybridization reactions with excess RNA in solution. DNA sequences complementary to the M13mp7 vector DNA were not detected in the cDNA product. Thus, these M13mp7-derived probes are the functional equivalent of cDNA copies to mRNAs and can be employed for quantitative measurements of mRNA concentration. This simple, rapid method probably can be used for most cloned DNA sequences to yield single-stranded radioactively labeled DNA, without contaminating vector DNA sequences, for virtually any hybridization requirement.
Presence of albumin mRNA precursors in nuclei of analbuminemic rat liver lacking cytoplasmic albumin mRNA.
Authors: Esumi H , Takahashi Y , Sekiya T , Sato S , Nagase S , Sugimura T
Published: May 2006

Analbuminemic rats, which lack serum albumin, were previously found to have no albumin mRNA in the cytoplasm of the liver. In the present study, the existence of nuclear albumin mRNA precursors in the liver of analbuminemic rats was examined by RNA X cDNA hybridization kinetics. Albumin mRNA precursors were present in the nuclei of analbuminemic rat liver at almost normal levels, despite the absence of albumin mRNA from the cytoplasm. Nuclear RNA of analbuminemic rat liver was subjected to electrophoresis on 1% agarose gel in parallel with nuclear RNA of normal rat liver. RNA was transferred from the gel to diazobenzyloxymethyl-paper and hybridized to cloned cDNA. Several bands of putative albumin mRNA precursors were obtained with nuclear RNA of analbuminemic rat liver and some of them were indistinguishable from those of normal rat liver. Nuclear RNA of analbuminemic rats was hybridized to 3'-end-labeled cloned cDNA under the conditions of RNA excess and then digested completely with S1 nuclease and subjected to electrophoresis on polyacrylamide gel. By this technique, nuclear RNA that could hybridized to cDNA was found to have the albumin mRNA sequence in at least the 3' half of the mRNa that was covered by cloned cDNA. For comparison of the structures of the albumin genes of analbuminemic and normal rats, DNAs from rat livers of both types were digested completely with EcoRI, HindIII, and Pst I; the fragments were separated by electrophoresis on 1% agarose gel, transferred to nitrocellulose paper, and hybridized to cloned cDNA. The intensities of the corresponding bands and the digestion patterns of the analbuminemic and normal rat genes were indistinguishable. From these data, it is concluded that analbuminemic rats have a unique type of mutation(s) affecting albumin mRNA maturation.
Analysis of histone gene expression during the cell cycle in HeLa cells by using cloned human histone genes.
Authors: Rickles R , Marashi F , Sierra F , Clark S , Wells J , Stein J , Stein G
Published: May 2006

Although it is generally agreed that histone protein synthesis is restricted to the S phase of the cell cycle--and therefore parallels DNA replication--both transcriptional and posttranscriptional levels of control have been invoked. Using blot hybridization with several cloned genomic human histone sequences representing different histone gene clusters as probes, we have assessed the steady-state level of histone RNAs in the nucleus and cytoplasm of G1 and S phase HeLa S3 cells. The representation of histone mRNA sequences of G1 compared with S phase cells was less than 1% in the cytoplasm and approximately 1% in the nucleus. These data are consistent with transcriptional control, but we cannot completely dismiss the possibility that regulation of histone gene expression is, to some extent, mediated posttranscriptionally. If histone gene transcription does occur in G1, the RNAs must either be rapidly degraded or be transcribed to a limited extent compared with S phase. An unexpected result was obtained when a blot of cytoplasmic RNA from G1 and S phase cells was hybridized with lambda HHG 41 DNA (containing H3 and H4 human genomic histone sequences). Although hybridization with histone mRNAs was observed for RNAs from S phase but not from G1 cells, hybridization with a nonhistone RNA of approximately 330 nucleotides present predominantly in G1 was also observed.
Murine erythroleukemia cell differentiation: DNase I hypersensitivity and DNA methylation near the globin genes
Authors: Sheffery Michael , Rifkind Richard A. , Marks Paul A.
Published: May 2006

