bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between the soluble glutathione-dependent delta 5-3-ketosteroid isomerase and the glutathione S-transferases of the liver.
Authors: Benson A M , Talalay P , Keen J H , Jakoby W B
Published: May 2006

Soluble, glutathione-stimulated delta 5-3-ketosteroid isomerase (EC 5.3.3.A) activity of human and rat liver resides in very basic proteins with molecular weights of about 45,000 which are present in high concentrations in these tissues. Physiochemical and immunological evidence is presented for the identity of the proteins responsible for this enzymatic activity with the glutathione S-transferases (RX:glutathione R-transferase, EC 2.5.1.18) that conjugate glutathione with a variety of electrophilic compounds. In the rat, the steroid isomerase is associated principally with the major transferase (B), which is also known as ligandin, and has the versatility to bind various hydrophobic compounds such as bilirubin, corticosteroids, and metabolites of a number of carcinogens. Other rat liver-glutathione S-transferase species are far less active in the steroid isomerization reaction. The delta 5-3-ketosteroid isomerase activity of human liver is more uniformly distributed among the five glutathione S-transferases that have been described. Steroid isomerization differs fundamentally from other reactions promoted by glutathione S-transferases in that glutathione is not consumed in the reaction. However, because the transferase enzymes promote nucleophilic attack by glutathione on a variety of largely foreign organic substrates, a similar mechanism may be involved in the isomerase reaction. Delta 5-3-ketosteroids are among the few known naturally occurring substrates for these enzymes.
Genetic control of mitochondrial malate dehydrogenases: evidence for duplicated chromosome segments.
Authors: Yang N S , Sorenson J C , Scandalios J G
Published: May 2006

The genetic control of the major mitochondrial isoenzymes of malate dehydrogenase (L-malate:NAD+ oxidoreductase; EC 1.1.1.37) has been investigated in Zea mays. The mitochondrial isozymes are coded at four nuclear gene loci. Two of the loci (mdh1 and mdh2) are diallelic and tightly linked. The other two loci (mdh3 and mdh4) appear to have arisen by duplication of the chromosome segment carrying mdh1 and mdh2, but are not linked to them. The segregation of such a duplicate segment can explain anomalous backcross and F2 segregation ratios.
Reconstitution of native human hemoglobin from separated globin chains and alloplex intermediates.
Authors: Yip Y K , Waks M , Beychok S
Published: May 2006

A complete experimental format is given for the reconstitution of human hemoglobin from the separated heme-free alpha- and beta-globin chains (alpha degrees, beta degrees) and hemin, by two alternative routes. Based on their oxygen binding properties, the reaction of the ferri-forms with reducing agent, and the response of the oxygen binding curves to pH variation and to the addition of the allosteric effector 2,3-diphosphoglycerate, the molecules are native. One reconstitution route uses direct addition of hemin to the separated globin chains with production of the separated subunits, which can then be recombined and reduced. This procedure occasions losses by precipitation in the heme-addition step except at high dilutions, and the yields are low. In the second pathway, either globin chain is mixed with the complementary untreated subunit to form the half-filled (with heme) intermediates, which combine stoichiometrically with hemin. No precipitation accompanies these reactions. For alpha-globin, the yield is about 50% because of incomplete combination with the heme-containing beta chain. For beta-globin, the yield is better than 70%. It is suggested that experiments intended to test either globin chain should use the second route in preparation for structural or functional comparisons with native hemoglobin.
Acyl group and electron pair relay system: A network of interacting lipoyl moieties in the pyruvate and α-ketoglutarate dehydrogenase complexes from
Escherichia coli
Authors: Collins Jimmy H. , Reed Lester J.
Published: May 2006

The dihydrolipoyl transacetylase component of the
Escherichia coli
pyruvate dehydrogenase complex [pyruvate:lipoate oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1] bears two sites on each of its 24 polypeptide chains that undergo reductive acetylation by [
2
-
14
C]pyruvate and thiamin pyrophosphate, acetylation by [
1
-
14
C]acetyl-CoA in the presence of DPNH, and reaction with
N
-ethyl[
2,3
-
14
C]maleimide in the presence of pyruvate and thiamin pyrophosphate. The data strongly imply that these sites are covalently bound lipoyl moieties. The results of similar experiments with the
E. coli
α-ketoglutarate dehydrogenase complex [2-oxoglutarate:lipoate oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2] indicate that its dihydrolipoyl transsuccinylase component bears only one lipoyl moiety on each of its 24 chains. Charging of the 48 acetyl acceptor sites on the transacetylase or the 24 succinyl acceptor sites on the transsuccinylase by pyruvate or α-ketoglutarate, respectively, and thiamin pyrophosphate was observed in the presence of only a few functionally active pyruvate dehydrogenase or α-ketoglutarate dehydrogenase chains. Extensive crosslinking of the transacetylase chains was observed when the pyruvate dehydrogenase complex was treated with pyruvate and thiamin pyrophosphate or with DPNH in the presence of
N,N′-o
- or
N,N′-p
-phenylenedimaleimide, respectively. When the α-ketoglutarate dehydrogenase complex was treated with DPNH in the presence of
N,N′-p
-phenylenedimaleimide, only transsuccinylase monomers and crosslinked transsuccinylase dimers were detected. It appears that the 48 lipoyl moieties in the transacetylase and the 24 lipoyl moieties in the transsuccinylase comprise an interacting network that functions as an acyl group and electron pair relay system through thiol-disulfide and acyl-transfer reactions among all of the lipoyl moieties.
Isolation of II-alcohol dehydrogenase of human liver: Is it a determinant of alcoholism?
Authors: Li Ting-Kai , Bosron William F. , Dafeldecker Werner P. , Lange Louis G.
Published: May 2006

