bluuebunny/crossref_metadata_2025 huggingface dataset:
Circular dichroic evidence for a conformational change in a cytochrome b--c1 complex by uncoupling agents.
Authors: Reed J , Reed T A , Hess B
Published: May 2006

Circular dichroic spectra of the cytochrome b--c1 complex exhibit bilobe formation typical of exciton splitting in the presence of uncoupler. Bilobe formation occurs if both cytochrome c1 and cytochrome b are fully reduced. The fully oxidized and ascorbate-reduced complexes are not altered dichroically by uncouplers. The exciton splitting induced by uncoupler is consistent with heme--heme interaction: specifically, interaction between the two cytochromes b in the complex.
Molecules in mammalian brain that interact with the colchicine site on tubulin.
Authors: Lockwood A H
Published: May 2006

Colchicine, a plant alkaloid, is a potent inhibitor of mitosis and other physiological processes that involve microtubules. These effects are mediated by the specific binding of colchicine to a high-affinity receptor site on tubulin, the major protein of microtubules. It seemed possible that the colchicine site on tubulin might also be the receptor for endogenous cellular molecules. We now report that mammalian brain does, in fact, contain a class of molecules that interact with the colchicine site on tubulin. Tubulin-agarose affinity chromatography has been used to isolate factors from soluble extracts of bovine brain that interact with tubulin. Certain of these factors inhibit the binding of [3H]colchicine to tubulin. Using the inhibition of colchicine binding as an assay, I have partially resolved at least two species. One is a protein and another may be a heat-stable peptide. In parallel to the action of colchicine, the smaller species inhibits the assembly of isolated microtubules. This class of molecules may include the endogenous ligands for which the colchicine site on tubulin is the receptor. They may have a significant role in the regulation of cellular microtubule function and assembly.
Measurement and purification of human chromosomes by flow cytometry and sorting.
Authors: Carrano A V , Gray J W , Langlois R G , Burkhart-Schultz K J , Van Dilla M A
Published: May 2006

The 24 human chromosome types of normal diploid fibroblast cell strain were classified into 15 groups by high-resolution flow cytometry on the basis of 33258 Hoechst fluorescence. Chromosomes associated with each group were flow sorted onto microscope slides and identified by quinacrine banding analysis. DNA cytophotometry of metaphase chromosomes from the same cell strain supported and extended this identification. Four of the groups purified were due to chromosomes of a single type--namely, chromosomes 5, 6, 13, and 17. Eight additional groups were also separated and found to contain the following chromosomes: 1 and 2; 3 and 4; 7, 8, and X; 9--12; 14 and 15; 16 and 18; 20 and Y; and 19, 21, and 22. The average purity for the 12 sorted fractions was 78%.
DNA fragments of the Mus musculus beta globin haplotypes Hbbs and Hbbd.
Authors: Weaver S , Haigwood N L , Hutchison C A , Edgell M H
Published: May 2006

Two alternative haplotypes at the complex locus controlling hemoglobin beta chain synthesis in Mus musculus were compared at the DNA level. As expected, Hbbd homozygotes--which as adults synthesize two species of beta chain--have two genes for beta globin. Adult mice homozygous for the Hbbs haplotype make only a single type of beta polypeptide, yet they also have two beta globin genes. Apparently the two Hbbs genes encode identical proteins, or one of the two genes is not detectably expressed. The Hbbs and Hbbd haplotypes are thus more similar at the DNA level than studies of their polypeptide products have indicated.
Inactivation of Ca conductance dependent on entry of Ca ions in molluscan neurons.
Authors: Tillotson D
Published: May 2006

Inactivation of the Ca channel of Aplysia neurons was studied in the absence of potassium current in cells that were cesium-loaded with the aid of the ionophore nystatin. Inactivation was substantially decreased by methods that limited Ca entry. Depolarizations commensurate with the equilibrium potential of Ca resulted in minimal inactivation. Replacement of extracellular Ca by Ba also decreased inactivation. It is concluded that inactivation of the Ca channel is a function of the extent of Ca entry rather than membrane potential, thus differing fundamentally from the purely voltage-dependent mechanism for sodium inactivation.
Stability and gap phenomena for Yang-Mills fields
Authors: Bourguignon Jean-Pierre , Lawson H. Blaine , Simons James
Published: May 2006

It is shown that any weakly stable Yang-Mills field of type
SU
2
or
SU
3
on the four-sphere must be self-dual or anti-self-dual. Any Yang-Mills field on

S
n
, n

≥ 5, is unstable. Examples of stable fields on
S
4
and

S
n

/Γ for
n
≥ 5 and Γ ≠ {
e
} are given. It is also shown that, for any Yang-Mills field
R
on
S
4
, the pointwise condition ∥
R
-
∥
2
< 3 (or ∥
R
+
∥
2
< 3) implies that
R
-
= 0 (or respectively that
R
+
= 0). In general, any Yang-Mills field
R
on

S
n
, n

≥ 3, that satisfies the pointwise condition ∥
R
∥
2
< ½(
2
n
) is trivial. If
n
= 3 or 4, the condition ∥
R
∥
2
≤ ½(
2
n
) implies that either
R
is the trivial field or it is the direct sum of a trivial field with a field of tangent spinors carrying the standard connection.
Conformation of an oligopeptide in phospholipid vesicles.
Authors: Wallace B A , Blout E R
Published: May 2006