The sensitivity to digestion by DNase I of chromatin containing the α- and β
major
-globin genes and the pattern of DNA methylation near these genes was examined during hexamethylenebisacetamide (HMBA)-mediated erythroid differentiation of murine erythroleukemia cells (MELC). In uninduced and induced cells, the chromatin regions containing the α- and β-
major
-globin genes are more sensitive to digestion by DNase I than is the region containing an immunoglobulin gene (Igα) not expressed during erythroid differentiation. However, at low concentrations of DNase I, a 6- to 10-fold increase in site-specific cleavages was generated in chromatin regions near both the α- and β
major
-globin genes in cells induced to differentiate by HMBA. The DNase I hypersensitive site near the β
major
-globin gene maps to a small region near the 5′ terminus of the gene. No detectable change in the pattern of DNA methylation around either the α- or β-globin genes was observed during HMBA-mediated erythroid differentiation. Of the potentially methylated sites assayed and mapped near the β
major
-globin gene, one site is fully methylated, one is partially methylated, and one is unmethylated both in uninduced and induced cells. Many (but not all) sites assayed near the α-globin genes are unmethylated in both uninduced and induced cells. These results show that specific alterations of chromatin structure occur during MELC differentiation and suggest that these changes may not involve alterations in the pattern of DNA methylation.
Mapping the structural genes coding for the major urinary proteins in the mouse: combined use of recombinant inbred strains and somatic cell hybrids.
Authors: Bennett K L , Lalley P A , Barth R K , Hastie N D
Published: May 2006

We have mapped the multiple (15-25) genes coding for the hormonally regulated major urinary proteins (MUPs) of the mouse by using a cloned cDNA probe. By Southern blot analysis of DNA from Chinese hamster-mouse somatic cell hybrids, all of the MUP genes were found to be on chromosome 4. Different inbred mouse strains showed DNA polymorphism in their MUP Southern hybridization pattern. Analysis of recombinant inbred strains derived from these parent strains has shown that all the polymorphisms are linked to the MUP-a locus on chromosome 4. The combination of these mapping techniques should be applicable to many cloned DNA sequences.
Expression of endogenous retroviral glycoprotein 70 by antigen-activated cytotoxic and suppressor T lymphocytes of nice.
Authors: Klenner D , Horak I , Schimpl A , Wecker E
Published: May 2006

Concanavalin A-stimulated murine spleen cells and antigen-stimulated B lymphocytes of normal mice express an antigen that reacts with goat antiserum against glycoprotein (gp) 70. Structural analysis of this antigen characterizes it as endogenous viral gp70 that is most likely of xenotropic origin. Activated nonspecific T suppressor cells and cytotoxic T lymphocytes express endogenous viral gp70, whereas nonactivated mouse T or B lymphocytes do not. The presence of endogenous retroviral gp70 is thus a novel marker for activated mouse lymphocytes in general.
Characterization and molecular cloning of the mRNA for the heavy (epsilon) chain of rat immunoglobulin E.
Authors: Hellman L , Pettersson U , Bennich H
Published: May 2006

We report a study of the mRNA for the heavy (epsilon) chain of rat IgE. Cytoplasmic RNA was prepared from the two rat immunocytomas IR2 and IR162 and fractionated by sucrose gradient centrifugation. An enriched fraction containing approximately 5% mRNA for the epsilon chain was obtained in this way. When translated in vitro, it produced a 59,000-dalton polypeptide, which in the presence of a membrane fraction yielded a 90,000-dalton polypeptide, presumably through posttranslational modification. Both polypeptides were precipitated by rabbit antisera that were monospecific for rat epsilon chains. The epsilon chain mRNA was estimated to be approximately 2200 nucleotides long and constitutes a minute fraction in the total mRNA both in the IR2 and the IR162 tumors, unlike the mRNA for light chains. Double-stranded cDNA copies prepared frm the RNA fraction, which was enriched for epsilon chain mRNA, were inserted into the Pst I cleavage site of the pBR322 vector. Twenty clones with inserts exceeding 1000 base pairs were used for selection of mRNA from the IR2 tumor. By in vitro translation of the selected mRNA, one clone was identified that yielded a polypeptide with the same size as the unprocessed epsilon chain. The nucleotide sequence was determined for part of the inserted cDNA in this candidate clone and was found to be homologous to a sequence in the constant region (C) of the human epsilon chain. In this communication we report a sequence from the C epsilon 3 domain of the rat IgE. When compared to the corresponding sequence of human IgE, 55% of the amino acids in the rat sequence were found to be conserved.
Molecular basis of bunyavirus per os infection of mosquitoes: role of the middle-sized RNA segment.
Authors: Beaty B J , Miller B R , Shope R E , Rozhon E J , Bishop D H
Published: May 2006