Human liver alcohol dehydrogenase (alcohol: NAD
+
oxidoreductase, EC 1.1.1.1), homogeneous by physicochemical criteria, has been available in quantity only recently [Lange, L. G. & Vallee, B. L. (1976)
Biochemistry
15, 4681-4686]. Until now, the biochemical basis of human alcohol metabolism had to be extrapolated from the properties and behavior of enzymes from other species, primarily horses and yeast. The biological determinants of human alcoholism have remained obscure, although recent evidence indicates a genetic predisposition, requiring delineation. A functionally distinct form of human liver alcohol dehydrogenase (ADH), which we have designated II-ADH, is provocative since, thus far, it seems to be unique to human beings. It has a high
K
m
for ethanol and is remarkably insensitive (apparent
K
I
, 500 μM) to pyrazole and its derivatives, which are usually potent ADH inhibitors (
K
I
, 1 μM), a property that is the basis for the isolation of II-ADH. The affinity resin 4-[3-(
N
-6-aminocaproyl)aminopropyl]pyrazole-Sepharose binds all other known forms of ADH but not II-ADH, thereby separating it selectively by affinity chromatography. In turn, this has led to the establishment of its identity with that enzyme form which was previously known as the anodic band and characterized by a high
K
m
for ethanol (20 mM at pH 7.5). The remarkable insensitivity of II-ADH to pyrazole inhibition has also permitted quantitation of its role in hepatic ethanol oxidation. At 5 mM ethanol, a saturating concentration for virtually all other forms of ADH, II-ADH contributes less than 15% to total ethanol oxidation. However, at intoxicating concentrations, e.g., 60 mM, it can account for as much as 40% of the total ethanol oxidation rate of liver, indicating a seemingly unique role for this enzyme form in ethanol elimination. Thus far, we have found the amount of II-ADH varies from liver to liver of individuals and is considerably more labile than the other molecular forms, phenomena whose inter- or independence requires further study. The isolation of human II-ADH advances efforts to recognize and understand biochemical mechanisms that may be biological determinants of alcoholism and alcohol-related disease states, now generally approached and managed largely as psychosocial disorders.
Replication of viral RNA by a defective interfering vesicular stomatitis virus particle in the absence of helper virus.
Authors: Johnson L D , Lazzarini R A
Published: May 2006

The genome of a defective interfering particle (DILT) derived from the heat-resistant strain of vesicular stomatitis virus is expressed in vivo without the assistance of infectious helper virus. The rates of RNA synthesis in the presence of cycloheximide (primary transcription) are the same when infections are with equal numbers of physical particles of DILT or virus. With this treatment, DILT synthesizes only 12-17S mRNAs as characterized by size, polarity, and polyadenylylation. In the absence of cycloheximide, DILT-infected cells produce not only these mRNAs but also a 28S RNA species. This RNA, which represents one half of the viral specific RNA, contains newly synthesized full-length (+) and (-) strand DILT RNA. Both strands are found intracellularly as ribonucleoprotein complexes. Without cycloheximide present, the rate of RNA synthesis by DILT was less than that by virus. This curtailment is most likely due to the inability of DILT to synthesize L protein mRNA. An expanded role for defective interfering particles in infection is discussed.
Simultaneous measurements of viscosity and density in solutions undergoing change
Authors: Beams J. W. , Kupke D. W.
Published: May 2006

The magnetic suspension densimeter-viscometer has been modified so that the variations of viscosity and density in solutions undergoing change are determined continuously as a function of time. The changes in values for both of these properties are also obtained when solutions of varying composition are passed through the sample cell (<0.5 ml). The new method measures the torque on the magnetically suspended buoy which is held stationary in a stable velocity gradient; this procedure also facilitates the measurement of the density with greater accuracy. Preliminary results relating to the conformation changes of ribonuclease A in the presence of guanidinium chloride and a disulfide cleaving agent are presented.
Synthesis and secretion of a high molecular weight form of nerve growth factor by skeletal muscle cells in culture.
Authors: Murphy R A , Singer R H , Saide J D , Pantazis N J , Blanchard M H , Byron K S , Arnason B G , Young M
Published: May 2006

Rat skeletal muscle cells and a cloned myogenic cell line synthesize and secrete in culture a molecule that is immunologically and biologically indistinguishable from the active form of nerve growth factor (NGF) from mouse submandibular gland. This protein can be detected in medium conditioned by muscle cells both before and after fusion and in the soluble fraction of muscle cell homogenates. Chromatographic data also reveal that the molecular properties of muscle cell NGF differ from those of the growth factor purified from mouse submandibular glands. Muscle cell NGF has a molecular weight between 140,000 and 160,000, whereas purified mouse gland NGF has a molecular weight of 26,000. The biologic function of muscle cell NGF is not known, although it could be that it plays some role relating to the association of nerves and muscle in vivo.
Evidence from UV transcription mapping in HeLa cells that heterogeneous nuclear RNA is the messenger RNA precursor.
Authors: Goldberg S , Schwartz H , Darnell J E
Published: May 2006