To demonstrate a method by which the conformation of membrane proteins may be determined spectroscopically in model membranes, we determined the structure of a hydrophobic oligopeptide, t-butyloxycarbonylprolylleucylvalylmethyl ester, in phospholipid vesicles by nuclear magnetic resonance, circular dichroism, and infrared spectroscopy. 13C nuclear magnetic resonance and circular dichroism techniques demonstrated that the conformation of this peptide in linear hydrocarbon solutions was essentially identical to its conformation in lipid vesicles. 1H nuclear magnetic resonance and infrared spectroscopy of the peptide in hydrocarbon solution then provided additional high-resolution information concerning the structure of the peptide as found in the hydrophobic portion of the lipid bilayer. The conformation of this peptide in hydrophobic media a differs from its structure in hydrophilic solvents, not only in bond angles and the proportion of cis/trans isomers about the X-proline bond, but also in its intermolecular associations.
Greatly decreased susceptibility of nonmetabolizing cells towards detergents.
Authors: Komor E , Weber H , Tanner W
Published: May 2006

The addition of different detergents to Chlorella cells that had previously accumulated 6-deoxyglucose causes a rapid release of the hexose analogue into the medium. This effect is independent of the nature of the detergent and is observed only when the cells are in an energized state. Thus, in the presence of the uncoupler p-trifluoromethoxycarbonylcyanide phenylhydrazone or of inhibitors such as N-ethylmaleimide, the cells show a greatly reduced susceptibility towards detergents. Similarly, the detergent-induced loss of accumulated alpha-aminoisobutyric acid from Saccharomyces cerevisiae and of potassium from Escherichia coli is also strongly affected by the energy state of the cells. The differential susceptibility of energized and nonenergized cells was observed at all detergent concentrations tested. Measurements of substrate efflux at different concentrations of Triton indicated that only Triton monomers are responsible for the increase in permeability. The absorption of [14C]Triton X-100 by Chlorella and the binding of detergent to the cells were measured in the presence of metabolic inhibitors. Again, nonenergized cells bound a significantly lower amount of Triton X-100. The amphiphilic antibiotic nystatin produced effects on cell permeability similar to those of detergents, whereas toluene, which is apolar, gave opposite results.
Superinduction of alpha 2u globulin by actinomycin D: evidence for drug-mediated increase in alpha 2u mRNA.
Authors: Chatterjee B , Hopkins J , Dutchak D , Roy A K
Published: May 2006

Actinomycin D, an inhibitor of DNA-dependent RNA synthesis, increased the hepatic concentration of alpha 2u globulin, an androgen-inducible protein in the rat. Spayed female rats with a marginally induced state of alpha 2u synthesis showed an approximately 5-fold increase in hepatic alpha 2u globulin within 3-6 hr after treatment with actinomycin D. Initial treatment of these animals with 5 alpha-dihydrotestosterone, followed by actinomycin D, resulted within 2-3 hr in a more than 2-fold increase in hepatic alpha 2u globulin compared to animals treated with the androgen alone. In spite of inhibition of hepatic synthesis of poly(A)-containing RNA to less than 25% of control, superinduction with actinomycin D resulted in a parallel increase in the translatable mRNA for alpha 2u globulin. These results showing increase in both alpha 2u globulin and its translatable mRNA after superinduction with actinomycin D support the concept of post-transcriptional repression of alpha2u synthesis.
Mutations in cyclic AMP-dependent protein kinase and corticotropin (ACTH)-sensitive adenylate cyclase affect adrenal steroidogenesis.
Authors: Rae P A , Gutmann N S , Tsao J , Schimmer B P
Published: May 2006

Two groups of mutant clones were isolated from YI adrenocortical tumor cells. One group, Y1(Kin), exhibited altered cytosolic cyclic AMP-dependent protein kinase activity; the second group, Y1(Cyc), exhibited diminished corticotropin-responsive adenylate cyclase activity. Steroidogenic responses to corticotropin and cyclic nucleotides closely paralleled cyclic AMP-dependent protein kinase activity in the Y1(Kin) mutants. In Y1(Cyc) mutants, corticotropin had little effect on steroidogenesis, whereas cyclic nucleotides were fully active. These data imply that adenylate cyclase and cyclic AMP-dependent protein kinase are obligatory components of the corticotropin-stimulated steroidogenic pathway.
Germ line integration of Moloney leukemia virus: Identification of the chromosomal integration site
Authors: Breindl Michael , Doehmer Johannes , Willecke Klaus , Dausman Jessica , Jaenisch Rudolf
Published: May 2006

The chromosomal integration site of the structural gene of Moloney murine leukemia virus (M-MuLV) in the genome of BALB/Mo mice was mapped genetically. These mice transmit the exogenous M-MuLV as an endogenous virus at a single Mendelian locus. Two independent experimental approaches were used: (
i
) Non-virus-producing fibroblasts prepared from homozygous BALB/Mo embryos were fused to Chinese hamster Wg3-h-o cells. In an analysis of 30 independent mouse-Chinese hamster cell hybrid clones, the segregation of the viral genome measured by molecular hybridization and enzymes assigned to 16 different mouse chromosomes were compared. We found a highly concordant segregation of M-MuLV sequences and the mouse enzyme triosephosphate isomerase (TPI, EC 5.3.1.1), whose gene has been assigned to chromosome 6. A further karyotype analysis of 9 clones, in which the chromosomes were identified cytochemically, supported this result. (
ii
) The segregation of the viral genome was studied in backcrosses of BALB/Mo with ABP/J mice. In the backcross ABP/J×(ABP/J×BALB/Mo) a linkage of the M-MuLV genome to the morphological marker
wa-1
on mouse chromosome 6 was found. This confirmed the conclusion that the M-MuLV genome is integrated in mouse chromosome 6. These experiments define the genetic locus
Mov-1
, denoting the genetically transmitted structural gene of M-MuLV in BALB/Mo mice.
Biliary transport of IgA: role of secretory component.
Authors: Fisher M M , Nagy B , Bazin H , Underdown B J
Published: May 2006

