bluuebunny/crossref_metadata_2025 huggingface dataset:
Viral src gene products are related to the catalytic chain of mammalian cAMP-dependent protein kinase.
Authors: Barker W C , Dayhoff M O
Published: May 2006

The transforming protein sequences translated from the Rous avian and Moloney murine sarcoma virus src genes are shown to be related to the catalytic chain of bovine cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). The avian transforming protein, also a protein kinase, shows greatest homology with the bovine protein kinase in the carboxyl-terminal half, where the protein kinase activity is localized. Moreover, lysine occurs in the inferred transforming protein sequences at the position homologous with the proposed ATP-binding lysine of the bovine protein kinase. This relationship is consistent with the hypothesis that the src genes originated in the host genomes, in which they are members of a superfamily of distantly related protein kinases that are normal constituents of mammalian cells. In the host, these sequences are much more highly conserved than in the viruses.
Oncogenes in human tumor cell lines: molecular cloning of a transforming gene from human bladder carcinoma cells.
Authors: Pulciani S , Santos E , Lauver A V , Long L K , Robbins K C , Barbacid M
Published: May 2006

The presence of dominant transforming genes in human tumor cell lines has been investigated. High molecular weight DNAs isolated from cell lines established from carcinomas and sarcomas of various organs as well as from a glioblastoma and two melanomas were utilized to transfect NIH/3T3 mouse fibroblasts. The DNAs of T24 and A2182, two cell lines derived from a bladder and a lung carcinoma, respectively, and of HT-1080, a cell line established from a fibrosarcoma, were able to transform recipient NIH/3T3 cells. First-cycle transformants exhibited anchorage-independent growth and were tumorigenic in athymic and immunocompetent mice. Moreover, they contained human DNA sequences and were able to transmit their malignant phenotype in additional cycles of transfection. Southern blot analysis of T24-derived transformants showed that a single fragment of human DNA specifically cosegregated with the malignant phenotype, suggesting that it contained the T24 oncogene. Therefore, these human sequences were molecularly cloned with lambda Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated lambda T24-15A, was shown to contain a 15-kilobase-pair EcoRI insert of human cellular DNA. lambda T24-15A DNA (either intact or EcoRI digested) transformed NIH/3T3 fibroblasts with a specific activity of 20,000 focus-forming units per pmol of cloned DNA. Our results indicate that we have molecularly cloned a biologically active oncogene present in T24 human bladder carcinoma cells.
Receptor-mediated endocytosis of diphtheria toxin by cells in culture.
Authors: Keen J H , Maxfield F R , Hardegree M C , Habig W H
Published: May 2006

The binding and uptake of fluorescently labeled diphtheria toxin by cells in culture has been examined by using epifluorescence video intensification microscopy. Rhodamine-labeled diphtheria toxin retained significant toxicity on bioassay and in cell culture and was tested for uptake by human WI-38 and mouse 3T3 fibroblasts grown in culture. When added to cells at 37 degrees C, toxin was observed to become concentrated and internalized in discrete vesicles in both cell lines. The appearance of fluorescent clusters could be prevented by addition of excess unlabeled diphtheria toxin to the medium or by addition of ATP (which has been shown to block toxin binding to cells), indicating that the rhodamine-labeled toxin was binding to diphtheria toxin-specific cell surface binding sites. When the simultaneous uptake of rhodamine-labeled diphtheria toxin and fluorescein-labeled alpha 2-macroglobulin was monitored, the two proteins appeared in the same clusters indicating that the toxin undergoes receptor-mediated endocytosis. Despite the difference in susceptibility to diphtheria toxin of cells derived from sensitive (human) and resistant (mouse) tissues, the behavior of the rhodamine-labeled derivative in both cell lines was indistinguishable in terms of toxin required for formation of clusters or inhibition by unlabeled toxin or by ATP. These results demonstrate that diphtheria toxin-specific cell surface binding sites occur on both insensitive and sensitive cells and suggest that toxin is processed similarly by both cell types during its initial cell surface binding and internalization by this pathway. The possible involvement of this uptake system in the mechanism of action of diphtheria toxin in cells is discussed.
Rapid thymomas induced by Abelson murine leukemia virus.
Authors: Cook W D
Published: May 2006

Abelson murine leukemia virus (A-MuLV) is derived from the thymotropic Moloney leukemia virus. However, injection of mice with A-MuLV by conventional routes results in rapidly arising peripheral and bone marrow lymphomas, not thymomas or T cell tumors. In this study thymomas have been induced by intrathymic injection of A-MuLV into BALB/c and C57BL/Ka mice. In both strains thymomas arose with short latent periods, comparable with the latencies of nonthymic tumors induced by intraperitoneal injection of A-MuLV and significantly shorter than those of thymomas induced by intrathymic injection of Moloney leukemia virus. Cells of the BALB/c thymomas were predominantly Thy-1-; those of C57BL/Ka thymomas were predominantly Thy-1+. Tissue culture lines were established and cloned. Some of these expressed low amounts of Thy-1 and one also expressed Lyt-1. Virus from cloned lines transformed 3T3 cells in vitro and induced Abelson disease in vivo when injected intraperitoneally into neonates. The A-MuLV p120 protein has been precipitated from metabolically labeled cell lysates of one cloned Thy-1+ line. These results show that A-MuLV can transform cells in the T lymphocyte lineage.
Association of a Mr 50,000 cap-binding protein with the cytoskeleton in baby hamster kidney cells.
Authors: Zumbé A , Stähli C , Trachsel H
Published: May 2006

