bluuebunny/crossref_metadata_2025 huggingface dataset:
Regularity of the Dirichlet problem for the complex Monge-Ampère equation
Authors: Moriyon Roberto
Published: May 2006

Regularity up to the boundary of the solutions of a boundary value problem for a complex Monge-Ampère equation on perturbations of an annulus in C
n
is proven. The result can be applied to the classification of such domains.
Aliphatic groups of sperm whale myoglobin: 13C NMR study.
Authors: Wittebort R J , Rothgeb T M , Szabo A , Gurd F R
Published: May 2006

The aliphatic region of the 13C NMR spectrum of sperm whale cyanoferrimyoglobin has been examined at 67.9 MHz. Fifty partially resolved or well-resolved resonances, representing at least half of the aliphatic carbons in the molecule, are observed in the spectral region from 9 to 29 ppm downfield of tetramethylsilane. Analyses of the spin lattice relaxation times (T1) and nuclear Overhauser enhancements for these resonances reveal considerable motion freedom of the aliphatic side chains. In the spectral region from 9 to 15 ppm, eight single carbon resonances are observed and tentatively assigned to Cdelta 1 of eight of the nine isoleucine residues. In at least five cases the reorientational motion of the isoleucine side chains could not be characterized solely by rotation of the Cdelta 1 methyl groups. The simplest model consistent with the data is a restricted diffusion model with two degrees of internal rotation [Wittenbort, R. J. & Szabo, A. (1978) J. Chem. Phys. 69, 1722--1736]. In light of the packing densities within the myoglobin molecule these results are taken to imply concerted motions of the buried aliphatic residues.
NBD-5-acylcholine: fluorescent analog of acetylcholine and agonist at the neuromuscular junction.
Authors: Jürss R , Prinz H , Maelicke A
Published: May 2006

We have studied the properties of N-7-(4-nitrobenzo-2-oxa-1,3-diazole)-omega-aminohexanoic acid beta-(N-trimethylammonium)ethyl ester, a fluorescent analog of acetylcholine at the cellular level by using pharmacological and electrophysiological techniques and at the molecular level by measuring the kinetics of interaction with solubilized acetylcholine receptor and with acetylcholine esterase (EC 3.1.1.7). The fluorescent drug is a powerful agonist of acetylcholine at the neuromuscular junction and also strongly desensitizes muscle fibers. Interaction with acetylcholine receptor is accompanied by large changes in the drug's fluorescence. From the kinetics of interaction studied by means of a stopped-flow fluorimeter with laser light source, we obtained a second-order forward rate constant in excess of 1 X 10(8) M-1 sec-1 and an initial dissociation rate constant (k1) of 0.5 sec-1 for receptor from Electrophorous electricus. Interaction of this analog with acetylcholine esterase from E. electricus is accompanied by a transient decrease in fluorescence followed by an increase leading to a stable plateau value at a level near the original one. The initial decrease in fluorescence followed second-order kinetics with k2 of the order of 10(9) M-1 sec-1. The slower consecutive reaction which could be blocked by phosphorylation of the esteratic site, was of first order with k1 = 0.05 sec-1.
Alkaline phosphatase of mouse teratoma stem cells: Immunochemical and structural evidence for its identity as a somatic gene product
Authors: Hass Philip E. , Wada H. Garrett , Herman Mary M. , Sussman Howard H.
Published: May 2006

The immunochemical and structural characteristics of the alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] from mouse teratoma stem cells derived from the OTT-6050 teratoma (ascitic and solid tumors and the F9 and PCC4 cell lines) have been compared to those of the alkaline phosphatases expressed in normal mouse placenta and several adult organs. Crossreactivity of the stem cell alkaline phosphatase with antisera reacting with placental, kidney, liver, and brain alkaline phosphatases indicated that the stem cell enzyme had common antigenic determinants. Structural studies utilizing two-dimensional electrophoresis of the
32
P-labeled alkaline phosphatase subunits showed that the stem cell, placental, and kidney alkaline phosphatases differed only in their sialic acid content and comigrated after removal of terminal sialic acid by neuraminidase digestion. Furthermore, one-dimensional peptide mapping of partial proteolysis fragments from
32
P-labeled enzymes demonstrated identical fragmentation patterns for the stem cell and somatic enzymes. These immunochemical and structural data indicate that the stem cell alkaline phosphatase is the same core enzyme as that produced in the mouse placenta and kidney, with different amounts of terminal sialic acid. The one mouse alkaline phosphatase examined that differed from the other enzymes was the intestinal alkaline phosphatase. This isoenzyme was not immunochemically crossreactive with the other alkaline phosphatases, did not comigrate in two-dimensional electrophoresis after neuraminidase digestion, and did not give identical peptide maps after partial proteolysis.
Dark field imaging of biological macromolecules with the scanning transmission electron microscope
Authors: Ohtsuki Mitsuo , Isaacson Michael S. , Crewe A. V.
Published: May 2006