Biliary transport of rat immunoglobulin was studied by perfusion of isolated rat liver with blood containing radiolabeled immunoglobulin. Transport to bile was selective for polymeric IgA. Between 15 and 27% of polymeric IgA was transported from blood to bile during a 210-min perfusion period, and approximately 60% of the IgA transported to bile bore secretory component. Small quantities of IgM (0.12%) were transported; transport of IgG2 alpha, IgE, or monomeric IgA was not detected. Purification of radiolabeled polymeric IgA by affinity chromatography on human secretory component-Sepharose yielded a fraction that was transported more efficiently (i.e., up to 40% transported). In contrast, secretory IgA (colostral or biliary) was transported 1/25th to 1/12th as well as polymeric IgA myeloma protein. Complexes of 125I-labeled secretory component and polymeric IgA formed in vitro were transported poorly (0.1%) compared to polymeric IgA (26%). It was concluded that biliary transport of polymeric IgA requires combination of it with secretory component in the liver. In support of this hypothesis, rabbit IgG anti-rat secretory component antibodies were also transported to bile but normal rabbit IgG was not.
Human placental immunoreactive corticotropin, lipotropin, and beta-endorphin: evidence for a common precursor.
Authors: Odagiri E , Sherrell B J , Mount C D , Nicholson W E , Orth D N
Published: May 2006

The concentrations and molecular sizes of immunoreactive corticotropin (ACTH), lipotropin (LPH, beta LPH plus gamma LPH), gamma LPH, and beta-endorphin (beta END) were determined in human placental extracts. Serial dilutions of a water extract of placenta generated competitive binding curves parallel with that of the standard in each assay. The concentrations of ACTH, LPH, gamma LPH, and beta END were 3.3, 0.8, 0.7, and 1.1 ng/g wet weight of tissue, respectively. A partially purified extract applied to a Sephadex G-50 column contained high Mr components with ACTH, LPH, gamma LPH, and beta END immunoreactivities. The extract was applied to an immune affinity chromatography column consisting of affinity-purified (1-24)ACTH antiserum covalently bound to agarose. The material that adsorbed to the column and eluted with buffer containing sodium dodecyl sulfate had ACTH, LPH, and beta END immunoreactivities, indicating that there was a component or components containing antigenic determinants for all of these peptides. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the affinity-purified placental extract revealed at least two high Mr components (Mr approximately 48,000 and 36,000) with all three immunoreactivities. These data suggest, but do not prove, that the placenta synthesizes ACTH, the LPHs, and beta END from a common precursor molecule.
Retrovirus sequences in a leukemic gibbon and its contact: evidence for partial provirus in the nonleukemic gibbon.
Authors: Wong-Staal F , Reitz M S , Gallo R C
Published: May 2006

Integrated viral DNA sequences were detected in tissues from two gibbon apes, a leukemic gibbon (6G-1) from whose leukocytes a distinct strain of gibbon ape leukemia virus (GaLVH) was isolated, and gibbon 6G-4, a contact of 6G-1 from the same colony that had uremia and cachexia of unknown origin. Although 6G-4 had no detectable neoplasia or viral proteins, its serum contained persistent antibody against GaLV antigens. Whereas DNA from most of the tissues of 6G-1 contained GaLV provirus, DNA from only three tissues (kidney, spleen, and liver) from 6G-4 showed detectable viral sequences, and the extent of hybridization in each case was lower than with 6G-1. After cleavage with BamHI, two virus-specific DNA fragments were detected in tissues of 6G-1. Only one of these fragments was detected in the positive tissues of 6G-4. The results indicate that: (i) 6G-4 was exposed to and infected by GaLV; (ii) early target sites for infection of gibbon by GaLV may be limited to a few tissues; and (iii) infection can be contained by integration of only partial provirus in a few tissues.
Bilirubin diglucuronide synthesis by a UDP-glucuronic acid-dependent enzyme system in rat liver microsomes.
Authors: Blanckaert N , Gollan J , Schmid R
Published: May 2006

Incubation of rat liver homogenate or microsomal preparations with bilirubin or bilirubin monoglucuronide with (BMG) resulted in formation of bilirubin diglucuronide (BDG). Both synthesis of BMG and its conversion to BDG were critically dependent on the presence of UDP-glucuronic acid. Pretreatment of the animals with phenobarbital stimulated both reactions. When 33 microM bilirubin was incubated with microsomal preparations from phenobarbital-treated rats, 80-90% of the substrate was converted to bilirubin glucuronides; the reaction products consisted of almost equal amounts of BMG and BDG. When phenobarbital pretreatment was omitted or when the substrate concentration was increased to 164 microM bilirubin, proportionally more BMG and less BDG were formed. Homogenate and microsomes from homozygous Gunn rats neither synthesized BMG nor converted BMG to BDG. These findings in vitro suggest an explanation for the observations in vivo that, in conditions of excess bilirubin load or of genetically decreased bilirubin UDP glucuronosyltransferase (EC 2.4.1.17) activity, proportionally more BMG and less BDG are excreted in bile.
Geometric and electronic structure of oxyhemocyanin: spectral and chemical correlations to met apo, half met, met, and dimer active sites.
Authors: Eickman N C , Himmelwright R S , Solomon E I
Published: May 2006