The effects of UV irradiation on the incorporation of [3H]uridine in HeLa (human) cell mRNA, rRNA, heterogeneous nuclear RNA (hnRNA) and early mRNA from adenovirus type 2 have been compared. The UV target size of cell mRNA is at least 3 times larger than the average size of the mRNA itself and larger than the adenovirus-2 early mRNA, which is known to derive from transcription units of about 1.5-5.0 kilobases. The UV target size of hnRNA, in contrast, is about the same as its size determined by sedimentation and overlaps with the target size of mRNA. It is concluded that most mRNA derives from a higher molecular weight hnRNA molecule.
Classification of methanogenic bacteria by 16S ribosomal RNA characterization
Authors: Fox George E. , Magrum Linda J. , Balch William E. , Wolfe Ralph S. , Woese Carl R.
Published: May 2006

The 16S ribosomal RNAs from 10 species of methanogenic bacteria have been characterized in terms of the oligonucleotides produced by T
1
RNase digestion. Comparative analysis of these data reveals the methanogens to constitute a distinct phylogenetic group containing two major divisions. These organisms appear to be only distantly related to typical bacteria.
Biochemical evidence that MCF murine leukemia viruses are envelope (env) gene recombinants.
Authors: Elder J H , Gautsch J W , Jensen F C , Lerner R A , Hartley J W , Rowe W P
Published: May 2006

Recently, a novel class of murine type C virus (MCF), some strains of which are highly oncogenic in the AKR acceleration test, has been isolated from premalignant and malignant thymuses of AKR mice. The biology of these viruses suggested that MCFs are the product of recombination between endogenous ecotropic and xenotropic viruses and, further, that the recombination has taken place within the envelope (env) gene which encodes the surface glycoprotein (gp70) of the virion. We have compared by tryptic peptide analysis, the gp70s of four MCF isolates with the gp70s of various possible parental viruses. In addition, we have compared the tryptic peptides of the gag gene products p30 and p15 from several of these viruses. The results allow the following conclusions: (i) the gp70s of the MCF viruses are not identical to one another and are different from the gp70s of the possible parental viruses tested; (ii) the MCF virus gp70s have tryptic peptides in common with xenotropic virus gp70s as well as with ecotropic virus gp70s; and (iii) the gap region protein, p30, of the MCFs tested is identical to p30 of AKR ecotropic virus (Akv-1 or Akv-2) and distinct from p30 of xenotropic viruses, suggesting that the 5' end of the recombinant viruses is of Akv origin. The findings are discussed with respect to the possible role a recombinant virus might play in leukemogenesis in AKR mice.
Multihormonal induction of hepatic α
2u
-globulin mRNA as measured by hybridization to complementary DNA
Authors: Kurtz David T. , Feigelson Philip
Published: May 2006

A procedure is presented for the preparation of a
3
H-labeled complementary DNA (cDNA) specific for the mRNA coding for α
2u
-globulin, a male rat liver protein under multihormonal control that represents approximately 1% of hepatic protein synthesis. Rat liver polysomes are incubated with monospecific rabbit antiserum to α
2u
-globulin, which binds to the nascent α
2u
-globulin chains on the polysomes. These antibody-polysome complexes are then adsorbed to goat antiserum to rabbit IgG that is covalently linked to
p
-aminobenzylcellulose. mRNA preparations are thus obtained that contain 30-40% α
2u
-globulin mRNA. A labeled cDNA is made to this α
2u
-globulin-enriched mRNA preparation by using RNA-dependent DNA polymerase (reverse transcriptase). To remove the non-α
2u
-globulin sequences, this cDNA preparation is hybridized to an RNA concentration × incubation time (R
0
t) of 1000 mol of ribonucleotide per liter × sec with female rat liver mRNA, which, though it shares the vast majority of mRNA sequences with male liver, contains no α
2u
-globulin mRNA sequences. The cDNA remaining single-stranded is isolated by hydroxylapatite chromatography and is shown to be specific for α
2u
-globulin mRNA by several criteria.

Good correlation was found in all endocrine states studied between the hepatic level of α
2u
-globulin, the level of functional α
2u
-globulin mRNA as assayed in a wheat germ cell-free translational system, and the level of α
2u
-globulin mRNA sequences as measured by hybridization to the α
2u
-globulin cDNA. Thus, the hormonal control of hepatic α
2u
-globulin synthesis by sex steroids and thyroid hormone occurs through modulation of the cellular level of α
2u
-globulin mRNA sequences, presumably by hormonal control of transcriptive synthesis.
Activation of lipoprotein lipase by native and synthetic fragments of human plasma apolipoprotein C-II.
Authors: Kinnunen P K , Jackson R L , Smith L C , Gotto A M , Sparrow J T
Published: May 2006

Apolipoprotein C-II (apoC-II), a protein constituent of human very low density lipoproteins, is the activator for lipoprotein lipase (LPL; triacylglycerol acyl-hydrolase, EC 3.1.1.3). The amino acid sequence of the 78 residues of apoC-II has recently been established in this laboratory. To determine the minimal sequence requirements for activation, we have prepared both native and synthetic fragments of apoC-II and tested them for their ability to activate LPL. Cyanogen bromide fragments of apoC-II corresponding to residues 1--9 and 10--59 had little ability to activate LPL. However, the COOH-terminal cyanogen bromide fragment corresponding to residues 60--78 increased hydrolysis 4-fold compared to an average of 9-fold activation for the same concentration of apoC-II. The synthetic peptide containing residues 60--78 prepared by solid-phase techniques enhanced the lipolysis 3-fold. Addition of five residues produced a synthetic fragment 55--78 that enhanced the release of fatty acid 12-fold compared to 13-fold for intact apoC-II. By contrast, the synthetic peptide containing residues 66--78 did not activate. Removal of the three COOH-terminal residues, Gly-Glu-Glu, from fragment 60--78 decreased the ability to activate LPL by greater than 95%. These studies suggest that the maximal activation of LPL by apoC-II requires a minimal sequence contained within residues 55--78.
Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate
Authors: Kun Ernest , Kirsten Eva , Sharma Manohar L.
Published: May 2006