A monoclonal antibody directed against eukaryotic mRNA 5'-cap-binding protein (anti-CBP antibody) was used to localize cap-binding protein (CBP) in BHK-21 baby hamster kidney cells by immunofluorescence microscopy. It was found that the antibody reacts with a fibrous network extending through the cytoplasm in a radial arrangement. The network behaves like intermediate filaments in colchicine-treated cells, suggesting a direct or indirect linkage of CBP with intermediate filaments. The association of CBP with a cytoskeletal element was further confirmed by isolation of proteins from Triton X-100-extracted cells and identification of CBP in the cytoskeletal fraction with anti-CBP antibody. The major polypeptide reacting with anti-CBP antibody is a Mr 50,000 component. Tryptic peptide mapping showed that this polypeptide is related to a Mr 24,000 polypeptide identified as CBP in earlier experiments [Sonenberg, N., Morgan, M. A., Testa, D., Colonna, R. J. & Shatkin, A. J. (1978) Proc. Natl. Acad. Sci. USA 75, 4843-4847].
Oligomeric structure of muscarinic receptors is shown by photoaffinity labeling: subunit assembly may explain high- and low-affinity agonist states.
Authors: Avissar S , Amitai G , Sokolovsky M
Published: May 2006

The potent muscarinic photoaffinity reagent N-methyl-4-piperidyl p-azidobenzilate (azido-4NMPB) was used to covalently label specific muscarinic binding sites in various brain regions and in the heart. In the cortex and hippocampus, a single specifically labeled protein with an apparent molecular mass of 86,000 daltons was detected by gel electrophoresis. In the medulla pons, cerebellum, and cardiac atria, there was a 160,000-dalton band in addition to the 86,000-dalton polypeptide. Under certain conditions, alkali or hydroxylamine treatment dissociated both macromolecules into a single 40,000-dalton polypeptide. These results suggest that the muscarinic receptor exists in oligomeric forms and that a dimer and tetramer of a basic 40,000-dalton peptide may exist as interconvertible species. We propose a model to explain the biological architecture of the muscarinic receptors and suggest a possible correlation between the azido-4NMPB-labeled polypeptides and the two states of the receptor observed in agonist binding experiments.
Anti-HLA antibodies of predetermined specificity: a chemically synthesized peptide induces antibodies specific for HLA-A,B heavy chain.
Authors: Church W R , Walker L E , Houghten R A , Reisfeld R A
Published: May 2006

A chemically synthesized dodecapeptide (Asp-Ala-Ala-Ser-Pro-Arg-Glu-Glu-Pro-Arg-Ala-Pro), consisting of residues 39 through 50 of the HLA-B7 heavy chain sequence and containing a highly hydrophilic hexapeptide segment (Pro-Arg-Glu-Glu-Pro-Arg), induced antibodies that bound specifically to free HLA-A, and HLA-B heavy chains and to the soluble dodecapeptide. Although these antibodies reacted with free HLA-A,B heavy chains in blots on nitrocellulose, they failed to immunoprecipitate the HLA-A,B-beta 2-microglobulin antigen complex solubilized from human lymphoblastoid cells by nonionic detergents. These observations suggest that the physical characteristics of this particular soluble hydrophilic dodecapeptide may closely resemble nonallotypic determinants expressed by free HLA-A,B heavy chains and that such antigenic sites may be modified by conformational changes in the native HLA-A,B heavy chain-beta 2-microglobulin complex.
Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library.
Authors: Helfman D M , Feramisco J R , Fiddes J C , Thomas G P , Hughes S H
Published: May 2006

A cDNA library of approximately equal to 9,000 members has been prepared from chicken smooth muscle mRNA by using the plasmid expression vector pUC8. Addition of Sal I and EcoRI linkers at different stages during the preparation of the cDNA resulted in a population of molecules, most of which had EcoRI linkers at the end of the cDNA that corresponded to the 5' end of the template mRNA and Sal I linkers at the end that corresponded to the 3' end of the mRNA. The cDNA molecules then were inserted into a EcoRI-Sal I-cut plasmid vector, pUC8, that contains the transcriptional and translational start sequences from the lacZ gene upstream of the EcoRI site. The sequential addition of the linkers to the cDNA ensured that most of the cDNAs were inserted into pUC8 in the proper orientation for expression. The colonies were replica plated onto nitrocellulose filters and lysed in situ with chloroform vapor. The library was screened for colonies producing products immunologically related to chicken tropomyosin by incubating the filters first with a rabbit antitropomyosin antibody and second with a 125I-labeled goat anti-rabbit IgG. Two colonies were detected that reacted specifically with the antisera. Plasmids from both clones have been partially subjected to sequence analysis; both plasmids contain cDNAs that encode tropomyosin. These protocols are potentially useful for the identification of cDNA clones for genes expressed at low levels from large cDNA expression libraries.
M13 procoat and a pre-immunoglobulin share processing specificity but use different membrane receptor mechanisms.
Authors: Watts C , Wickner W , Zimmermann R
Published: May 2006

Bacteriophage M13 procoat is accurately processed to transmembrane coat protein by salt-washed or N-ethylmaleimide-treated rough microsomes from dog pancreas. These treatments inhibit the processing of eukaryotic secreted protein precursors. M13 procoat can assemble into dog pancreas microsomes post-translationally. Thus, the microsomal proteins needed for assembly may be determined by the nature of the precursor protein itself. These results, and our finding that the mouse IgG kappa chain fragment precursor is processed by Escherichia coli leader peptidase, also suggest that the cleavage specificity of leader (signal) peptidases and the properties of preproteins that render them suitable for cleavage have been conserved during evolution.
Perturbation of hydrogen bonds in the adenine . thymine base pair by Na+: A quantum chemical study.
Authors: Hobza P , Sandorfy C
Published: May 2006