The chemical and spectral properties of a series of hemocyanin derivatives were systematically compared to provide insight into the geometric and electronic structure of the oxyhemocyanin active site. The binuclear copper site is characterized as two tetragonal Cu(II) atoms bridged by both an endogenous protein ligand and the exogenous ligand (i.e., peroxide), with the lack of an electron paramagnetic resonance signal being the result of antiferromagnetic exchange via the endogenous bridge. A transition dipole-vector coupling model is used to assign the unique absorption spectral properties of oxyhemocyanin: the bands at 570 and 486 nm are assigned as components of the peroxide pi v* to copper dx2-y2 charge transfer. The 345-nm band is one component of the pi sigma* leads to dx2-y2 charge transfer. The model also predicts an end-to-end bridging geometry for the peroxide in oxyhemocyanin.
Cellular energy metabolism, trans-plasma and trans-mitochondrial membrane potentials, and pH gradients in mouse neuroblastoma
Authors: Deutsch C. , Erecińska M. , Werrlein R. , Silver I. A.
Published: May 2006

A method for quantitative evaluation of transmembrane electrical potential and pH gradients across a subcellular compartment in an intact cell is presented. This approach has been applied in studies of mouse neuroblastoma C-1300 clone NB41A3, in which the transmembrane electrical potential and pH gradients and the mitochondrial volume percent have been determined. Membrane potentials and pH gradients were measured by two different methods. Equilibrium distributions of [
3
H]triphenylmethyl phosphonium and [
14
C]-thiocyanate ions gave calculated apparent membrane potentials of -77.0 and -29.6 mV, respectively, at 20-25°C; a value of -60.8 mV was obtained from microelectrode measurements. Equilibrium distributions of weak acids ([
14
C]trimethylacetic acid and 5,5-di[
14
C]methyl-2,4-oxazolidine-dione) and of weak bases ([
14
C]dimethylamine and [
14
C]trimethylamine) gave calculated upper and lower limits of the pH gradient (Δ pH = pH
e
- pH
i
) of -0.14 and -0.21 pH unit, respectively. The microelectrode measurements showed that the intracellular pH is within 0.1 of a pH unit or less of the extracellular pH over the extracellular pH range of 7.35-6.85. The mitochondrial volume percent calculated on the basis of the measured cytochrome
c
content is 5.6 ± 1.2% and compares well with estimates of 5.4 ± 1.1% obtained from 25 electron micrographs. Measurements of the cellular energetic parameters gave values within the range found in other cells and perfused organs. Comparison of the results of the microelectrode and equilibrium measurements permits estimates of the electrical potential and pH gradients across the mitochondrial membrane (mitochondria-to-cytoplasm gradients) to be made and suggests that the trans-mitochondrial membrane protonmotive force in the
intact cell
cannot be greater than -143 mV.
Cellular retinol-binding protein allows specific interaction of retinol with the nucleus in vitro.
Authors: Takase S , Ong D E , Chytil F
Published: May 2006

Purified cellular retinol-binding protein (CRBP), a potential mediator of vitamin A action, was found to enable retinol to bind in a specific manner to isolated nuclei from livers of vitamin A deficient rats. Binding was followed after complexing [3H]retinol with CRBP. The binding was specific, saturable, and temperature dependent. CRBP charged with unlabeled retinol or CRBP without retinol diminished binding of radioactivity whereas free retinol did not. No specific binding sites could be detected for free retinol. Purified cellular retinoic acid binding protein (CRABI) complexed with retinoic acid did not diminish the amount of retinol bound to nuclei. Approximately 3 x 10(5) specific binding sites per nucleus could be detected. Fewer binding sites were found in nuclei isolated from livers of control (chow-fed) rats and also from livers of vitamin A-deficient rats 2 hr after refeeding with retinylacetate.
Steady-state coupling of four membrane systems in mitochondrial oxidative phosphorylation.
Authors: Hill T L
Published: May 2006

According to Alexandre, Reynafarje, and Lehninger, four different membrane systems are involved, with definite stoichiometry, in the mitochondrial synthesis of ATP by electron transport, via proton transport. We adopt this model and pursue some of its thermodynamic consequences. At steady state, each of the four systems must have the same flux J through the membrane and the overall thermodynamic force X for oxidative phosphorylation is the sum of the four separate forces. From these properties, using an empirical linear flux-force relation for each system, it is easy to obtain J as a function of X. In turn, X depends on the inside [NAD+]/[NADH] and the outside [ATP]/[ADP][Pi] quotients (and on the pH inside). Thus, J is related to these quotients. The relationship we derive is similar to that described by Erecińska and Wilson, as deduced from a quite different model of oxidative phosphorylation. Proton transport is involved explicitly in three of the four systems of the present model. However, because of the steady-state stoichiometric coupling of the four systems, proton transport does not appear in the overall reaction. On the other hand, Erecińska and Wilson use, in their model, a direct connection between electron transport and ATP synthesis. The present paper demonstrates that J can be related to the quotients mentioned above without this direct connection.
Genetic variation and relative catalytic efficiencies: lactate dehydrogenase B allozymes of Fundulus heteroclitus.
Authors: Place A R , Powers D A
Published: May 2006