The molecular basis of bunyavirus per os infection of mosquitoes was determined; La Crosse (LaC), snowshoe hare (Ssh), and LaC-Ssh reassortment viruses were compared for their ability to infect Aedes triseriatus, the natural vector of the LaC virus. The viruses were comparable in their ability to infect midgut cells; 115 of 117 (98%) mosquitoes ingesting viruses containing the LaC middle-sized RNa segment and 92/100 (92%) of mosquitoes ingesting viruses containing the Ssh middle-sized RNA segment became infected. However, those viruses containing the LaC middle-sized RNA segments disseminated efficiently (113/115, 98%) from the midgut to infect secondary target organs. Those viruses containing the Ssh middle-sized RNA segment efficiently infected the midgut and large amounts of viral antigen were detected in the midgut cells but antigen was detected in the secondary target organs only in 26% (24/92) of the mosquitoes with midgut infection. Thus, the middle-sized RNA segment seems to be the major determinant for successful dissemination of LaC virus from infected A. triseriatus midgut cells.
Analysis of the sequence of amino acids surrounding sites of tyrosine phosphorylation.
Authors: Patschinsky T , Hunter T , Esch F S , Cooper J A , Sefton B M
Published: May 2006

We have identified the single phosphorylated tyrosine in p60src, the transforming protein of Rous sarcoma virus, as part of the sequence. NH2-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg-COOH. Therefore, this is a sequence that is recognized efficiently by a tyrosine protein kinase in vivo. Phosphorylation of tyrosine in cellular proteins appears to play a role in malignant transformation by four classes of genetically distinct RNA tumor viruses. Phosphorylated tyrosines in several other proteins resemble of the tyrosine in p60src in that they are located 7 residues to the COOH-terminal side of a basic amino acid and either 4 residues to the COOH-terminal side of, or in close proximity to, a glutamic acid residue. Therefore it is possible that these features play a role in the selection of sites of phosphorylation by some tyrosine protein kinases. However, several clear exceptions to this rule exist.
Verdohemochrome IX alpha: preparation and oxidoreductive cleavage to biliverdin IX alpha.
Authors: Saito S , Itano H A
Published: May 2006

Several studies have shown that both terminal oxygen atoms of biliverdin are derived from molecular oxygen. Since the conversion of verdohemochrome to biliverdin has been assumed to be hydrolytic, these findings seemed to exclude verdohemochrome as an intermediate in the degradation of heme to biliverdin. Coupled oxidation of myoglobin and ascorbate yielded a pure preparation of verdohemochrome IX alpha. The structure and ferrous state of this product were determined from its composition, ligand reactions, 1H NMR spectrum, and conversion to biliverdin IX alpha dimethyl ester. Reaction with ascorbate and 18O2 converted this compound to biliverdin that contained an atom of 18O. Successive treatment of verdohemochrome, first oxidation with H2O2 and then reduction with phenylhydrazine, yielded the iron complex of biliverdin. These results showed that hydrolysis is not an obligatory step in the conversion of verdohemochrome to biliverdin and, moreover, indicated how heme can be converted, with verdohemochrome as an intermediate, into biliverdin in which the two terminal oxygen atoms are derived from different O2 molecules.
Characterization of a porcine genomic clone encoding a major histocompatibility antigen: expression in mouse L cells.
Authors: Singer D S , Camerini-Otero R D , Satz M L , Osborne B , Sachs D , Rudikoff S
Published: May 2006