Ab initio self-consistent field calculations with 4-31G and STO-3G basis sets show that Na+ are able to damage the H bonds in the formamidine . formamide complex, modeling the adenine . thymine base pair (both H bonds in the complex investigated are the same as in adenine . thymine). If a water molecule is placed between the complex and Na+, the H bonds of the model are fully protected. Covalent as well as noncovalent binding of polycyclic aromatic hydrocarbons to nucleic acids should decrease the local concentration of water as a result of the insertion of the hydrophobic hydrocarbon into the DNA and thereby increase the exposure of the DNA H bonds to Na+ attack. Thus, Na+ reaching the base pair could provoke or interfere with cell division.
In vitro synthesis of the tryptophan operon leader peptides of Escherichia coli, Serratia marcescens, and Salmonella typhimurium.
Authors: Das A , Urbanowski J , Weissbach H , Nestor J , Yanofsky C
Published: May 2006

We used an in vitro DNA-dependent protein-synthesizing system to demonstrate de novo synthesis of the leader peptide specified by the tryptophan (trp) operons of several bacterial species. Peptide synthesis was directed by self-ligated short restriction fragments containing the trp promoter and leader regions. Synthesis of leader peptides was established by demonstrating that they were labeled in vitro only by those amino acids predicted to be present in the peptides. Leader peptide synthesis was abolished by the addition of the Escherichia coli trp repressor. The E. coli trp leader peptide was found to be extremely labile in vitro; it had a half-life of 3-4 min. In a highly purified DNA-dependent peptide-synthesizing system, synthesis of the di- and tripeptides predicted from the Salmonella typhimurium trp operon leader sequence, fMet-Ala and fMet-Ala-Ala, also was observed. Using this dipeptide synthesis system, we demonstrated that translation initiation at the ribosome binding site used for trp leader peptide synthesis was reduced 10-fold when the transcript contained a segment complementary to the ribosome binding site.
Rearrangement and mutagenesis of a shuttle vector plasmid after passage in mammalian cells.
Authors: Razzaque A , Mizusawa H , Seidman M M
Published: May 2006

A shuttle vector plasmid that contains sequences from simian virus 40, pBR322, and a bacterial marker gene, galactokinase, has been constructed. After replication in cells permissive for virus progeny, plasmid DNA was introduced into a galactokinase-deficient bacterial strain and the relative frequency of colonies with plasmids but without galactokinase activity was determined. This assay showed that 1% of the plasmids were defective after passage in the mammalian cells. Individual mutant plasmids were examined and found to contain deletions, duplications, point mutations, and insertions of cell DNA.
Specific labeling of mouse brain membrane phospholipids with [20-3H]phorbol 12-p-azidobenzoate 13-benzoate, a photolabile phorbol ester.
Authors: Delclos K B , Yeh E , Blumberg P M
Published: May 2006

As part of our effort to characterize receptors for the phorbol ester tumor promoters, a phorbol ester photoaffinity probe, [20-3H]phorbol 12-p-azidobenzoate 13-benzoate (PaBzBz), was synthesized. In the dark, PaBzBz bound reversibly to brain particulate fractions with a dissociation constant (Kd) of 0.81 +/- 0.09 x 10(-9) M. Specific binding of PaBzBz, at a concentration equal to its Kd, represented 85% of the total bound. At saturation, 24 +/- 5 pmol of PaBzBz were bound per mg of brain protein, a level similar to that observed with [20-3H]phorbol 12,13-dibutyrate. Under the conditions used (concentrations greater than the Kd for PaBzBz), irradiation caused 45% of the PaBzBz binding to become irreversible. Most of the binding (approximately equal to 60%), including most of the specific irreversible binding, was to phospholipid rather than to protein. Based on susceptibility to enzymatic digestion and on chromatographic mobility, the specifically labeled phospholipids were identified as phosphatidylserine, phosphatidylethanolamine, and phosphatidylethanolamine plasmalogen. Although the PaBzBz specifically labeled lipid, labeling was blocked by pretreatment of membranes at 100 degrees C for 5 min or by papain digestion. Therefore, it seems likely that the identified lipids are specifically associated with a protein receptor and are preferentially labeled either because of the location or reactivity of the nitrene generated on the photoaffinity probe.
Demonstration of a neurotrophic factor for the maintenance of acetylcholinesterase and butyrylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.
Authors: Koelle G B , Ruch G A
Published: May 2006

Under sodium pentobarbital anesthesia, the superior cervical ganglia of cats were preganglionically denervated bilaterally. The following day cats were reanesthetized, the external carotid and lingual arteries were ligated bilaterally, and the right common carotid artery was infused for 24 hr with an extract prepared from cat brain, spinal cord, and sciatic nerves, with and without the incorporation of aprotinin, an inhibitor of proteases. They were sacrificed 48 hr after denervation, and the acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) and butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) contents of the superior cervical ganglia were compared with those of similarly denervated control ganglia. Both types of extract produced a significant reduction in the loss of both enzymes from the superior cervical ganglia, as did infusions of aprotinin alone. These findings demonstrate the presence of an endogenous neurotrophic factor for the maintenance of ganglionic acetylcholinesterase and butyrylcholinesterase. Its possible mechanisms of action, and those of aprotinin, are discussed.
Self-similar self-avoiding structures: Models for polymers
Authors: Klein D. J. , Seitz W. A.
Published: May 2006