A scanning transmission electron microscope (STEM) equipped with a field emission gun has been employed for the examination of biological macromolecules at high resolution. The quality of micrographs obtained with the STEM is dependent upon the quality of the substrate used to support biological objects because the image contrast in dark field is proportional to the mass density of the specimen. In order to reduce deleterious effects of the substrates on the image quality, we have developed a method of fabricating substrates consisting of very thin, very clean carbon films supported on very clean fenestrated plastic films. These films are approximately 15 Å thick. Well-known biological macromolecules such as glutamine synthetase and tobacco mosaic virus (both stained) and low-density lipoprotein and ferritin (both unstained were placed on these substrates and examined with the STEM by using various modes of contrast. The micrographs obtained by using the dark field mode of contrast employing an annular detector were free from phase contrast, as expected. Using this contrast mode, we have been able to directly observe (in-focus) 2.5- to 4.4-Å lattice spacings in the ferritin core. The effect of electron radiation damage on the helical structure of tobacco mosaic virus was also examined. Micrographs as well as corresponding optical diffraction patterns obtained with moderately low doses showed very clear helical structure from both sides of the virus. In addition, the (11.5 Å)
-1
layer lines indicated the effective resolution attained on these particles.
Nerve growth factor-induced transformation of immature chromaffin cells in vivo into sympathetic neurons: effect of antiserum to nerve growth factor.
Authors: Aloe L , Levi-Montalcini R
Published: May 2006

Pre- and postnatal injections of nerve growth factor, initiated with one dose on day 17 of gestation and continued after birth with daily subcutaneous administration until day 10 of life, produce massive transformation of chromaffin in sympathetic nerve cells in the rat adrenal medulla. Large sympathetic ganglia, absent in controls, adhere to the medial external surface of the gland. Nerve fibers produced by the transformed chromaffin cells invade the inner and outer cortical zones of the organ, producing cell depletion and substantial alteration of the structure of the cortical layers. When the growth factor treatment is initiated after birth, only a partial replacement of chromaffin with nerve cells takes place. The treatment is ineffective after the second postnatal week. Injections of a specific antiserum to nerve growth factor in 17-day-old rat fetuses, which were continued after birth, produce progressive and massive destruction of chromaffin cell precursors and of immature chromaffin cells in the adrenal medullary gland.
Genetic control of chromosome breakage and rejoining in Drosophila melanogaster: spontaneous chromosome aberrations in X-linked mutants defective in DNA metabolism.
Authors: Gatti M
Published: May 2006

Eight X-linked recombination-defective meiotic mutants (representing five loci) and 12 X-linked mutagen-sensitive mutants (representing seven loci) of Drosophila melanogaster have been examined cytologically in neuroblast metaphases for their effects on the frequencies and types of spontaneous chromosome aberrations. Twelve mutants, representing five loci, significantly increase the frequency of chromosomal aberrations. The mutants at these five loci, however, differ markedly both in the types of aberrations produced and the localization of their effects along the chromosome. According to these criteria, the mutants can be assigned to four groups: (i) mutants producing almost exclusively chromatid breaks in both euchromatin and heterochromatin; (ii) mutants producing chromatid and isochromatid breaks in both euchromatin and heterochromatin; (iii) mutants producing chromatid mutants producing chromatid and isochromatid breaks clustered in the heterochromatin.
Target—effector interaction in the natural killer cell system: Isolation of target structures
Authors: Roder John C. , Rosén Anders , Fenyö Eva Maria , Troy Frederic A.
Published: May 2006