A porcine genomic clone encoding a major histocompatibility, (MHC) antigen was isolated by direct screening of a swine genomic library with a heterologous human MHC cDNA probe. Mouse L cells transformed with DNA from the clone stably express swine MHC antigen. Pig alloantisera specifically lyse transformant but not control cell lines in a complement-mediated cytotoxicity assay. Direct immunoprecipitation of radiolabeled cellular protein from transformed lines by pig alloantiserum results in the coprecipitation of swine MHC heavy chain and mouse beta 2-microglobulin, demonstrating the association of heterologous subunits of MHC antigens.
Right-handed and left-handed DNA: studies of B- and Z-DNA by using proton nuclear Overhauser effect and P NMR.
Authors: Patel D J , Kozlowski S A , Nordheim A , Rich A
Published: May 2006

We have differentiated between syn and anti glycosidic torsion angles in nucleic acid duplexes by measuring the transient nuclear Overhauser effect (NOE) between the sugar H-1' protons and the purine H-8 and pyrimidine H-6 base protons. The transient NOE measurements demonstrate a syn glycosidic torsion angle at guanosine and an anti glycosidic torsion angle at cytidine in poly(dG-dC) in 4 M NaCl and in poly(dG-m5dC) in 1.5 M NaCl solution. These features have been observed previously in the left-handed Z-DNA conformation of (dC-dG)3 in the crystalline state. By contrast, transient NOE studies demonstrate that both guanosine and cytidine residues adopt the anti conformation about the glycosidic bond for the right-handed poly(dG-dC) and poly(dG-m5dC) conformation in a low-salt solution. We have used P NMR to monitor the equilibrium between B- and Z-DNA forms of poly(dG-dC) in LiCl solutions; at high temperatures, the equilibrium shifts from B- to Z-DNA.
Regulation of lactose permease activity by the phosphoenolpyruvate:sugar phosphotransferase system: evidence for direct binding of the glucose-specific enzyme III to the lactose permease.
Authors: Osumi T , Saier M H
Published: May 2006

Interaction between the glucose-specific enzyme III (enzyme IIIglc) of the phosphoenolpyruvate:sugar phosphotransferase system and the lactose permease was studied with membrane fragments from an Escherichia coli strain that overproduces the lactose permease. Substrates of the permease markedly and specifically stimulated binding of enzyme IIIglc to the membranes. The sugar-stimulated binding of enzyme IIIglc was concluded to the specific to the lactose permease because it (i) was dependent on the amount of the permease, (ii) was promoted only by sugar substrates of the permease, and (iii) was completely eliminated by treatment of the membranes with N-ethylmaleimide in the absence (but not the presence) of thio-beta-D-digalactoside. The pH dependence of binding was similar to that reported for the binding of thio-beta-D-digalactoside to the permease. Phosphoenolpyruvate prevented the binding of enzyme IIIglc to the lactose permease in the presence (but not the absence) of the other phosphate transfer components of the phosphotransferase system. These results support the hypothesis that enzyme IIIglc, in its dephosphorylated form, modulates the activity of the lactose permease by a direct protein-protein interaction.
RNA ligation via 2'-phosphomonoester, 3'5'-phosphodiester linkage: requirement of 2',3'-cyclic phosphate termini and involvement of a 5'-hydroxyl polynucleotide kinase.
Authors: Konarska M , Filipowicz W , Gross H J
Published: May 2006

Extracts of wheat germ contain a RNA ligase activity that catalyzes the conversion of linear polyribonucleotides into covalently closed circles. As reported previously, this enzyme joins two ends of a RNA substrate via a 2'-phosphomonoester, 3',5'-phosphodiester linkage. In the present work we provide evidence that a 2',3'-cyclic phosphate group at the 3' terminus is required for RNA ligation and that the 5'-hydroxyl end is phosphorylated before the two RNA ends are joined. We report on the presence of 5'-hydroxyl polynucleotide kinase and polynucleotide 2',3'-cyclic phosphate 3'-phosphodiesterase activities in wheat germ extracts. A possible involvement of these enzymes in the ligation process and a potential role of the newly described ligation pathway in RNA processing are discussed.
Dolichyl phosphate-mediated mannosyl transfer through liposomal membranes.
Authors: Haselbeck A , Tanner W
Published: May 2006