The hierarchy of
m,n
-self-similar self-avoiding structures on a lattice is introduced as a model for linear and branched polymers. The self-similarity condition permits exact solutions that use a generating-function renormalization procedure with full accounting for volume exclusion. Illustrative results are given for the 1,2-linear-chain model and for the 1,2-branched-structure model on a honeycomb lattice. Exponents for the dependence of polymer size on monomer number are obtained for these models, with that for branched structures being independent of the (nonzero) fraction of branch points. Exponents also are found involving branching probability and branching activity; the fact that those quantities are found to be distinct even in the dilute branching limit is a manifestation of the long-range nature of the excluded volume interaction.
Rational approximations to linear forms of exponentials and binomials
Authors: Chudnovsky G. V.
Published: May 2006

Mahler proved the following quantitative result supplementing the Lindemann-Weierstrass theorem: ǀΣ

i
=0

n

C
i

e

ri

ǀ >

H
-n-ε

for any distinct rational numbers
r
0
,
r
1
,...,

r
n

and rational integers
C
0
,
C
1
,...,

C
n

with
H
= max

0≤
i
≤
n

ǀ

C
i

ǀ. We improve Mahler's estimate by replacing exponentials

e
ri

by linearly independent linear forms

L
i

= Σ

L
ij
e
sij

with rational

L
ij
,s
ij

i
= 0,1,...,
n
. Similar results are obtained for binomials (
a/b
)

ri

or Σ

L
ij

(
a/b
)

sij

with integers
a,b
and logǀ
b
ǀ/logǀ
a
ǀ > 1 - ε. The simplest examples of new numbers with the irrationality exponent “2 + ε” are sinh 1 or sin 1.
Biological expression of an Escherichia coli consensus sequence promoter and some mutant derivatives.
Authors: Rossi J J , Soberon X , Marumoto Y , McMahon J , Itakura K
Published: May 2006

A prokaryotic consensus sequence promoter has been chemically synthesized and cloned in bacterial plasmid vectors. This designed sequence is biologically active and promotes efficient expression of the genes to which it is fused. It is an unusually strong promoter in vitro, capable of specifying multiple rounds of transcription even when there is a large molar excess of heparin present prior to the addition of RNA polymerase. These properties make this a useful sequence for the in vitro production of RNAs. A 2-base-pair spacer mutant and a -35 region transversion mutant have been created in vitro in the synthetic promoter by synthetic-DNA-mediated, site-specific mutagenesis. The spacer mutant has a marginal in vivo effect on promoter strength but virtually abolishes the in vitro heparin resistance. The -35 region transversion changes a highly conserved nucleotide into the statistically least preferred base. This mutation has no marked effect on in vivo or in vitro promoter strength.
cDNA cloning and transcriptional mapping of nine polyadenylylated RNAs encoded by the genome of human respiratory syncytial virus.
Authors: Collins P L , Wertz G W
Published: May 2006

We have isolated cDNA clones representing nine unique poly(A)+ RNAs transcribed from the genome of human respiratory syncytial virus, a paramyxovirus. A cDNA library was constructed by using poly(A)+ RNA from virus-infected cells as template and the Escherichia coli plasmid pBR322 as vector. Viral cDNA clones were identified by hybridization with cDNA probes prepared from viral genomic RNA. The viral clones were grouped into nine different families by hybridization with individual size-selected reverse transcripts representing the major classes of poly(A)+ RNA from virus-infected cells. The largest clone from each family was selected for analysis. These nine clones, molecular sizes ranging from 520 to 2,600 base pairs, were shown to be unrelated on the basis of reciprocal hybridization using dot-blots. These cDNA clones were then used as hybridization probes to analyze intracellular viral RNAs that had been separated by gel electrophoresis and transferred to diazobenzyloxymethyl-paper. All nine clones hybridized with intracellular viral genomic RNA, confirmation of virus specificity. Nine unique intracellular viral poly(A)+ RNAs were identified [molecular sizes ranging from 720 to 7,500 nucleotides, including poly(A)]. Comparison of the sizes of these major RNAs and the cDNA clones indicated that a number of the clones represented nearly complete copies of the corresponding RNAs. Several other intracellular viral poly(A)+ RNAs appeared to be polycistronic by the criteria of molecular weights and homologies to various combinations of cDNA clones. The sizes and sequence contents of these polycistronic RNAs were used to prepare a transcriptional map whose significance is discussed.
Localization of DNA sequences promoting RNA polymerase I activity in Drosophila.
Authors: Kohorn B D , Rae P M
Published: May 2006

We used BAL-31 nuclease to delete sequences that surround the transcription initiation site of Drosophila ribosomal DNA. A series of deletions was used as templates for in vitro transcription in a Drosophila cell-free system to identify sequences that influence the activity of RNA polymerase I. Sequences that lie upstream of the site of transcription initiation (nucleotide + 1) affect ribosomal RNA synthesis. We show that the major promoter of polymerase I involves the sequence -43 to -27 and that the region between nucleotides -18 and +20 contains sequences capable of sustaining a low level of accurate transcription.
Acidification of macrophage and fibroblast endocytic vesicles in vitro.
Authors: Galloway C J , Dean G E , Marsh M , Rudnick G , Mellman I
Published: May 2006