In order to evaluate whether functional differences exist between allelic variants of a B type lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) in the teleost fish Fundulus heteroclitus (Linnaeus), the kinetic properties of pyruvate reduction were examined. While the pH dependence and the temperature dependence for maximal catalysis were indistinguishable among the allozymes, reaction velocities at low pyruvate concentrations were significantly different. At pH values below 8.00, the LDH-BbBb allozyme showed a greater reaction rate at lower temperatures (e.g., 10 degrees C) than LDH-BaBa. The phenomenon was reversed at higher temperatures (e.g., greater than 25 degrees C) for pH values between 6.50 and 7.00. The rates for the heterozygous phenotype, LDH-BaBb, were not the arithmetic average of the two homotetrameric allozymes. When reaction rates were compared at constant relative alkalinity, that is, a constant [OH-]/[H+] ratio, the findings were similar. The differences in the temperature dependence and the pH dependence for pyruvate reduction found between the LDH-B allozymes may reflect a selective adaptation and help explain the geographical variation in the Ldh-B gene frequencies of F. heteroclitus.
Down syndrome: Gene dosage at the transcriptional level in skin fibroblasts
Authors: Kurnit David M.
Published: May 2006

Chromosome imbalance (aneusomy) is the leading known cause of both spontaneous abortion and mental retardation in human beings. The primary abnormality is thought to result from quantitative changes of transcription products from the unbalanced genetic material. To document this point, I compared chromosome 21-specific transcription in skin fibroblasts from subjects with monosomy 21, disomy 21 (normal), and trisomy 21 (Down syndrome). Polyadenylylated RNA [poly(A)-RNA], which is enriched in messenger and messenger-precursor RNA sequences, was isolated from the above fibroblast lines. Radioactive DNA (cDNA) complementary to these RNAs was synthesized with reverse transcriptase (RNA-dependent DNA polymerase). These cDNAs were hybridized with (
i
) DNA from a cell line with a mouse genome plus human chromosome 21 and (
ii
) mouse DNA. Subtraction of the amount of hybridization in experiment
ii
from that in experiment
i
yielded a measure of human chromosome 21-specific RNA sequences. The results were consistent with gene dosage at the transcriptional level; for monosomy 21-derived cDNA, 0.6% (of the total cDNA) hybridized specifically to human chromosome 21; for disomy 21-derived cDNA, 2% hybridized; and for trisomy 21-derived cDNA, 3% hybridized. Thus, for DNA sequences on chromosome 21 in human skin fibroblasts, transcription depends on DNA dosage. Characterization of the chromosome 21-specific RNA sequences quantitated in these experiments could help to elucidate the mechanisms by which abnormal karyotypes result in abnormal phenotypes.
Molecular signals for initiating protein synthesis in organ hypertrophy.
Authors: Hammond G L , Wieben E , Markert C L
Published: May 2006

When chronically provoked to increased physiologic activity, organs increase in mass through augmented protein protein synthesis. This process of compensatory hypertrophy can involve cell division as well as cell growth. To test for molecules that might regulate organ size, by inducing hypertrophy, we performed a series of experiments using isolated, perfused, canine hearts in which the left ventricle was beating but performed no work. Hypertrophying hearts and kidneys as well as normal control organs were extracted and the extracts were perfused through isolated heart preparations. Before and after perfusion, RNA was extracted from fragments of the isolated hearts and translated in cell-free media containing [35S]methionine. Incorporation of methionine into protein was measured by liquid scintillation spectrometry. When perfused through normal hearts, extracts from hypertrophying heart and kidney were able to increase greatly the translational ability of RNA extracted from the normal hearts; corresponding perfusates from nonhypertrophying hearts and kidneys had no effect. Our results indicate that molecules that initiate hypertrophic organ growth are extractable, are generated by the cells of the organ under stress, and are probably similar in heart and kidney and perhaps in many other organs as well.
Cyclized dipeptide model for a beta-bend.
Authors: Deslauriers R , Leach S J , Maxfield F R , Minasian E , McQuie J R , Meinwald Y C , Némethy G , Pottle M S , Rae I D , Scheraga H A , Stimson E R , Van Nispen J W
Published: May 2006

A cyclic dipeptide in which L-Ala-Gly was cyclized with epsilon-aminocaproic acid has been synthesized as a model for a beta-bend. Its conformational properties have been examined by means of conformational energy calculations and nuclear magnetic resonance, infrared, Raman, and circular dichroism spectroscopy in various solvents. These calculations and experiments suggest that a type II beta-bend exists in the Ala-Glymoiety, with an NH...O = C hydrogen bond in the epsilon-aminocaproic acid portion of the molecule, and that the molecule adopts a unique conformation in solution. In contrast, an open-chain analog of this compound exists in solution as an ensemble of conformations but with a significant amount of a type II beta-bend structure in the ensemble.
Enzyme activities in endothelial cells and smooth muscle cells from swine aorta.
Authors: Hayes L W , Goguen C A , Stevens A L , Magargal W W , Slakey L L
Published: May 2006