A soluble extract of the mitochondrial compartment composed of the inner membrane and matrix catalyzes the enzymatic synthesis and hydrolysis of the 1:1 adduct of citric acid and glutathione. The adduct was identified as the thioester by isolation with single and double isotope labeling ([
14
C]citric acid and [
35
S]glutathione) and by conversion to the monohydroxamate of citric acid and comparison with the synthetic product by thin layer chromatography and high voltage electrophoresis. The enzymatic formation of the thioester (pH optimum 7.39 at 30°) requires oxidized glutathione and citrate; both substrates exhibit a Michaelis-Menten kinetics. During the enzymatic reaction equimolar quantities of thioester and glutathione sulfinic acid are formed. After gel filtration or salt fractionation the enzyme system requires Mn
2+
(or Mg
2+
, which is less effective) for maximal activity. When extracts of mitoplast are tested, the time course of reaction is biphasic due to the rapid synthesis of the product by the thioester-forming system (molecular weight 171,000) followed by its decay by the hydrolase (molecular weight 71,000). The two systems were separated by molecular filtration on Sephadex G-200 and by precipitation with (NH
4
)
2
SO
4
. The thioester-forming system is inhibited by preincubation with 0.5 mM mersalyl. Other inhibitors are 1,2,3-propane tricarboxylic acid, 10 mM Ca
2+
, 200 mM K
+
, and the free radical trapping agent, phenazine methosulfate. The citrate-glutathione thioester formation is irreversibly and specifically inhibited by (-)erythrofluorocitrate (50% inhibition at 25 pmol of added fluorocitrate per mg of protein), which forms a trichloroacetic acid-stable adduct with the enzyme protein (at 50% inhibition, 0.8 pmol is bound to 1 mg of protein). Synthesis of malyl-glutathione thioester by inner membrane vesicles is selectively inhibited by (-)erythrofluoromalate.
Structure of murine Ia antigens: partial NH2-terminal amino acid sequences of products of the I-E or I-C subregion.
Authors: McMillan M , Cecka J M , Murphy D B , McDevitt H O , Hood L
Published: May 2006

Partial amino acid sequence of the Ia molecule encoded by the I-E or I-C (I-EC) subregion of the major histocompatibility complex of the mouse are presented. The Ia molecule appears to be comprised of two noncovalently associated polypeptides. The larger subunit, alpha, has an approximate molecular weight of 35,000 and the smaller subunit, beta, an approximate molecular weight of 28,000. Several interesting homology relationships (or the lack thereof) are apparent when the Ia polypeptides from the I-EC subregion are compared both with their counterparts from man and guinea pig and with the molecules encoded in the I-A subregion. Clearly the most impressive homology relationship is that seen between the alpha polypeptide from the I-EC subregion of mouse and its human counterpart. This is in striking contrast to the beta polypeptide, which bears no apparent homology to its human counterpart.
Regional dissociation of beta-endorphin and enkephalin contents in rat brain and pituitary.
Authors: Rossier J , Vargo T M , Minick S , Ling N , Bloom F E , Guillemin R
Published: May 2006

beta-Endorphin and enkephalin in extracts of whole brain, various brain regions, adenohypophysis, and combined pars intermedia and neurohypophysis of the rat were measured by radioimmunoassay. In brain extracts, the immunoreactive substances were further separated according to molecular size by gel filtration. beta-Endorphin was found in the diencephalon but not in the hippocampus, cerebral cortex, cerebellum, and striatum. Enkephalin was found predominantly in the striatum and diencephalon. Attention is called to possible artifactual interference by myelin basic protein in the immunoassays for beta-endorphin in some regions of the brain. In the pituitary, enkephalin was mainly restricted to the pars intermedia-neurohypophysis. Neither adrenalectomy nor hypophysectomy significantly altered levels of beta-endorphin in brain extracts. Adrenalectomy increased the levels of beta-endorphin in adenohypophysis and pars intermedia-neurohypophysis; after adrenalectomy, enkephalin was also increased in the adenohypophysis but less so in the pars intermedia-neurohypophysis. These results show that brain endorphin levels are independent of pituitary endorphin levels; they suggest that beta-endorphin-containing neurons and those containing enkephalin constitute two separate groups of brain cells.
Storage and release of acetylcholine by a clonal cell line.
Authors: Schubert D , Klier F G
Published: May 2006

The nerve cell line PC12, in its morphologically undifferentiated state, synthesizes, stores, and secretes catecholamines and acetylcholine. At least 60% of the basal level of neurotransmitter release is due to a calcium-dependent mechanism, and the rate of secretion is enhanced by increasing external potassium. A minimum of 80% of the intracellular acetylcholine and catecholamines are stored in particulate structures. The storage site for acetylcholine is dense core vesicles that can be distinguished from those containing catecholamines on the basis of vesicle density on sucrose gradients, vesicle size, and reserpine sensitivity. These results are discussed in relation to what are thought to be the early stages of synapse formation in cell culture.
Generalizing Fisher's "reproductive value": linear differential and difference equations of "dilute" biological systems.
Authors: Samuelson P A
Published: May 2006