A partially purified (up to 1000-fold) mannosyl transferase that catalyzed the reversible reaction GDP-Man + Dol-P in equilibrium Dol-P-Man + GDP was incorporated into liposomes consisting of soybean lecithin and dolichyl phosphate (Dol-P). The enzyme transferred the mannosyl moiety from external GDP-Man to liposome-associated Dol-P. However, when the liposomes were preloaded with GDP, mannosyl residues were also transferred to the inside, giving rise to internal GDP-Man by the reverse reaction. This transfer of an activated sugar through a membrane required the presence of Dol-P and the enzyme in the liposome. Mannosyl residues were not transferred to the inside when the liposomes were preloaded with ADP or GMP. Amphomycin completely inhibited the formation of Dol-P-Man as well as the transfer of mannose into the liposomes. The results are taken as evidence for the open postulated role of dolichols in sugar translocation through membranes. The data are discussed in relation to glycoprotein synthesis at the endoplasmic reticulum.
Growth inhibitory and stimulatory effects of retinoic acid on murine 3T3 cells.
Authors: Schroder E W , Rapaport E , Kabcenell A K , Black P H
Published: May 2006

All-trans-beta-retinoic acid (RA) has both comitogenic and antiproliferative effects on murine Swiss 3T3 cells. Treatment of quiescent 3T3 cells for less than 24 hr with micromolar concentrations of RA potentiates subsequent mitogenic response of those cells to phorbol 12-myristate 13-acetate. Longer exposures of 3T3 cells to RA result in inhibition of DNA replication as measured by [3H]thymidine incorporation and decreased growth rates and saturation densities for cells grown in either 2% or 10% serum. Both the comitogenic and antiproliferative activities of RA for 3T3 cells are RA-dose dependent. RA-induced decreases in the 3T3 cell saturation density are reversible only after resuspension of cells by trypsinization and replating. Treatment of 3T3 cells for 48 hr with RA inhibits the rate of [3H]thymidine incorporation by 35--50%, while autoradiographic data show that labeling indices are similar to control values. Equal percentages of control and 48-hr RA-treated quiescent 3T3 cells respond to subsequent stimulation with 10% serum as determined by autoradiographic and flow cytometric analyses. However, the progression of RA-treated cells through the S phase of the cell cycle is slowed. These data suggest that inhibition of 3T3 cell proliferation by RA is established after a minimum 24-hr treatment and that this inhibition is the result of a decreased rate of DNA replication in S-phase cells.
Amplified DNA sequences in Y1 mouse adrenal tumor cells: association with double minutes and localization to a homogeneously staining chromosomal region.
Authors: George D L , Powers V E
Published: May 2006

DNA associated with double minutes (dm) of the Y1-DM mouse adrenocortical tumor cell line has been cloned in Charon 4A and a preliminary characterization has been made of a recombinant clone, lambda Y1dm-1, isolated from this dm DNA library [George, D. L. & Powers, V. E. (1981) Cell 24, 117--123]. Cloned sequences in lambda Y1dm-1 are amplified in the genome of the Y1-DM cells. They are also amplified in the genome of a related Y1 subline (Y1-HSR), which has a homogeneously staining chromosomal region (HSR). Here we report that the amplified sequences complementary to lambda Y1dm-1 are localized in the HSR, as determined by in situ hybridization. In addition, we found that a population of Y1-DM cells originally containing only dm later consisted of two cell types. Some cells retained dm; others had lost dm but gained a HSR-bearing chromosome morphologically distinct from that in the Y1-HSR cell line. Subclones isolated from this mixed culture have either dm or a HSR, but not both. Southern blotting studies revealed that genomic DNA samples from subclones with a HSR, like subclones with dm, still possess amplified copies of DNA homologous to our recombinant probe. These experiments provide direct evidence that the dm and HSRs in these Y1 cells are structurally related and further support the hypothesis that these chromosomal anomalies result from a process of gene amplification.
Substance P-immunoreactive peripheral branches of sensory neurons innervate guinea pig sympathetic neurons
Authors: Matthews Margaret R. , Cuello A. Claudio
Published: May 2006