A sensitive target binding assay has recently been shown to detect natural killer (NK) cells in the mouse. Preincubation of NK cells with detergent-solubilized cell-surface proteins of YAC lymphoma cells prevented subsequent binding to intact YAC targets. The NK target structures (NK-TS) consisted of three molecular species tentatively assigned molecular weights of 130,000, 160,000, and 240,000 based on electrophoretic mobility in sodium dodecyl sulfate/polyacrylamide gels. Moloney cell surface antigen (MCSA), gp71, p30, H-2, and NK-TS were localized in distinct fractions of gels. The NK-TS bound to concanavalin A-Sepharose columns and could be eluted with the specific sugar, suggesting that the target structures may be glycosylated. NK-TS molecules could not be detected in gels of NK-insensitive target cells such as P815, A9HT, YWA, or EL-4. The quantity obtained from the gels varied directly with the NK sensitivity of YAC which is more sensitive when grown
in vitro
than when grown
in vivo
. The NK-TS molecules specifically inhibited the binding of NK cells but not alloimmune T cells to their appropriate targets. Additional NK-sensitive tumor cells also expressed some or all of the target molecules exhibited by YAC. Some of these structures shared specificities in the case of MPC-11 or were unique in the case of Molt-4 and K562, as shown by cross-inhibition studies. These results suggest that NK-sensitive cell lines express distinct target structures with possible relevance to natural tumor resistance.
Protein-lipid interactions in bilayer membranes: A lattice model
Authors: Pink David A. , Chapman Dennis
Published: May 2006

A lattice model has been developed to study the effects of intrinsic membrane proteins upon the thermodynamic properties of a lipid bilayer membrane. We assume that only nearest-neighbor van der Waals and steric interactions are important and that the polar group interactions can be represented by effective pressure—area terms. Phase diagrams, the temperature
T
0
, which locates the gel—fluid melting, the transition enthalpy, and correlations were calculated by mean field and cluster approximations. Average lipid chain areas and chain areas when the lipid is in a given protein environment were obtained. Proteins that have a “smooth” homogeneous surface (“cholesterol-like”) and those that have inhomogeneous surfaces or that bind lipids specifically were considered. We find that
T
0
can vary depending upon the interactions and that another peak can appear upon the shoulder of the main peak which reflects the melting of a eutectic mixture. The transition enthalpy decreases generally, as was found before, but when a second peak appears departures from this behavior reflect aspects of the eutectic mixture. We find that proteins have significant nonzero probabilities for being adjacent to one another so that no unbroken “annulus” of lipid necessarily exists around a protein. If
T
0
does not increase much, or decreases, with increasing
c
, then lipids adjacent to a protein cannot all be all-
trans
on the time scale (10
-7
sec) of our system. Around a protein the lipid correlation depth is about one lipid layer, and this increases with
c
. Possible consequences of ignoring changes in polar group interactions due to clustering of proteins are discussed.
Restriction enzyme analysis of mouse cellular type C viral DNA: emergence of new viral sequences in spontaneous AKR/J lymphomas.
Authors: Canaani E , Aaronson S A
Published: May 2006

The topography of endogenous type C viral sequences in mouse cellular DNA was investigated by EcoRI nuclease restriction and application of the Southern blotting technique. The DNAs from one outbred and five inbred strains were resolved into 20-35 fragments containing viral sequences, distributed in unique, though related, patterns for each mouse strain. Different normal tissues from the same animal were indistinguishable in their DNA patterns, suggesting that tissue differentiation is not associated with gross alteration in the topography of endogenous type C virus sequences. Tumor tissues from spontaneous lymphomas of AKR/J mice were similarly analyzed. In four out of seven individual tumors we detected the emergence of one or two new virus-containing DNA fragments. The mass of these fragment varied, indicating different insertion sites of the new viral sequences. The detection of these new viral sequences suggests that each tumor was composed of descendents of only one or a few cells.
Tryptic dissection and reconstitution of translocation activity for nascent presecretory proteins across microsomal membranes.
Authors: Walter P , Jackson R C , Marcus M M , Lingappa V R , Blobel G
Published: May 2006