In populations of cultured arterial endothelial and smooth muscle cells grown under the same conditions, we have measured the total activity per cell of 10 enzymes commonly used as "markers" for subcellular organelles: NADH: ferricyanide reductase, NADH:cytochrome c reductase (rotenone insensitive). NADPH:cytochrome c reductase, alpha-glucosidase, 5'-nucleotidase, alkaline phosphodiesterase I, cytochrome oxidase, monoamine oxidase, cathepsin D, and N-acetyl-beta-glucosaminidase. Significant differences between the cell types were found for 7 of the 10 enzymes tested. The total activity of 5'-nucleotidase in cultured smooth muscle cells was 17 times that of cultured endothelial cells. Comparison of the activities in the two cell types freshly collected and in culture showed that the difference in 5'-nucleotidase in cultured cells is due principally to loss of activity from endothelial cells, suggesting that this activity is regulated differently in the two cell types. In both cell types cathepsin D activity rose during culture.
Protein kinase activity associated with simian virus 40 T antigen.
Authors: Griffin J D , Spangler G , Livingston D M
Published: May 2006

Incubation of simian virus 40 (SV40) tumor (T) antigen-containing immunoprecipitates with [gamma-32P]ATP results in the incorporation of radioactive phosphate into large T antigen. Highly purified preparations of large T antigen from a SV40-transformed cell line, SV80, are able to catalyze the phosphorylation of a known phosphate acceptor, casein. The kinase activity migrates with large T antigen through multiple purification steps. Sedimentation analysis under non-T-antigen-aggregating conditions reveals that kinase activity and the immunoreactive protein comigrate as a 6S structure. The kinase activity of purified preparations of large T antigen can be specifically adsorbed to solid-phase anti-T IgG, and partially purified T antigen from a SV40 tsA transformation is thermolabile in its ability to phosphorylate casein when compared to comparably purified wild-type T antigen. These observations indicate that the SV40 large T antigen is closely associated with protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) activity.
Defective splicing of mitochondrial rRNA in cytochrome-deficient nuclear mutants of Neurospora crassa.
Authors: Mannella C A , Collins R A , Green M R , Lambowitz A M
Published: May 2006

Recent studies have shown that the gene encoding the large (25 S) mitochondrial rRNA of Neurospora crassa contains an intervening sequence of 2-2.5 kilobases that is not present in the mature 25S mitochondrial rRNA. Earlier studies had provided evidence that mitochondrial rRNAs in Neurospora are synthesized via a 32S precursor RNA that contains sequences for both the mature 19S and 25S RNA species. The present work shows that the intervening sequence is not present in 32S RNA. However, we have identified two temperature-sensitive nuclear mutants that fail to excise the intervening sequence at the nonpermissive temperature (37 degrees C). When grown at 37 degrees C, the mutants show decreased ratios of 25S to 19S RNA and accumulate a novel 35S RNA that appears to consist of 25S RNA plus most or all of the intervening sequence. The mutants are allelic but can be distinguished in temperature shift-up experiments, mitochondrial rRNA processing turning off more rapidly in one than in the other. These mutants should provide powerful new tools for studying RNA processing in eukaryotic cells.
Conversion of glutathione to glutathione disulfide by cell membrane-bound oxidase activity.
Authors: Tate S S , Grau E M , Meister A
Published: May 2006

An apparently specific glutathione oxidase activity is present in renal cortex, epididymal caput, jejunal villus tip cells, choroid plexus, and retina (but not in liver). The activity is membrane-bound and is localized on the luminal surface of the brush border membranes of the kidney and jejunum. The distribution and localization of the oxidase are similar to those of gamma-glutamyl transpeptidase, suggesting that there is a significant relationship among the translocation of intracellular glutathione, the extracellular oxidation of glutathione to glutathione disulfide, and the reactions of the gamma-glutamyl cycle. Thus, both glutathione present in the blood plasma and intracellular glutathione translocated to the cell surface are accessible to oxidation and transpeptidation. Acceptor substrates of the transpeptidase (e.g., L amino acids) promote transpeptidation and decrease oxidation of glutathione. Conversion of glutathione to glutathione disulfide is followed by utilization of the latter compound by gamma-glutamyl transpeptidase and dipeptidase. Although intracellular oxidation of glutathione to glutathione disulfide is readily reversed by the action of glutathione reductase, glutathione disulfide formed extracellularly cannot be reduced; instead, it undergoes hydrolytic and transpeptidation reactions leading to gamma-glutamyl amino acid and amino acid products which may be recovered by being transported into the cell.
Functional characteristics of dispersed rat submandibular cells.
Authors: Quissell D O , Redman R S
Published: May 2006

Rat submandibular gland cells have been obtained through enzymatic dispersion using chromatographically purified collagenase (EC 3.4.24.3) and hyaluronidase (EC 3.2.1.35) and gentle mechanical force. The recovery of viable cells after the isolation procedure was 59% on the basis of total glandular DNA content. Approximately 60% of the total cell population consisted of acinar cells; less than 8% were immature granular duct cells; and the remainder were intercalated duct, striated duct, and myoepithelial cells. Most of the acinar cells were in acinar-intercalated duct complexes. The integrity of the isolated cells was substantiated by their exclusion of trypan blue, intracellular electrolyte composition, incorporation of [14C]glucosamine into trichloroacetic acid + phosphotungstic acid precipitable material at a linear rate for 1.5 hr, secretory responses to parasympathomimetic and sympathomimetic stimulation, and morphologic integrity as determined by light and electron microscopy. The cholinergic receptors were characterized through investigation of the net transmembrane flux of K+ in response to carbamoylcholine. The alpha-adrenergic receptors were characterized by investigating the net transmembrane flux of K+ in response to norepinephrine stimulation and the beta-adrenergic receptors were characterized by determining the rate of secretion of 14C-labeled mucin after isoproterenol stimulation. A high degree of sensitivity to both cholinergic and adrenergic secretagogues was observed.
Endogenous oncornaviral antigen in the bursa of Fabricius of 15B X 7(2) chickens.
Authors: England J M , Halpern M S
Published: May 2006