We have used the pH-dependent fluorochrome fluorescein-dextran (FD) to study the acidification of prelysosomal vacuoles (endosomes) and lysosomes isolated from cultured macrophages and fibroblasts. FD was internalized by pinocytosis under conditions that allowed its selective localization in endosomes (1- to 5-min pulse) or in lysosomes (5-min pulse, 30-min chase). Fibroblasts were also exposed to FD at 20 degrees C, at which temperature endosome-lysosome fusion is inhibited. Cells were homogenized and labeled organelles were separated by centrifugation in Percoll density gradients. The addition of ATP rapidly decreased the internal pH of both endosomes and lysosomes, as indicated by a decrease in fluorescence intensity. The pH gradient was dissipated by H+ ionophores and ammonium chloride. Acidification was not affected by inhibitors of the mitochondrial F1, F0-ATPase or the Na+, K, K+-ATPase and did not require permeant anions, Na+, or K+. Of the inhibitors tested, only N-ethylmaleimide prevented the ATP-dependent acidification of both compartments. These findings provide direct support for the existence of an acidic prelysosomal compartment that may be acidified via the same type of H+ pump believed to operate in lysosomes and secretory granules.
Development of basal and induced aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity in the chicken embryo in ovo.
Authors: Hamilton J W , Denison M S , Bloom S E
Published: May 2006

The development of the hepatic microsomal mixed-function oxidase system was studied to determine the basal level of embryonic enzyme activity and the inducibility of this system throughout growth and differentiation. Chicken embryo livers were assayed for basal and inducible hepatic aryl hydrocarbon hydroxylase (AHHase; designated elsewhere as AHH) activity from the first appearance of the liver as a discrete organ at 5 days of incubation (DI) through day 10 after hatching. In addition, whole-embryo and viscera preparations were assayed at 3 and 4 DI. Basal AHHase activity was equal to or greater than adult levels from 3 DI through hatching in all preparations (approximately 0.3-0.5 nmol/min per mg). A 3-fold increase in basal activity above adult values occurred at hatching. The onset of inducibility in chicken embryo liver between 5 and 6 DI was concomitant with hepatocyte differentiation. A developmental profile of 24-hr 3,4,3', 4'-tetrachlorobiphenyl-induced AHHase activity showed 15- to 30-fold induction over controls from 7 DI through day 10 after hatching, with a maximum of 15 nmol/min per mg at 14 DI and day 1 after hatching, a specific activity greater than 50% greater than maximal induction in the adult. Embryonic AHHase activity was also induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, beta-naphthoflavone, and sodium phenobarbital. Induction kinetics throughout embryonic development were similar to those reported for the adult chicken and other animals. These findings demonstrate development of a mixed-function oxidase system in very early embryogenesis and then in the liver as it differentiates. Liver AHHase activity is inducible throughout development and perinatally but such activity is under strict developmental regulation. The chicken embryo has adult levels of AHHase activity which would be sufficient to achieve metabolic activation of promutagens/carcinogens before and after hepatocyte differentiation.
Fluorescence-activated cell sorting of mouse-human hybrid cells aids in locating the gene for the Leu 7 (HNK-1) antigen to human chromosome 11.
Authors: Schröder J , Nikinmaa B , Kavathas P , Herzenberg L A
Published: May 2006

Leu 7 (HNK-1) is a membrane antigen expressed on human natural killer cells and some other lymphoid cells. Starting with two clones of mouse-human hybrid lymphoid cells that had 1.6% and 35% Leu 7-positive cells, respectively, we viably sorted Leu 7-positive and -negative cells using a fluorescence-activated cell sorter (FACS). Short-term progeny of the sorted cells were then karyotyped. Chromosome 11 was the only human chromosome that was absent from the Leu 7-negative population and present in nearly all of the progeny of the Leu 7-positive selected cells. Thus, we assigned the Leu 7 gene to chromosome 11.
Rates of receptor-dependent and -independent low density lipoprotein uptake in the hamster.
Authors: Spady D K , Bilheimer D W , Dietschy J M
Published: May 2006

By using a constant infusion technique in the hamster, rates of uptake of [14C]sucrose-labeled hamster low density lipoprotein (hamLDL) and methylated hamster LDL (MehamLDL) were directly measured in 15 tissues. From these measurements the magnitude of LDL receptor-dependent and receptor-independent lipoprotein transport was calculated. The whole-animal clearance of hamLDL equaled 547 microliters/hr per 100 g of body weight. LDL clearance per g of tissue was highest in the liver (114 microliters/hr per g), ovary (43), spleen (36), adrenal gland (29), and intestine (24) and was lowest in fat (0.75), brain (0.35), and muscle (0.26). When adjusted for organ weight, the sum of the absolute clearance rates in all of the tissues examined equaled the rate of whole-animal LDL turnover. Liver accounted for 73%, and the jejunum and ileum combined accounted for 7% of whole-animal clearance. The 12 other tissues each accounted for only a minor portion of LDL clearance. Rates of uptake of Me-hamLDL were much less in many tissues and accounted for only 6-12% of the uptake of LDL in the liver, ovary, adrenal gland, lung, and kidney. However, this receptor-independent uptake was quantitatively more important in the intestine (44%) and spleen (72%) and accounted for essentially all LDL uptake in organs such as muscle, skin, and brain. Thus, in the hamster, most LDL is taken up and degraded by the liver. This uptake process is greater than 90% mediated by the LDL receptor and manifests saturation kinetics. Finally, cholestyramine feeding increases receptor-mediated LDL transport in the liver but in no other tissue studied.
Circular differential scattering can be an important part of the circular dichroism of macromolecules.
Authors: Bustamante C , Tinoco I , Maestre M F
Published: May 2006