R. A. Fisher's 1930 "reproductive value" is defined as the contribution made by a population's initial age elements to its asymptotically dominating exponential growth mode. For the Leslie discrete-time model, it is the characteristic row vector of the Leslie matrix, and for the integral-equation model of Lotka the similar eigenfunction. It generalizes neatly to a 2-sex model of linear differential equations, and to general n-variable linear systems. However, when resource limitations end the "dilute" stage of linearity, reproductive value loses positive definability. The present linear analysis prepares the way for generalizing reproductive value to nonlinear systems involving first-degree-homogeneous relationships.
"Viral" expansion of enzyme flux and use of quasi-chemical approximation for two-state enzymes with enzyme-enzyme interactions.
Authors: Hill T L
Published: May 2006

Two examples of enzyme systems with interactions, at steady state, are treated here. In both cases, the enzyme cycle has two states and quasi-equilibrium in spatial distributions obtains at steady state (because f alpha + f beta = 1). The first example is a dilute solution of enzyme molecules in a solvent. The flux (turnover) per molecule is expanded in powers of the enzyme concentration (a "viral" expansion). Aggregation of the enzyme molecules in solution is considered as a special case. In the second example, we treat an arbitrary lattice of enzyme molecules, with nearest-neighbor interactions, using the well-known quasi-chemical approximation. The flux per molecule is obtained. Critical behavior and hysteresis are illustrated.
Identification of the
recA
(
tif
) gene product of
Escherichia coli
Authors: Gudas Lorraine J. , Mount David W.
Published: May 2006

Treatments that inhibit DNA synthesis in
recA
+
lexA
+
Escherichia coli
stimulate synthesis of a 40,000 molecular weight protein species (protein X). The protein X molecules produced by wild-type and mutant
E. coli
strains have been compared by two-dimensional gel electrophoresis. One
recA
mutant (DM1415
spr recA1
) produced a protein X with a more acidic isoelectric point than protein X from the wild type, demonstrating that protein X is probably the product of the
recA
gene. Additional mutants carrying the
recA
-linked
tif-1
mutation yielded a protein X that was more basic than the wild-type protein, indicating that the
tif-1
mutation also alters the
recA
protein. Protein X molecules from the above mutants and wild-type
E. coli
have been shown to yield similar partial products upon limited proteolysis in sodium dodecyl sulfate, indicating they are the same protein species. These and additional studies suggest that (
i
) the
tif-1
mutation alters a site on the
recA
protein that is sensitive to DNA synthesis inhibition, (
ii
) synthesis of
recA
protein is self-regulated, and (
iii
) synthesis of
recA
protein is also regulated by the
lexA
product with
lexA
-suppressor mutations such as
spr
resulting in constitutive synthesis of
recA
protein.
Probing of β-adrenergic receptors by novel fluorescent β-adrenergic blockers
Authors: Atlas Daphne , Levitzki Alexander
Published: May 2006

The synthesis of two high-affinity fluorescent β-adrenergic blockers is described:
dl-N
1
-[2-hydroxy-3-(1-naphthyloxy)propyl]-
N
2
-(9-acridyl)-1,2-propanediamine (9-aminoacridylpropanolol, 9-AAP) and
dl-N
-[2-hydroxy-3-(1-naphthyloxy)propyl]-
N
′-dansylethylenediamine (dansyl analogue of propranolol, DAPN). Both 9-AAP and DAPN inhibit competitively the
l
-epinephrine-dependent adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in turkey erythrocyte membranes without affecting the fluoride-stimulated adenylate cyclase activity. Similarly, 9-AAP and DAPN inhibit in a competitive manner the binding of [
125
I]-iodohydroxybenzylpindolol to these β-adrenergic receptors. The two fluorescent β-adrenergic blockers 9-AAP and DAPN probe specifically β-adrenergic receptors in the central nervous system as well as in other organs when injected into rats. The fluorescence pattern can be monitored by fluorescence microscopy performed on cryostat slices of these organs. The appearance of the characteristic fluorescence pattern can be blocked in a stereospecific fashion by a prior injection of
l
-propranolol and not by a prior injection of
d
-propranolol. These compounds therefore offer a powerful means to map β-adrenergic receptors
in vivo
. The stereospecific displacement of 9-AAP from the β-adrenergic receptors of turkey erythrocyte membranes by
l
-propranolol and by
l
-epinephrine can be detected
in vitro
using front-face fluorescence. The potential use of these compounds to probe β-receptors
in vitro
and
in vivo
is discussed.
Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth
Authors: Wehland Jürgen , Osborn Mary , Weber Klaus
Published: May 2006

Phalloidin, the toxic drug from the mushroom
Amanita phalloides
, was injected into the cytoplasm of tissue culture cells and the changes in intracellular actin distribution were followed by immunofluorescence microscopy with actin antibody. At low concentrations, phalloidin recruits the non- or less highly polymerized forms of cytoplasmic actin into stable “islands” of aggregated actin polymers and does not interfere with the preexisting thick bundles of microfilaments (stress fibers). Differential focusing shows that these islands of phalloidin-induced actin polymers occur at a level in the cytoplasm that is above the submembranous bundles of microfilaments present on the adhesive side of the cells. The pattern of cytoplasmic microtubules remains unaffected by the injection of phalloidin; however, filamin, a protein usually associated with actin in the cytoplasm, is also recruited into the islands. At higher phalloidin concentrations, contraction of the cell is observed. These results are discussed in the light of previous biochemical studies by Wieland and Faulstich and their coworkers [for a review see Wieland, T. (1977)
Naturwissenschaften
64, 303-309] on the
in vitro
interaction of phalloidin with muscle actin, which have documented that phalloidin reacts stoichiometrically with actin, promotes actin polymerization, and stabilizes actin polymers.