The ability of microsomal membranes to translocate nascent presecretory proteins across their lipid bilayer into the intravesicular space was investigated by using trypsin as a proteolytic probe. We found that under defined conditions trypsin is able to dissect the translocation activity of microsomal membranes into components that can be separated into two fractions, one soluble and the other membrane bound. The trypsinized membrane fraction has lost its translocation activity. Addition of the trypsin-generated soluble fraction, however, results in reconstitution of translocation activity. These results are compatible with the notion proposed in the signal hypothesis that the translocation activity of the microsomal membrane resides in transmembrane protein(s). We propose that trypsin effects solubilization from the membrane of cytosol-exposed domain(s) involved in recognition of the signal sequence or ribosome or both, leaving behind membrane-integrated domain(s) that provide the environment for the passage of the nascent chain across the membrane. Signal peptidase activity was unaffected by trypsinization of microsomal vesicles consistent with a localization of the active site of this enzyme on the cisternal side of the vesicles.
Catabolic control of the enhanced alanine-preferring system for amino acid transport in glucose-starved hamster cells requires protein synthesis
Authors: Christopher C. William , Nishino Hoyoku , Schiller Robert M. , Isselbacher Kurt J. , Kalckar Herman M.
Published: May 2006

In cultured hamster cells starved for glucose for 24 hr there is an enhancement of the rate of α-aminoisobutyric acid transport (“shiftup”). When the starved cells are re-fed with glucose, the rate of transport shifts back down to the low, “regulated” rate typical of cells continuously fed with medium containing glucose (“shiftdown”). The high, deregulated rate of transport is maintained, however, when cycloheximide is present for 24 hr during the re-feeding with glucose. Maintenance of the high transport rate is evident only when the cells are incubated in amino acid-free medium just prior to the transport assay or when the assays are conducted with isolated membrane vesicles. A premature, pseudoshiftdown was observed in intact cells within as little as 2 hr after re-feeding when care was not taken to deplete the amino acid pool prior to the transport assay. In addition, a cycloheximide-insensitive increase in transport was observed when cultures were re-fed for 2 hr with amino acid-free medium containing fresh serum. These results emphasize the often overlooked precautions that should be taken to guard against artifacts that could mislead interpretations of amino acid transport data. More important, however, is the finding that Na
+
-dependent amino acid transport in cultured animal cells is regulated in part by a factor (or factors) that becomes inactivated when the cells are maintained under nonglycolytic culture conditions. In order to reactivate the control mechanism, starved cells that have been re-fed with glucose must resynthesize the regulatory factor(s). Thus, in at least cultured hamster cells, Na
+
-dependent amino acid transport regulation is much like the hexose transport regulation in that catabolic control (shiftdown) requires protein synthesis.
Biochemical and electron microscopic evidence that cell nucleus negatively controls mitochondrial genomic activity in early sea urchin development.
Authors: Rinaldi A M , De Leo G , Arzone A , Salcher I , Storace A , Mutolo V
Published: May 2006

Enucleated halves of sea urchin eggs obtained by centrifugation contain almost all the mitochondrial population of the egg. Removal of the nucleus followed by parthenogenetic activation stimulates the incorporation of [3H]thymidine into the mitochondrial DNA, whereas no such incorportion is observed in activated whole eggs. The block is not the result of a modification in the permeability of the mitochondrial membrane. Electron microscopic observations demonstrated duplication of mitochondrial DNA molecules in activated enucleated halves. No duplication was found in the mitochondrial DNA from activated whole eggs or from nonactivated enucleated halves. We conclude that the cell nucleus exerts a negative control on the activity of the mitochondrial genome through some short-lived nuclear substance(s).
Separation of human T lymphocyte subpopulations (Tμ, Tγ) by density gradient electrophoresis
Authors: Platsoucas Chris D. , Good Robert A. , Gupta Sudhir
Published: May 2006