Oncornaviral antigen was detected in the bursal epithelium and in a subpopulation of bursal follicular cells of 15B X 72 chickens. This antigen is present in the bursal epithelium at 11 days of embryogenesis and persists there for at least 3 weeks after hatching. The absence of detectable antigen in the intestinal epithelium contiguous to the bursal epithelium indicates that the accumulation of viral antigen is a specific property of the bursal epithelium. The observation of C-type particles in the intraepithelial spaces suggests that the viral antigen in synthesized and assembled into virions by the bursal epithelial cells. In embryonic bursas, viral antigen-positve cells radiate from the surface epithelium toward the central region of the follicles. In bursas from post-hatch chickens, viral antigen-positive cells, including intrafollicular epithelial cells and cells resembling lymphocytes, are confined to the medullary region of the follicles.
Phospholipid methylation in macrophages is inhibited by chemotactic factors
Authors: Pike Marilyn C. , Kredich Nicholas M. , Snyderman Ralph
Published: May 2006

Chemotaxis by human monocytes has been shown to require methylation mediated by
S
-adenosyl-L-methionine(AdoMet), but the specific transmethylation reaction necessary for this function was not elucidated. In an attempt to define the methylation requirement for chemotaxis, we examined the effect of chemotactic agonists and antagonists on protein carboxy-
O
-methylation of protein and methylation of phospholipid in guinea pig macrophages. Chemotactic agents tested over a wide dose and time range produced no alteration in carboxy-
O
-methylation. However, these agents did produce an effect on the methylation of phosphatidylethanolamine by macrophages. AdoMet-mediated phospholipid methylation was inhibited by as much as 73% by chemotactic factors, and there was excellent correlation (
r
= 0.99) between their concentrations for producing half-maximal chemotactic responses and for inhibiting phospholipid methylation. The inhibition of methylation by chemotactic factors was observed at all incubation times and could not be explained by an increased turnover of membrane phospholipid. Neither the chemotaxis antagonist fPhe-Met nor the nonchemotactic tripeptide Met-Met-Met significantly depressed phospholipid methylation. Immune phagocytosis by macrophages similarly did not alter phospholipid methylation. The chemotactic factors produced no alteration in total macrophage phospholipid synthesis or in the phospholipid methylation in a nonchemotactic cell type. The formation of newly methylated derivatives of phosphatidylethanolamine in macrophages was decreased by a biologically active dose of chemotactic factor. These findings indicate that chemotactic factors are capable of altering the methylation of phosphatidylethanolamine in chemotactically responsive cells. The inhibition of phospholipid methylation by chemotactic factors may be necessary for the translation of a chemotactic signal on the surface of the cell into directional cell movement.
Isolation and characterization of somatostatin from pigeon pancreas.
Authors: Spiess J , Rivier J E , Rodkey J A , Bennett C D , Vale W
Published: May 2006

Most of the somatostatin-like activity from pigeon pancreas was found to correspond to small species with an apparent molecular weight of 1500--2500. This species was isolated under conditions minimizing intermolecular interactions and protease activities. The isolated product was characterized by two somatostatin radioimmunoassays, a bioassay, endgroup determination, and amino acid analysis. The structure of the isolated compound was determined to be H-Ala-Gly-cyclo-(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys)-OH. Additionally, small amounts of des-Ala1-somatostatin, a possible degradation product of pancreatic somatostatin, and a large somatostatin-like species with an apparent molecular weight of 11,000--12,500 were detected. It is concluded that the main somatostatin-like polypeptide isolated from pigeon pancreas is identical to the mammalian hypothalamic tetradecapeptide somatostatin.
Structural studies of human urinary kallikrein (urokallikrein).
Authors: ole-MoiYoi O K , Spragg J , Austen K F
Published: May 2006

Human urinary kallikrein (urokallikrein) has been purified by affinity chromatography with aprotinin coupled to CH-Sepharose and by gel filtration. The isolation procedure, which was performed under mild conditions, was completed in a 36-hr period and yielded an overall recovery of more than 75% and a purification of 1727-fold. Homogeneity of the urokallikrein was demonstrated by three criteria: the coincidence of the stained protein band and functional urokallikrein in duplicate gels after alkaline disc gel electrophoresis; the appearance of a single stained band of molecular weight 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis of reduced and unreduced enzyme; and the finding of a single amino-terminal residue, namely alanine after dansylation and acid hydrolysis of purified enzyme. The Km of urokallikrein on N alpha-p-tosyl-L-arginine methyl ester was 400 microM, and the Vmax was 194 mumol/min per mg of protein, which is higher than that observed with any previous preparations. The molecular weight of 48,700 determined on gel filtration and the molecular weight of 48,000 observed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis are in good agreement with the molecular weight of 48,213 calculated from the amino acid composition. The finding of a molecular weight higher than those previously reported, namely 27,000-43,500, the increased functional activity on tosylarginine methyl ester, and the detection of a single amino-terminal residue are consistent with the isolation of a more native protein by the procedure described in this paper.
Vitamin K-dependent gamma-carbon-hydrogen bond cleavage and nonmandatory concurrent carboxylation of peptide-bound glutamic acid residues.
Authors: Friedman P A , Shia M A , Gallop P M , Griep A E
Published: May 2006