In addition, we show that microinjection of phalloidin interferes in a concentration-dependent manner with cell locomotion and cell growth. These results indicate that a well-balanced controlled reversible equilibrium between different polymerization states of actin may be a necessary requirement for cell locomotion and may also influence other cellular functions such as growth.
Interactions of C-reactive protein and complement with liposomes
Authors: Richards Roberta L. , Gewurz Henry , Osmand Alexander P. , Alving Carl R.
Published: May 2006

Interactions between C-reactive protein (CRP) and liposomal model membranes containing phosphatidylcholine were investigated. These interactions, in the presence of human serum, resulted in consumption of each of the components of the classical complement pathway (C1-C9) and also resulted in complement-dependent damage and release of trapped glucose from certain types of liposomes. CRP-initiated lysis of liposomes was strongly dependent upon membrane lipid composition. Optimal activity occurred with positively charged liposomes containing galactosylceramide (galactocerebroside); positively charged liposomes lacking galactocerebroside released much less glucose, while negatively charged liposomes, either with or without galactocerebroside, did not release glucose at all. Glucose release was inhibited by free phosphocholine. Lesser, but significant, “background” glucose release independent of the presence of CRP also was observed with positively charged liposomes containing galactocerebroside, and this was associated with marked preferential consumption of the later-acting complement components (C3-C9). C2-deficient human serum failed to support CRP-dependent glucose release, but glucose release was observed upon reconstitution of the serum with C2. Guinea pig complement also did not support CRP-mediated glucose release, but upon addition of human C1q substantial glucose release was observed. We conclude that (
i
) CRP can sensitize appropriate liposomes for complement-dependent damage via the primary complement pathway starting at the level of C1q; (
ii
) of those studied, liposomes that are most susceptible to membrane damage contain phosphatidylcholine, have a positive charge, and contain a ceramide glycolipid; and (
iii
) such liposomes also are sensitive, although to a much lesser degree, to complement-dependent lysis initiated in the absence of CRP and involving consumption of terminal in excess of early acting complement components.
Kinetic polarization deficiency in electrolyte solutions.
Authors: Hubbard J B , Onsager L , van Beek W M , Mandel M
Published: May 2006

The prediction and experimental confirmation of a previously unsuspected kinetic effect occurring in electrolyte solutions are presented herein. Kinetic polarization deficiency may be described as a reduction, with respect to the pure solvent, in the static permittivity of the solution; the decrement in epsilon0 is shown to be proportional to the product of the dielectric relaxation time of the solvent and the low frequency conductivity of the solution. The kinetic ion-solvent interaction affects the capacitive admittance in two closely related ways: as an ion migrates, the surrounding volume elements of the liquid tend to rotate according to the laws of hydrodynamics, and although dielectric relaxation tends to restore an equilibrium polarization appropriate to the local electric field, this adjustment is not instantaneous; rather it lags behind by the dielectric relaxation time. Conversely, the force that an external field exerts on an ion does not develop its full strength instantly because the ion is driven partly by the external field and partly by the polarization that develops in response to the applied field, the polarization field evolving with a time constant that is the relaxation time for the orientation of solvent dipoles.
Functional acetylcholine receptor--electroplax membrane microsacs (vesicles): purification and characterization.
Authors: Hess G P , Andrews J P
Published: May 2006

Kinetic analysis of the flux of sodium ions in a heterogeneous population of acetylcholine receptor-rich microsacs (vesicles) formed by membrane fragments of electroplax indicated that functional microsacs, which on average comprise only 15% of the preparation, can be filled with 190 mM sodium chloride while nonfunctional microsacs are filled by 190 mM cesium chloride. The functional microsacs have then been successfully separated from nonfunctional microsacs on the basis of their density differences with a continuous sucrose-190 mM cesium chloride density gradient. In the presence of acetylcholine analogs all the internal sodium ions in these microsacs rapidly exchange with external ions. The efflux of sodium ions follows a single exponential decay. The isolation of functional microsacs opens up at least two new avenues of investigation of the molecular mechanism of receptor-mediated processes. The first deals with the efficiency of the process, and the second with the characterization of membrane components important in this process. The conclusions reached so far are: (i) The efficiency of the receptor-mediated process that allows inorganic ions to equilibrate across the membranes of the microsacs can adequately account for electrophysiological results obtained with muscle and nerve cells. (ii) In the receptor-rich heterogeneous population of microsacs the concentration of receptor sites in functional and nonfunctional microsacs is about the same and is therefore not the only factor determining functionality. Significant differences between functional and nonfunctional microsacs have been found so far in the concentrations of acetylcholinesterase and Na+-K+ ATPase.
Isolation of a multi-enzyme complex of fatty acid oxidation from Escherichia coli.
Authors: Binstock J F , Pramanik A , Schulz H
Published: May 2006