Purified human peripheral blood T lymphocytes were fractionated by density gradient electrophoresis on the basis of their surface charge. The high mobility cell fractions were found to be enriched in T cells having receptors for IgM (Tμ cells) with only minor proportions of T cells having receptors for IgG (Tγ cells). In contrast, the low mobility cell fractions were enriched in Tγ cells with very low numbers of contaminating Tμ cells. Both populations were of higher mobility than human peripheral blood B lymphocytes. High-affinity sheep erythrocyte resette-forming cells (E-RFC) were relatively enriched in the high and intermediate mobility fractions and appear to include the Tμ cells and the T cells without receptors for IgG or IgM (Tϕ). The low affinity E-RFC were found only in the lower mobility fractions that included the Tγ cell population. A direct correlation was observed between the number of Tγ lymphocytes and the low affinity E-RFC in all the fractions. The separated cell fractions were treated
in vitro
with different concentrations of concanavalin A (Con A) and examined for the numbers of Tμ and Tγ cells. Low concentrations of Con A (2.5 μg/ml) significantly increased the number of Tμ cells, whereas high concentrations of Con A (20 μg/ml and 40 μg/ml) markedly reduced the number of Tμ cells and increased the number of Tγ cells. Furthermore, all fractions (both Tμ and Tγ cell enriched) responded by proliferation to Con A, whereas only the high and intermediate mobility fractions (enriched in Tμ cells) responded to phytohemagglutinin. Fractions enriched in Tγ cells responded very poorly to phytohemagglutinin. This method provides another technique for separating human Tμ- and Tγ-enriched lymphocyte subpopulations and does not modulate the Fc receptors of the cells, in contrast to the rosetting techniques currently in use for the separation of these lymphocytes.
Albumin microspheres as carrier of an inhibitor of leukocyte elastase: potential therapeutic agent for emphysema.
Authors: Martodam R R , Twumasi D Y , Liener I E , Powers J C , Nishino N , Krejcarek G
Published: May 2006

Methods are described for the covalent attachment of succinoyl-Ala-Ala-Pro-ValCH2Cl, an active site-directed inhibitor of human leukocyte elastase (EC 3.4.21.11), to microspheres of human albumin. The insertion of side arms of various lengths revealed that maximum inhibition of this enzyme was obtained when the spacer arm was at least 24.3 A in length. Approximately 30 molecules of the inhibitor could be attached to each molecule of albumin. Such derivatized microspheres were capable of inhibiting approximately one mole of elastase per mole of albumin, which is comparable to the inhibitory activity of alpha 1-antitrypsin. Experiments in vivo in which rats were injected intravenously with radiolabeled microspheres to which the inhibitor had been attached showed a rapid and exclusive uptake by the lungs. About 40--50% of the injected microspheres subsequently remained in the lungs with a half-life of approximately 17 days. These derivatized microspheres thus appear to offer promise as a therapeutic agent for emphysema.
Thermotropic behavior of some fluorodimyristoylphosphatidylcholines.
Authors: Sturtevant J M , Ho C , Reimann A
Published: May 2006

The thermotropic behavior of three fluorinated phospholipids has been studied by means of high-sensitivity differential scanning calorimetry. The three lipids are 1,2-di(X,X-difluoromyristovyl)phosphatidylcholine with X = 4,8, or 12. The compound with X = 4 has a higher transition temperature than dimyristoylphosphatidylcholine whereas the other two have lower values. All three lipids have transition enthalpies approximately twice that of the unsubstituted lipid. It was found the substituted lipids form strikingly nonideal mixtures with the parent compound. In view of these observations it is evident that results obtained using these or related compounds as probes of bilayer or membrane behavior must be interpreted with due caution. Some of the properties exhibited by geminal fluorine atoms in fluorinated phospholipids appear to be similar to those of cis double bonds in lipids.
The ovalbumin gene: cloning and molecular organization of the entire natural gene.
Authors: Dugaiczyk A , Woo S L , Colbert D A , Lai E C , Mace M L , O'Malley B W
Published: May 2006

We report the analyses of recently cloned restriction fragments of the natural ovalbumin gene that overlap in part with previously cloned DNA fragments but extend further into the flanking sequences of the gene. These clones now permit us to identify the DNA sequence that codes for the 5' end of ovalbumin mRNA. Based on these and previous results, the molecular organization of the entire ovalbumin gene was established. The entire gene is composed of eight structural DNA sequences separated by seven intervening sequences that are not present in the mature mRNA. In addition, an ovalbumin gene clone has been obtained from a chicken gene library. Analysis of DNA isolated from this particular clone by molecular hybridization and electron microscopic mapping revealed that it contains the entire ovalbumin gene a single segment of DNA and its structure was consistent with that predicted from our physical map constructed from individually cloned fragments of the gene.
Antibodies raised against purified β-adrenergic receptors specifically bind β-adrenergic ligands
Authors: Caron Marc G. , Srinivasan Yogambal , Snyderman Ralph , Lefkowitz Robert J.
Published: May 2006