Differential scattering of incident left and right circularly polarized light can be an important contribution to the circular dichroism of macromolecules. In principle both differential absorption and differential scattering of circularly polarized light contribute to circular dichroism, but differential scattering is increasingly important for particles whose dimensions are greater than 1/20th the wavelength of light. The scattering contribution is probably not important for unaggregated proteins and nucleic acids in solution. It can be very important for viruses, membranes, and other protein-nucleic acid complexes. Outside the absorption bands of the scattering, chiral particle, only differential scattering contributes to the circular dichroism. The sign and magnitude of the differential scattering is quantitatively related to the relative orientations and the distances between the scattering units of the particle. The interpretation of the circular differential scattering depends on a simple, classical method. Thus, in understanding a measured circular dichroism, it often will be easier to relate the differential scattering to the structure of a particle (such as a virus) than it is to relate the differential absorption to the structure.
Cytosolic activator of adenylate cyclase: Reconstitution, characterization, and mechanism of action.
Authors: Sahyoun N , LeVine H , Stenbuck P , Cuatrecasas P
Published: May 2006

Rat liver and isolated hepatocytes contain high levels of a soluble adenylate cyclase stimulator, whereas rat erythrocytes lack this activity. Accordingly, a reconstitution system was developed with adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from erythrocyte ghosts and the soluble activator from liver cytosol. Pretreatment of erythrocyte ghosts with the cytosolic factor resulted in a 5- to 15-fold activation of adenylate cyclase in the presence or absence of NaF, 5'-guanylyl imidodiphosphate, or isoproterenol and GTP. The sequence of addition of the cytosolic component and the other activators was critical in determining the maximal activity of the enzyme. The cytosolic factor appears to be a heat-labile Mr 105,000 protein, which activates adenylate cyclase in a saturable reaction involving binding of the protein to the erythrocyte ghosts. This molecular interaction was accompanied by stabilization of a labile thiol group that was essential for catalytic activity. The cytosolic component also unmasks latent adenylate cyclase activity in human erythrocyte ghosts and in cytoskeletal preparations from rat erythrocyte ghosts. These observations suggest that the cytosolic activator may also occur as a native, peripheral membrane component of adenylate cyclase systems and may be required for the expression and stabilization of catalytic activity.
A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions.
Authors: Dürst M , Gissmann L , Ikenberg H , zur Hausen H
Published: May 2006

DNA from one biopsy sample of invasive cancer of the cervix contained sequences hybridizing with human papillomavirus (HPV) type 11 DNA only under nonstringent conditions. This DNA was molecularly cloned in lambda phage. Under stringent conditions of hybridization it cross-hybridized to a minor extent (less than 0.1%) with HPV types 10, 14, and 15 and showed no homology with DNA of other human HPV types. We therefore propose to designate it tentatively as HPV 16. HPV 16 DNA was used as a probe to test additional cancer biopsy samples from cervical, vulval, and penile cancer, as well as benign genital warts (condylomata acuminata) and cervical dysplasias for the presence of homologous sequences. In 61.1% (11/18) of cervical cancer samples from German patients sequences were found hybridizing with HPV 16 DNA under conditions of high stringency. In contrast, only 34.8% (8/23) of cancer biopsy samples from Kenya and Brazil revealed this DNA. Vulval and penile cancer biopsy samples hybridized to 28.6% (2/7) or 25% (1/4), respectively. Only 2 out of 33 condylomata acuminata contained HPV 16 DNA. Both positive tumors harbored in addition HPV 6 or HPV 11 DNA. The data thus indicate that HPV 16 DNA prevails in malignant tumors, rendering an accidental contamination with papillomavirus DNA from adjacent papillomas rather unlikely. The rare presence in benign genital papillomas in addition to common genital papillomaviruses suggests a dependence of HPV 16 replication on helper virus.
Isolation and characterization of RNA ligase from wheat germ.
Authors: Furneaux H , Pick L , Hurwitz J
Published: May 2006

A RNA ligase from wheat germ has been extensively purified. In the presence of ATP these enzyme preparations catalyze the covalent linkage of 5'-phosphate and 2',3'-cyclic phosphate termini of RNA chains. Concomitant with the formation of a 3',5'-phosphodiester linkage, the 2',3'-cyclic phosphate is converted to a 2'-phosphate ester, in accord with the findings of Konarska et al. [Konarska, M., Filipowicz, W. & Gross, H. J. (1982) Proc. Natl. Acad. Sci. USA 79, 1474-1478]. The action of the purified enzyme is totally dependent on ATP and on RNA substrates containing a 5'-phosphate terminus at one end and either a 2',3'-cyclic phosphate or a 2'-phosphate terminus at the other end. In the latter case, the reaction is about 30% as active as with the cyclic derivative. In contrast, RNA chains containing 3'-phosphate ends are less than 5% as active as those with the cyclic ends. Purified preparations of RNA ligase have an intrinsic ability to hydrolyze 2',3'-cyclic phosphate termini to 2'-phosphate termini. This reaction is readily detectable in the absence of ATP.
Gene structure of a phenobarbital-inducible cytochrome P-450 in rat liver.
Authors: Mizukami Y , Sogawa K , Suwa Y , Muramatsu M , Fujii-Kuriyama Y
Published: May 2006