A multi-enzyme complex of fatty acid oxidation has been isolated from E. coli B cells and has been purified to near homogeneity by a simple two-step procedure. The complex exhibits thiolase (EC 2.3.1.9), enoyl-CoA hydratase (EC 4.2.1.17), and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities towards short-, medium-, and long-chain substrates. The complex has been estimated to have a molecular weight of approximately 300,000 and is apparently composed of two types of subunits with molecular weights of 78,000 and 42,000.
Specific changes in the pattern of protein synthesis during meiotic maturation of mammalian oocytes in vitro.
Authors: Schultz R M , Wassarman P M
Published: May 2006

High resolution two-dimensional electrophoresis has been used to examine the pattern of protein synthesis during meiotic maturation of mouse oocytes in vitro. Fluorograms of [35S]methionine-labeled oocyte proteins have revealed that meiotic progression from dictyate to metaphase II (meiotic maturation) is accompanied by marked changes in the pattern of proteins synthesized by oocytes. Virtually all of the changes observed take place subsequent to the breakdown of the oocyte's germinal vesicle, but are not dependent upon the occurrence of other morphological events, such as spindle formation or polar body emission. These changes in protein synthesis do not take place in oocytes that fail to undergo breakdown of germinal vesicles spontaneously or in oocytes arrested at the germinal vesicle stage by dibutyryl 3':5'-cyclic AMP. These data suggest that mixing of the oocyte's nucleoplasm and cytoplasm may trigger many of the changes in protein synthesis that accompany meiotic maturation of mouse oocytes in vitro.
Identification of the iron-sulfur center in trimethylamine dehydrogenase.
Authors: Hill C L , Steenkamp D J , Holm R H , Singer T P
Published: May 2006

Trimethylamine dehydrogenase [trimethylamine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.7] from a facultative methylotroph bacterium has a molecular weight of 147,000 and contains two types of prosthetic groups, one a covalently bound organic chromophore of uncertain structure and the other containing iron and labile sulfur (S*). The structure of the Fe-S* center has been investigated by reactions of the enzyme with sodium mersalyl, o-xylyl-alpha,alpha'-dithiol, and p-methoxybenzenethiol in a 4:1 vol/vol hexamethylphosphoramide/water reaction medium, which destabilizes tertiary structure. Mersalyl treatment results in reduction of visible absorbance consistent with the presence of a 4-Fe center of the ferredoxin type. Reaction with thiols effects partial bleaching of the organic chromophore, as established by separate studies of a detached chromophore peptide, and results in removal (extrusion) of the core unit of the Fe-s* center in the form of the complexes [Fe4S*4(S2-o-xylyl)2]n2n- and [Fe4S*4(SC6H4OMe)4]2-, which were identified by absorption spectra. These results, in conjunction with control extrusion reactions of oxidized ferredoxins from spinach and Clostridium pasteurianum, establish that trimethylamine dehydrogenase contains one Fe4S*4 core unit most probably present as a ferredoxin-type, cysteinate-ligated cluster [Fe4S*4(S-Cys)4].
A rapid procedure for selective enrichment of photosynthetic electron transport mutants.
Authors: Schmidt G W , Matlin K S , Chua N H
Published: May 2006

Metronidazole (2-methyl-5-nitroimidazole-l-ethanol) is shown to be effective for the selective enrichment of mutants of Chlamydomonas reinhardtii that posses impaired photosynthetic electron transport. More than 99.9% of wild-type cells are killed when incubated in the presence of 6-10 mM metronidazole for 24 hr under illumination of 7500 lux. Survival of wild-type cells in darkness and of mutants that are blocked at different steps in photosynthetic electron transport is nearly 100% when incubated in the presence of the drug under identical conditions. The toxicity of metronidazole is demonstrated to depend upon its reduction by photosynthetic electron transport. Light-dependent oxygen uptake mediated by metronidazole is shown to require active photosystem l in vitro and in vivo. Ferredoxin is necessary for metronidazole reduction by thylakoid membrane fractions enriched in photosystem l activity. We propose that the toxicity of metronidazole is due to the formation of lethal derivatives of the drug or to the accumulation of hydrogen peroxide, which could occur upon autooxidation of metronidazole reduced by one electron. The results indicate that mutants of C. reinhardtii, and probably other photosynthetic organisms, with any lesion in photosynthetic electron transport from the oxidizing side of photosystem ll to ferredoxin can be isolated by metronidazole treatment of mutagenized cultures.
Lymphocyte mitogenesis induced by a mammalian liver protein that specifically binds desialylated glycoproteins.
Authors: Novogrodsky A , Ashwell G
Published: May 2006

A purified rabbit liver membrane protein that binds desialylated glycoproteins has been shown to be a mitogen for human peripheral lymphocytes. The mitogenic activity is specific for desialylated thymus-derived (T)-cells. The loss of mitogenicity upon exposure of the binding protein to neuraminidase and the inhibitory potency of asialo-orosomucoid support the conclusion that the site involved in the binding of asialoglycoproteins is also responsible for the mitogenic effect on desialylated lymphocytes. The potential relevance of this phenomenon to the etiology of hepatocellular necrosis is considered.
Changes in the composition of plasma membrane proteins during differentiation of embryonic chick erythroid cell.
Authors: Chan L L
Published: May 2006