Antibodies raised against purified β-adrenergic receptors themselves specifically bind β-adrenergic ligands. Digitonin-solubilized frog (
Rana pipiens
) erythrocyte β-adrenergic receptors, purified 100- to 200-fold by adsorption to an alprenolol-agarose affinity support and specifically eluted from the affinity resin by 1-100 mM (±)-isoproterenol, were used to immunize six rabbits. All immune sera, in contrast to preimmune sera, bound the β-adrenergic antagonist [
3
H]Dihydroalprenolol binding activity was due to immunoglobulins. By competition studies, antibody [
3
H]dihydroalprenolol binding was found to display a specificity and stereoselectivity resembling that of the β-adrenergic receptor, [i.e., (-)-isoproterenol > (-)-epinephrine > (-)-norepinephrine; alprenolol ≈ propranolol ≫ phentolamine = aloperidol; and (-) isomers of both agonists and antagonists 10-100 times more potent than (+) isomers]. A portion of the [
3
H]dihydroalprenolol binding antibodies could be specifically adsorbed onto purified frog erythrocyte membranes, whereas
Xenopus
and human erythrocyte membranes, both of which are almost devoid of β-adrenergic receptors, were ineffective in adsorbing [
3
H]dihydroalprenolol binding antibodies. We suggest that the likely immunogen was a β-adrenergic receptor-isoproterenol complex and that immunization with drugs noncovalently bound to their receptors might be a means of raising antibodies to biologically active otherwise nonimmunogenic small molecules. Such antibodies, whose specificity mimics that of a receptor, should also provide useful models for the study of the structure of the receptor binding sites.
Functional roles of DNA polymerases beta and gamma.
Authors: Hübscher U , Kuenzle C C , Spadari S
Published: May 2006

The physiological functions of DNA polymerases (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) beta and gamma were investigated by using neuronal nuclei and synaptosomes isolated from rat brain. UV irradiation of neuronal nuclei from 60-day-old rats resulted in a 7- to 10-fold stimulation of DNA repair synthesis attributable to DNA polymerase beta which, at this developmental stage, is virtually the only DNA polymerase present in the nuclei. No repair synthesis could be elicited by treating the nuclei with N-methyl-N-nitrosourea, but this way probably due to the inability of brain tissues to excise alkylated bases from DNA. The role of DNA polymerase gamma was studied in synaptosomes by using a system mimicking in vivo mitochondrial DNA synthesis. By showing that, under these conditions, DNA replication occurs in mitochondria, and exploiting the fact that DNA polymerase gama is the only DNA polymerase present in mitochondria, evidence was obtained for a role of DNA polymerase gamma in mitochondrial DNA replication. Based on these results and on the wealth of literature on DNA polymerase alpha, we conclude that DNA polymerase alpha is mainly responsible for DNA replication in nuclei, DNA polymerase beta is involved in nuclear DNA repair, and DNA polymerase gamma is the mitochondrial replicating enzyme. However, minor roles for DNA polymerase alpha in DNA repair or for DNA polymerase beta in DNA replication cannot be excluded.
Roles of actin during sporocarp culmination in the simple mycetozoan
Planoprotostelium aurantium
Authors: Spiegel Frederick W. , Olive L. S. , Brown R. Malcolm
Published: May 2006

Developing unicellular fruiting bodies of the protostelid
Planoprotostelium aurantium
examined by electron microscopy are shown to have a cortical zone of microfilaments that surrounds the lower portion of the sporogenic cell and extends into a cytoplasmic plug that fills the lumen of the tubular, microfibrillar stalk. Labeling with the S-1 fragment of myosin indicates that the microfilaments are actin. A model is proposed in which the actin is involved both in a contractile process that serves to raise the sporogen off the substrate at the tip of the stalk and in a cytoskeletal role by either directly or indirectly localizing the synthesis and orientation of the stalk tube microfibrils. The process of culmination in protostelids is compared to that of the multicellular dictyostelid cellular slime molds.
Actin polymerization induced by a motility-related high-affinity cytochalasin binding complex from human erythrocyte membrane
Authors: Lin Diane Chang , Lin Shin
Published: May 2006