The pentapeptide Phe-Leu-Glu-Glu-Leu, tritiated at the gamma carbon of each Glu residue, has been synthesized. In a system using microsomal preparations derived from rat liver, vitamin K-dependent tritium release from the L-Glu residues of this substrate can occur without the concurrent gamma-carboxylation of Glu. This tritium release reaction, which indicates cleavage of the gamma C-H bond, although easily uncoupled from CO2-dependent gamma C carboxylation, does require the reduced (hydroquinone) form of vitamin K and oxygen. The data argue against a concerted mechanism for the cleavage of the gamma C-H bond and carboxylation and against a mechanism in which the vitamin functions solely to transfer or activate CO2. Although the tritium release is related clearly to the oxidation of vitamin KH2, it is not yet established how the subsequent carboxylation proceeds. However, two carboxylation mechanisms compatible with the results are discussed.
A normal cell protein similar in structure and function to the avian sarcoma virus transforming gene product.
Authors: Collett M S , Erikson E , Purchio A F , Brugge J S , Erikson R L
Published: May 2006

This report extends our previous studies concerning the identification and characterization of a protein from normal cells that is closely related to the avian sarcoma virus (ASV) transforming gene product pp60src. This normal cellular protein, which we have found in both avian and mammalian cells and have tentatively designated pp60sarc, was detected by immunoprecipitation of radiolabeled cell extracts with serum derived from both mice and rabbits bearing ASV-induced tumors. The normal cell pp60sarc is a 60,000-dalton phosphoprotein that is structurally similar, but not identical, to viral pp60src. The phosphorylation patterns of the normal cell and viral proteins are also similar: both contain two major phosphorylated residues, a phosphoserine located on the NH2-terminal 60% of the polypeptide and a phosphothreonine present on the COOH-terminal 40% of the molecule. In addition, the normal cell pp60sarc from both chicken and mammalian cells appears to have an associated protein kinase activity analogous to that previously described for the viral pp60src. The possible roles played by the normal cell protein pp60sarc and the ASV transforming protein pp60src in normal cellular growth and neoplastic disease, respectively, are discussed.
The rat serum albumin gene: analysis of cloned sequences.
Authors: Sargent T D , Wu J R , Sala-Trepat J M , Wallace R B , Reyes A A , Bonner J
Published: May 2006

The rat serum albumin gene has been isolated from a recombinant library containing the entire rat genome cloned in the lambda phage Charon 4A. Preliminary R-loop and restriction analysis has revealed that this gene is split into at least 14 fragments (exons) by 13 intervening sequences (introns), and that it occupies a minimum of 14.5 kilobases of genomic DNA.
Differential sensitivity of gene expression in vitro to inhibitors of DNA gyrase.
Authors: Yang H L , Heller K , Gellert M , Zubay G
Published: May 2006

We have used the antibiotics coumermycin A1, novobiocin, and oxolinic acid, which are specific inhibitors of DNA gyrase, to study the coupled transcription and translation of several bacterial and plasmid genes in a DNA-directed cell-free system. The expression of different genes is reduced to different extents by inhibition of DNA gyrase activity. Among the genes tested, the lac operon, an rRNA gene, and the colicin gene of colicin E1 plasmid were found to be most sensitive, while the trp operon and some other genes in colicin E1 plasmid were relatively unaffected by the inhibitors. These results, together with earlier work on the transcription of circular DNA templates, indicate that DNA supercoiling can significantly enhance transcription from certain promoters.
Acoustic microscopy: resolution of subcellular detail.
Authors: Johnston R N , Atalar A , Heiserman J , Jipson V , Quate C F
Published: May 2006

Recent advances now permit the use of scanning acoustic microscopy for the analysis of subcellular components. By sequential viewing of identified fixed cells with acoustic, light, and electron microscopy, we have established that the acoustic microscope can readily detect such features as nuclei and nucleoli, mitochondria, and actin cables. Under optimal conditions, images can even be obtained of filopodia, slender projections of the cell surface that are approximately 0.1-0.2 micron in diameter. Small objects separated by as little as 0.5-0.7 micron can successfully be resolved. Three aspects of the acoustic micrographs prepared in this preliminary survey seem especially prominent. These are, first, the extraordinary level of acoustic contrast that can differentiate the various cytoplasmic organelles, even in regions of very thin cytoplasm; second, the reversals in acoustic contrast that occur when altering the plane of focus; and third, the sensitivity of the acoustic response to overall cytoplasmic thickness. The acoustic microscope uses a novel source of contrast that is based on local mechanical properties. In addition, it can provide a degree of resolution that is comparable to that of the light microscope.
Growth of Madin-Darby canine kidney epithelial cell (MDCK) line in hormone-supplemented, serum-free medium.
Authors: Taub M , Chuman L , Saier M H , Sato G
Published: May 2006

Madin-Darby canine kidney (MDCK) cells can grow in synthetic medium supplemented with five factors--insulin, transferrin, prostaglandin E1, hydrocortisone, and triiodothyronine--as a serum substitute. These five factors permit growth for 1 month in the absence of serum and a growth rate equivalent to that observed in serum-supplemented medium. Dibutyryl adenosine 3',5'-cyclic monophosphate substitutes for prostaglandin E1 in the medium. Potential applications of the serum-free medium are discussed. The medium permits a defined analysis of the mechanisms regulating hemicyst formation by hormones and permits the growth of primary kidney epithelial cell cultures in the absence of fibroblast overgrowth.