Erythroid cells which are homogeneous with regard to stage of maturation are naturally available from the circulation of chick embryos at various times of development. This provides a convenient system for examining the changes in plasma membrane protein composition during red cell maturation. Plasma membranes are isolated from chick embryonic erythroid cells at various stages of maturation. Extensive characterization of the isolated membranes show that they are pure and their proteins undegraded. Analyses by sodium dodecyl sulfate/polyacrylamide gel electrophoresis show that both qualitative and quantitative changes occur in membrane protein composition during the early stage of erythroid differentiation. Specific proteins of red cell membrane such as "spectrin" and band three proteins are present in low levels in early erythroblasts but increase in their relative amounts with maturation. A steady-state membrane protein composition seems to be established by the late polychromatophilic erythroblast stage.
Endotoxin-induced serum factor controlling differentiation of bone-marrow-derived lymphocytes.
Authors: Hoffmann M K , Oettgen H F , Old L J , Chin A F , Hammerling U
Published: May 2006

Serum from bacillus Calmette-Guerin-infected mice injected with endotoxin induces the appearance of surface immunoglobulin, Ia antigen, and complement receptor on the surface of precursor bone-marrow-derived (B) cells. While endotoxin itself causes phenotypic conversion of both thymus-derived (T) cells and B cells in vitro, the endotoxin-induced serum factor was found to be a selective inducer of B cell differentiation. Spleen cells rendered immunodeficient by removal of B cells bearing the complement receptor regained the capacity to cooperate with helper T cells and to produce antibody against red cell antigens in vitro upon upon addition of the serum factor to the culture medium. Thus, a factor that controls selective phenotypic and functional differentiation of B cells has been identified and can now be characterized,
Feline oncornavirus-associated cell-membrane antigen (FOCMA): distinction between FOCMA and the major virion glycoprotein.
Authors: Stephenson J R , Essex M , Hino S , Hardy W D , Aaronson S A
Published: May 2006

The humoral antibody response of feline leukemia virus (FeLV)-exposed cats to the feline oncornavirus-associated tumor cell-membrane antigen (FOCMA) is directly correlated with immunosurveillance against tumor development under natural conditions. By means of membrane immunofluorescence and radioimmunoprecipitation, the antibody response to FOCMA was found to be independent of the antibody response to the major envelope and core proteins of FeLV, gp70 and p30. This was especially true for healthy viremic cats, where antigenemia with circulating FeLV gp70 and p30 apparently binds any free antibody to these proteins, but high levels of FOCMA antibody are often concurrently present. Exhaustive in vitro absorption of highly immune nonviremic serum with gp70 and p30 also failed to remove FOCMA antibody activity. These results indicate that FOCMA is not one of these major FeLV structural proteins.
Facilitation at neuromuscular junctions: contribution to habituation and dishabituation of the Aplysia gill withdrawal reflex.
Authors: Jacklet J W , Rine J
Published: May 2006

The gill withdrawal reflex of Aplysia has been used as a model for studying the neuronal mechanisms of habituation, a behavioral plasticity. We have assessed the contribution of neuromuscular facilitation, an elementary synaptic plasticity, during habituation of the reflex by recording gill muscle potentials, which we show are caused by excitatory junctional potentials. These potentials show systematic frequency-dependent changes in amplitude. The gill withdrawal evoked by central motor neuron firing during each habituation trial is determined by facilitation of the excitatory junctional potentials during the trial and the facilitated state of the initial excitatory junctional potential in a trial, determined by neuron activity prior to the trial. The neuromuscular junctions, therefore, act like a frequency-dependent amplifier of central motor activity. They are fully responsive to the dynamic changes of motor neuron firing that occurs during habituation and especially after dishabituation.
Calcium modulation in brain extracellular microenvironment demonstrated with ion-selective micropipette.
Authors: Nicholson C , Bruggencate G T , Steinberg R , Stöckle H
Published: May 2006

Changes in extracellular Ca2+ concentration were directly measured in the rat cerebellum, using an ion-selective micropipette. Extracellular K+ was measured simultaneously with a second ion-selective micropipette. The potential reference barrels of the ion electrodes also provided fast field and slow potentials. During repetitive stimulation of the parallel fiber--Purkinje cell cerebellar circuit, extracellular Ca2+ fell to about 80% of base line concentration. During the spreading depression of Leão, extracellular Ca2+ fell to about 10% of base line; decreases of this magnitude also occurred during terminal anoxia. In all cases extracellular K+ increased substantially. These results show that extracellular Ca2+ is modulated during neuronal activity in the central nervous system and that under some conditions the Ca2+ change can be extreme. Given the well-established and antagonistic effects of reduce extracellular Ca2+ on axonal excitability and synaptic transmission, these results suggest that Ca2+ modulation in the brain cell microenvironment may be a significant parameter in the behavior of neuronal ensembles.
Detection in bovine adrenal cortex of a lipoidal substance that yields pregnenolone upon treatment with alkali.
Authors: Hochberg R B , Bandy L , Ponticorvo L , Lieberman S
Published: May 2006

Bovine adrenal cortical tissue contains a lipoidal derivative of pregnenolone (3beta-hydroxy-pregn-5-en-20-one) from which the free steroid can be liberated by treatment with alkali. Evidence for the presence of such an entity comes from examination of a nonpolar extract of tissue from which pregnenolone and its sulfate had been removed by chromatography. Treatment of the nonpolar fraction with alkali followed by exhaustive chromatographic analysis led to the detection of pregnenolone. The steroid was identified by both gas chromatography/mass spectrometry and double isotope procedures. Quantitative analysis indicated that the three forms of pregnenolone are present in bovine adrenal cortical tissue in the following amounts (mug/kg): lipoidal derivative, 290; free steroid, 435; and sulfate, 65. Because the only known metabolic function of pregnenolone is to serve as a precursor of the steroid hormones, these findings have far-reaching implications for steroid hormone biochemistry.