The presence of substance P-immunoreactive (SPI) varicose nerve networks and nerve fiber bundles in guinea pig prevertebral sympathetic ganglia has been confirmed by fluorescence immunohistochemistry. No SPI neurons have been found in sympathetic ganglia, including lumbar paravertebral ganglia. Peroxidase-antiperoxidase immunocytochemical methods have shown that SPI nerve terminal varicosities in the inferior mesenteric ganglion (IMG) form morphologically identifiable synapses on dendritic shafts. Cutting the intermesenteric nerve produces no obvious change in SP immunoreactivity in the IMG; cutting the lumbar splanchnic nerves produces nearly total depletion which becomes virtually complete if the two lesions are combined; SP immunoreactivity accumulates in the central ends of the lumbar splanchnic nerves and in the cranial end of the intermesenteric nerve. Cutting hypogastric nerves or colonic branches of the IMG leads to accumulation of SP immunoreactivity in their ganglionic stumps and to build-up (colonic nerve lesion) rather than depletion of SP immunoreactivity in the IMG. Capsaicin treatment leads to total loss of SP immunoreactivity from the prevertebral ganglia and dorsal root ganglia, severe depletion in laminae I and II and dorsolateral fasciculus of the spinal cord, and total loss from perivascular and paravascular networks of the ileum and mesentery, with sparing of the SP immunoreactivity of the enteric nerve plexuses. Capsaicin is thought to deplete sensory neurons selectively. Removal of the spinal cord below T7 without damage to the dorsal root ganglia leaves the intraganglionic SPI nerve networks and bundles intact. We conclude that these are derived from peripheral processes of sensory neurons and we propose that the SPI synapses in the IMG arise from collateral branches of these sensory peripheral processes. This implies a novel role for these processes, in forming intraganglionically in the prevertebral ganglia synapses which may take part in the reflex control of the viscera, independently of the central nervous system.
Oxygen transport parameter in membranes as deduced by saturation recovery measurements of spin-lattice relaxation times of spin labels.
Authors: Kusumi A , Subczynski W K , Hyde J S
Published: May 2006

Spin-lattice relaxation time (T1) measurements of nitroxide radical spin labels in membranes have been made by using the saturation-recovery technique. Stearic acid and sterol-type labels were used as probes of dimyristoylphosphatidylcholine liposomes from 0 degrees C to 36 degrees C. In the absence of oxygen, the range of variation of T1 over all samples and conditions is about a factor of 3. Heisenberg exchange between oxygen and spin labels is an effective T1 mechanism for the spin labels. The full range of variation of T1 in the presence of air is about a factor of 100. It is suggested that the oxygen transport parameter W = T1(-1) (air) - T1(-1) (N2) is a useful new monitor of membrane fluidity that reports on translational diffusion of small molecules. The values of W change at the prephase and main phase transitions and vary in complex ways. Arguments are advanced that the data are indicative of anisotropic translational diffusion of oxygen.
Liver mitochondria contain an ATP-dependent, vanadate-sensitive pathway for the degradation of proteins.
Authors: Desautels M , Goldberg A L
Published: May 2006

A large fraction (30-50%) of the various proteins synthesized within isolated rat liver mitochondria were degraded to amino acids within 60 min after synthesis. Incomplete mitochondrial polypeptides resulting from the incorporation of puromycin were degraded even more extensively (80% per hr). Protein breakdown was measured by the appearance of acid-soluble radioactivity and by the disappearance of labeled polypeptides detected on NaDodSO4/polyacrylamide gel electrophoresis. The amino acids generated by proteolysis were transported rapidly out of the mitochondria and no peptide intermediates accumulated in the organelle. This degradative process did not involve lysosomes or lysosomal enzymes and was markedly stimulated by ATP either generated within the mitochondria or supplied exogenously. An inhibitor of respiration (cyanide) or uncouplers of oxidative phosphorylation (oligomycin, dinitrophenol) reduced proteolysis when mitochondria were provided substrates for ATP generation. When exogenous ATP was provided, these agents did not affect proteolysis, but degradation was then sensitive to atractyloside, an inhibitor of adenine nucleotide transport. Vanadate, an inhibitor of various ATPases, blocked proteolysis even in the presence of ATP and caused a marked stabilization of nearly all polypeptide bands. Thus, mitochondria--like bacteria or the cytosol of animal cells--contain a pathway for complete degradation of proteins which seems to selectively remove polypeptides with abnormal structures. Within this organelle, ATP hydrolysis appears necessary for an initial step in this degradative process.
Broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli host cells.
Authors: Figurski D H , Pohlman R F , Bechhofer D H , Prince A S , Kelton C A
Published: May 2006