The gene structure of a phenobarbital-inducible rat liver cytochrome P-50 was elucidated by sequence analysis of two isolated genomic clones. The total length of the gene was approximately 14 kilobases and was separated into nine exons by eight intervening sequences. Nucleotide sequences of all exon/intron boundaries follow the G-T/A-G rule. A putative transcription initiation site was assigned to an A, 30 base pairs upstream from the initiation codon by S1 nuclease protection mapping. A possible "TATA" equivalent sequence, C-A-T-A-A-A, was found 27 base pairs further upstream from this initiation site. A poly(A) attachment site was determined to be 386 or 387 base pairs downstream from the termination codon by comparison with the cDNA sequence. Detailed comparison with two cDNA sequences determined previously showed the coding nucleotide sequence of the genomic clones to concur with that of the pcP-450pb2 cDNA clone coding for cytochrome P-450e except for three neutral base substitutions. Therefore, we conclude that the gene sequence determined here is for the cytochrome P-450e gene or a similar gene. On the other hand, 40 base substitutions were found in about 1,900 base pairs compared between the sequences of the genomic clone and the other cDNA clones (pcP-450pbl and 4) coding for cytochrome P-450b, and 15 of them result in 14 amino acid replacements in the total 491 amino acid residues. These base substitutions occur in relatively limited regions of the sequences. Most of them are found in exons 6, 7, 8, and 9; most frequently in exon 7.
Kpn I family of long interspersed repeated DNA sequences in primates: polymorphism of family members and evidence for transcription.
Authors: Lerman M I , Thayer R E , Singer M F
Published: May 2006

An approximately equal to 2-kilobase-pair-long member (Kpn I-LS1) of the African green monkey Kpn I family of repeated sequences has been cloned, subjected to sequence analysis, and compared to other family members which are over 6 kilobase pairs (Kpn I-alpha 7) and 829 base pairs (Kpn I-RET) long. Both Kpn I-LS1 and Kpn I-RET lack sequences found at the ends of the longer family member and their structures resemble those of processed genes. Kpm I-LS1 sequences are colinear with part of the long family member, Kpn I-alpha 7. However, although all sequences in Kpn I-RET are represented in Kpn I-LS1, the two are not colinear; Kpn I-RET is missing 731 base pairs found in Kpn I-LS1 and one segment flanking the deletion is inverted. The results demonstrate that Kpn I family members are not only of different lengths but may also contain scrambled arrangements of common sequences. Sequences in Kpn I-LS1 hybridize to RNA from monkey and human cells, indicating that some family members are transcribed.
Photosynthetic electron transport from water to NADP
+
driven by photosystem II in inside-out chloroplast vesicles
Authors: Albertsson Per-Åke , Hsu Ban-Dar , Tang George M.-S. , Arnon Daniel I.
Published: May 2006

It is now widely held that the light-induced noncyclic (linear) electron transport from water to NADP
+
requires the collaboration in series of the two photosystems that operate in oxygen-evolving cells: photosystem II (PSII) photooxidizes water and transfers electrons to photosystem I (PSI); PSI photoreduces ferredoxin, which in turn reduces NADP
+
(the Z scheme). However, a recently described alternative scheme envisions that PSII drives the noncyclic electron transport from water to ferredoxin and NADP
+
without the collaboration of PSI, whose role is limited to cyclic electron transport [Arnon, D. I., Tsujimoto, H. Y. & Tang, G. M.-S. (1981)
Proc. Natl. Acad. Sci. USA
78, 2942-2946]. Reported here are findings at variance with the Z scheme and consistent with the alternative scheme. Thylakoid membrane vesicles were isolated from spinach chloroplasts by the two-phase aqueous polymer partition method. Vesicles, originating mainly from appressed chloroplast membranes that are greatly enriched in PSII, were turned inside-out with respect to the original sidedness of the membrane. With added plastocyanin, ferredoxin, and ferredoxin-NADP
+
reductase, the inside-out vesicles enriched in PSII gave a significant photoreduction of NADP
+
with water as electron donor, under experimental conditions that appear to exclude the participation of PSI.
alpha 2u-Globulin is present in the rat anterior pituitary.
Authors: Antakly T , Pelletier G , Feigelson P
Published: May 2006

The possibility that the pituitary gland may contain as yet undiscovered regulatory factors is intriguing. Recent reports have suggested the presence, in the anterior pituitary, or a number of proteins of extrapituitary origin. alpha 2u-Globulin, a rat serum and urinary protein, previously shown to be synthesized in the submaxillary gland and in the liver under anterior pituitary control, has now been localized by immunocytochemistry in the cytoplasm of some cells of the anterior pituitary. No alpha 2u-globulin could be detected in either the intermediate or posterior pituitary. The presence of alpha 2u-globulin was confirmed and quantitated by radioimmunoassay. Using RNA blot analysis and cloned alpha 2u-globulin cDNA probes, we could not detect alpha 2u-globulin mRNA sequences in pituitary RNA, indicating that alpha 2u-globulin is not synthesized therein. The presence of alpha 2u-globulin, presumably of circulatory origin, in certain anterior pituitary cells suggests that it may play a role in anterior pituitary function.
Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.
Authors: Nadakavukaren K K , Chen L B , Habliston D L , Griffith O H
Published: May 2006