Cloning of specific regions of RK2, a broad host range incompatibility group P plasmid, has revealed three genes: kilA, kilB, and kilC. Each of these genes can cause loss of viability of an Escherichia coli host. This effect on the host is normally prevented by the functions of three additional RK2 genes: korA, korB, and korC. Each kor gene is specific for a particular kil gene. The kil and kor genes are located in four distinct regions of the RK2 genome. The three kil genes are not clustered and, with the possible exception of kilA, they are also well separated from their corresponding kor genes. We have found that the korA and korB determinants are not peculiar to RK2 but instead are highly conserved throughout the incompatibility group P plasmids.
5'-Untranslated sequences of two structural genes in the qa gene cluster of Neurospora crassa.
Authors: Alton N K , Buxton F , Patel V , Giles N H , Vapnek D
Published: May 2006

The coding regions of two genes (qa-2 and qa-3) in the qa gene cluster of Neurospora crassa have been localized by nucleotide sequence analysis combined with data on previously determined NH2-terminal amino acid sequences for the proteins that these genes encode. The start point of transcription for each of these genes has been determined by nuclease S1 mapping experiments with poly(A)+RNA isolated from quinic acid-induced cultures of N. crassa. The sequences of approximately 200 nucleotides 5' to the start point of transcription have been compared with each other and with those of other eukaryotes. The results show that neither of these regions for the qa-2 nor the qa-3 genes share any significant homology with sequences apparently conserved in higher eukaryotic promoters (-25 and -70 regions). However, the qa-2 and qa-3 sequences do show homology with each other in these regions. Comparison of the 5'-flanking regions of these Neurospora genes with those of several Saccharomyces cerevisiae genes reveals a number of similarities in the region preceding the translation initiation codons.
Recognition and surprise alter the human visual evoked response.
Authors: Neville H , Snyder E , Woods D , Galambos R
Published: May 2006

Event-related brain potentials (ERPs) to colored slides contained a late positive component that was significantly enhanced when adults recognized the person, place, or painting in the photograph. Additionally, two late components change in amplitude, corresponding to the amount of surprise reported. Because subjects received no instructions to differentiate among the slides, these changes in brain potentials reflect natural classifications made according to their perceptions and evaluations of the pictorial material. This may be a useful paradigm with which to assess perception, memory, and orienting capacities in populations such as infants who cannot follow verbal instructions.
Construction of plasmids for expression of Rous sarcoma virus transforming protein, p60src, in Escherichia coli.
Authors: Gilmer T M , Parsons J T , Erikson R L
Published: May 2006

We have constructed plasmids that direct the synthesis of the Rous sarcoma virus transforming gene (src) product (p60src) in Escherichia coli. A 203-base-pair lac promoter-operator DNA encoding the first eight amino acids of beta-galactosidase was ligated to the 5' end of the src gene from the Prague A strain of Rous sarcoma virus (PrA-RSV) which had been cloned in pBR325. Antiserum, from a tumor-bearing rabbit, directed against pp60src was used to screen bacteria containing the recombinant plasmid for a protein of approximately 60,000 daltons, and several colonies producing a protein immunologically related to pp60src were detected. Partial proteolytic cleavage analysis revealed that the src-related protein produced in bacteria is structurally similar to pp60src immunoprecipitated from PrA-RSV-infected chicken cells. Partially purified src protein from E. coli can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase. Tryptic phosphopeptide analysis demonstrated that the catalytic subunit phosphorylated a serine-containing tryptic peptide in the bacterial src protein that comigrated with the phosphoserine-containing tryptic peptide of pp60src immunoprecipitated from 32P-labeled PrA-RSV-infected chicken cells.
Intermembrane contact affects calcium binding to phospholipid vesicles.
Authors: Ekerdt R , Papahadjopoulos D
Published: May 2006

Binding of Ca2+ to liposomes composed of phosphatidylserine (PtdSer) was analyzed by potentiometric titrations. Ca2+ binding to large unilamellar PtdSer vesicles was saturable at a stoichiometry of 1:2 (Ca2+/PtdSer). At approximately 6 X 10(-4) M [Ca2+]free, the binding curve exhibited a discontinuity that can be attributed to the formation of a Ca2+/PtdSer complex with a higher affinity. When both Ca2+ and Mg2+ are present, depending on the relative concentrations, Mg2+ can either complete or can enhance Ca2+ binding. Concomitant to the enhanced binding, the vesicle suspension was found to aggregate, suggesting that close contact of membranes is a prerequisite for the abrupt change in affinity. This concept was tested by binding studies with liposomes of mixed composition. It was found that the incorporation of 50 mol% phosphatidylethanolamine (PtdEtn) into PtdSer liposomes produced a similar binding pattern to that of pure PtdSer with a saturable stoichiometry of 1:2 (Ca2+/PtdSer). However, incorporation of 50 mol% phosphatidylcholine (PtdCho) completely abolished the discontinuous shift in affinity and apparent saturation was reached at a stoichiometry of 1:4 (Ca2+/PtdSer). In addition, Ca2+ binding to PtdSer liposomes with 10 mol% galactosylcerebroside was not altered when compared to pure PtdSer, whereas 10 mol% of the glycolipid GL-4 abolished the increased binding. The results are closely correlated with recent findings on the role of the membrane composition in Ca2+-induced fusion of liposomes and argue in favor of a specific Ca2+/PtdSer complex (with 1:2 stoichiometry) forming only at points of close contact between membranes and serving as the trigger for membrane fusion.
Identification of the stereospecific hexose transporter from starved and fed chicken embryo fibroblasts.
Authors: Pessin J E , Tillotson L G , Yamada K , Gitomer W , Carter-Su C , Mora R , Isselbacher K J , Czech M P
Published: May 2006

When deprived of D-glucose for 24 hr, chicken embryo fibroblasts exhibit a marked increase in hexose transport activity compared with that of control cells. Scatchard analysis of [3H]cytochalasin B binding to starved cell plasma membranes (46 pmol/mg) indicated a six-fold increase compared with fed cell plasma membranes (7.5 pmol/mg). Irradiation of starved cell plasma membranes with high-intensity UV light in the presence of 0.5 microM [3H]cytochalasin B resulted in covalent labeling of polypeptides of Mr 52,000 and 46,000. In fed cell plasma membranes irradiated under the same conditions, both polypeptides were labeled but at greatly decreased levels. In fact, labeling of the Mr 52,000 polypeptide was barely detectable. The amount of D-glucose-sensitive [3H]cytochalasin B covalent insertion into these membrane components was increased 11 +/- 2 (n = 4)-fold in starved versus fed cell plasma membranes. Photoaffinity labeling of both polypeptides in starved cell plasma membranes was inhibited by D-glucose, 3-O-methylglucose, 2-deoxyglucose, cytochalasin B, and cytochalasin A but not by D-sorbitol, L-glucose, or cytochalasin E. Half-maximal inhibition of labeling of the Mr 52,000 polypeptide occurred at 8 mM D-glucose whereas, for the Mr 46,000 polypeptide, half-maximal inhibition occurred at 40 mM D-glucose. It is concluded that (i) two hexose transport proteins, one of Mr 46,000 and one of Mr 52,000, have been identified in chicken embryo fibroblasts and (ii) the increased affinity labeling of these transporter components after cell starvation may reflect increased numbers of transporters in the plasma membrane.