Pt K2 rat kangaroo epithelial cells and Rat-1 fibroblasts were grown on conductive glass discs, fixed, and permeabilized, and the cytoskeletal elements actin, keratin, and vimentin were visualized by indirect immunofluorescence. After the fluorescence microscopy, the cells were postfixed and dehydrated for photoelectron microscopy. The contrast in these photoelectron micrographs is primarily topographical in origin, and the presence of fluorescent dyes at low density does not contribute significantly to the material contrast. By comparison with fluorescence micrographs obtained on the same individual cells, actin-containing stress fibers, keratin filaments, and vimentin filaments were identified in the photoelectron micrographs. The apparent volume occupied by the cytoskeletal network in the cells as judged from the photoelectron micrographs is much less than it appears to be from the fluorescence micrographs because the higher resolution of photoelectron microscopy shows the fibers closer to their true dimensions. Photoelectron microscopy is a surface technique, and the images highlight the exposed cytoskeletal structures and suppress those extending along the substrate below the nuclei. The results reported here show marked improvement in image quality of photoelectron micrographs and that this technique has the potential of contributing to higher resolution studies of cytoskeletal structures.
Nucleotide sequence of the S-2 mitochondrial DNA from the S cytoplasm of maize
Authors: Levings Charles S. , Sederoff Ronald R.
Published: May 2006

Mitochondria from the S male-sterile cytoplasm (
cms-S
) of maize contain two plasmid-like DNAs, S-1 and S-2, that appear to be prominently involved with the cytoplasmic male sterility trait. The complete nucleotide sequence of the S-2 DNA molecule was determined by the chain termination method. The linear S-2 DNA molecule contains 5,452 base pairs and is terminated by exact 208-base-pair inverted repetitions. Two large open reading frames were identified in the S-2 DNA, suggesting the possibility of protein-encoding genes. The nucleotide sequence of the S-2 termini are discussed with regard to models proposed for the replication of linear DNA molecules.
Purification of binding protein for Tityus gamma toxin identified with the gating component of the voltage-sensitive Na+ channel.
Authors: Norman R I , Schmid A , Lombet A , Barhanin J , Lazdunski M
Published: May 2006

The gating component associated with the voltage-sensitive Na+ channel from electroplax membranes of Electrophorus electricus has been purified by using toxin gamma from the venom of the scorpion Tityus serrulatus serrulatus. The toxin-binding site was efficiently solubilized with Lubrol PX, resulting in an extract of high initial specific activity. Purification was achieved by adsorption of the toxin-binding component to DEAE-Sephadex A-25 followed by desorption at high ionic strength and chromatography on either wheat germ agglutinin-Ultrogel or Sepharose 6B. Maximal final specific activities were at least 42% of the specific activity expected for a pure toxin-binding component. The purified material exhibited a Stokes radius of 85 A, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated a single polypeptide component of Mr 270,000. Furthermore, tetrodotoxin binding activity and Tityus gamma toxin binding activity copurified, suggesting that the selectivity filter and the gating component of the Na+ channel are carried by the same polypeptide chain.
The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli.
Authors: Cox M M
Published: May 2006

The FLP gene of the yeast 2-microns plasmid is involved in a site-specific recombination event that results in the inversion of a set of sequences within the plasmid. This gene has been cloned and expressed in Escherichia coli. Expression of the FLP gene results in efficient recombination within the bacterial cell, which is specific for plasmids containing at least one 2-microns plasmid recombination site. This work demonstrates that (i) FLP protein is actively involved in 2-microns plasmid recombination; (ii) no other factors specific to yeast are required for the reaction; (iii) FLP protein acts efficiently in trans; (iv) FLP protein will promote site-specific insertion and deletion reactions in addition to the inversion reaction; and (v) FLP-promoted recombination is not dependent upon any DNA structural features unique to yeast chromatin.
Molecular cloning of a vitamin D-dependent calcium-binding protein mRNA sequence from chick intestine.
Authors: Hunziker W , Siebert P D , King M W , Stucki P , Dugaiczyk A , Norman A W
Published: May 2006

We have constructed a recombinant cDNA library to facilitate study of the genomic actions of vitamin D3 and its hormonally active metabolite 1,25-dihydroxyvitamin D3 in initiation of the de novo biosynthesis of a 28,000-dalton vitamin D-dependent calcium binding protein (CaBP) present in chick intestine. The recombinant plasmids were prepared by the homopolymeric tailing and hybridization method using as a starting template poly(A)-enriched mRNA obtained from the intestinal mucosa of vitamin D3-replete (+D) chicks. Screening of 9,516 clones in this library was effected by using a comparative in situ colony hybridization technique with two [32P]cDNA probes; these probes were prepared from total poly(A)-RNA from chick intestinal mucosa of vitamin D-deficient (-D) chicks and a poly(A)-RNA specifically enriched for chick intestinal CaBP mRNA by immunoprecipitation of polysomes derived from vitamin D-replete (+D) chicks. We identified 26 clones that consistently displayed a significantly increased hybridization signal when comparing the -D vs. CaBP-enriched probe. Further evaluation of these clones by hybrid-selected translation showed the presence of CaBP-specific sequences. By "RNA gel" analysis of poly(A)-RNA, three independent mRNA species were found to hybridize to a CaBP clone; none of these RNA species were found in -D poly(A)-RNA. With this comparative colony hybridization procedure, we were able to identify CaBP-specific clones corresponding to a mRNA that is 0.1% of the total poly(A)-mRNA. The differential colony hybridization procedure using an enriched vs. a nonenriched probe should be of value in screening for other cDNA clones complementary to rare mRNA species.