A high molecular weight complex (sedimentation coefficient ≈27 S) containing high-affinity binding site(s) for [
3
H]dihydrocytochalasin B has been isolated from a low ionic strength extract of human erythrocyte membranes by sucrose density gradient centrifugation. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed that actin, spectrin, and other minor components, including two polypeptides with the electrophoretic mobility of band 4.1, were present in the complex-containing fraction. Addition of this complex to a solution of muscle monomeric actin (G-actin) in a low ionic strength medium resulted in a rapid increase in viscosity to a level comparable to that of a solution of filamentous actin (F-actin). Electron microscopy showed that the viscosity increase reflected actin filament formation. The rate of induced actin polymerization was dependent on the amount of complex added to the G-actin; in less than 1 hr, less than 1 μg of protein from the complex-containing fraction induced the conversion of 0.4 mg of G-actin to the “F” from. Binding studies indicated that, upon polymerization of the actin, the cytochalasin binding complex became associated with the actin filaments. Low concentrations of cytochalasins D and E and dihydrocytochalasin B inhibited actin polymerization induced by the complex; the relative potencies of the drugs in inhibiting this process corresponded to their relative affinities for the complex, as well as their relative potencies in affecting cell motility. These results suggest that the cytochalasin binding complex functions as a regulatory site for cell motility by controlling formation and membrane attachment of actin-containing microfilaments in the cell.
Detection by complementation of defective or uninducible (herpes simplex type 1) virus genomes latent in human ganglia.
Authors: Brown S M , Subak-Sharpe J H , Warren K G , Wroblewska Z , Koprowski H
Published: May 2006

Reconstruction experiments have shown that temperature-sensitive (ts) mutants of herpes simplex virus type 1 (HSV-1)(Glasgow strain 17) grow, complement, and recombine with similar efficiency in human nerve ganglion cells, human brain cells, normal human fibroblasts (WI38), and baby hamster kidney (BHK) 21/C13 hamster cells. Cultures of human trigeminal, superior cervical, and vagus ganglia that had failed to release herpes simplex virus spontaneously were superinfected with a range of ts mutants of HSV-1 and incubated at both permissive (31 degrees C) and nonpermissive (38.5 degrees C) temperatures. Progeny virus was assayed at both temperatures to determine if complementation of or recombination with the input genomes had occurred. The results showed that the ganglia from 8 of 14 individuals, which had been consistently negative for spontaneous release of virus, contained information that could be detected or rescued following superinfection with ts mutants of herpes simplex virus. In two additional cases, positive results were obtained after the superinfection of negative ganglia explants, but in each of these herpes simplex virus had previously been spontaneously released from one of six ganglia explanted.
Lymphoma development in mice and humans: diversity of initiation is followed by convergent cytogenetic evolution.
Authors: Klein G
Published: May 2006

Human B cell lymphoma and murine T cell leukemia can be initiated by several agents. The present paper formulates some thoughts on the role of cytogenetic changes in the subsequent neoplastic process. Initiation creates long-lived preneoplastic cells. In some respects, they are comparable to in vitro-transformed ("immortalized") cell lines that maintain a diploid karyotype and are not tumorigenic in vivo. The development of a tumorigenic ("autonomous") clone is dependent on additional changes at the genetic level. In human B and murine T cell lymphoma, there are characteristic nonrandom chromosomal changes. The 14q+ marker appears to play a key role in human B cell lymphomas. The reciprocal 8;14 translocation in Burkitt lymphoma is a specialized subclass within this category. In murine T cell leukemia, trisomy 15 is the predominant change. The clustering of these nonrandom changes to tumors derived from a certain cell type rather than to tumors induced by a given etiological agent has important implications for the understanding of the genetic control of cellular responsiveness to growth-regulating forces in vivo.
Multiple transitions induced by light intensity fluctuations in illuminated chemical systems
Authors: Lefever R. , Horsthemke W.
Published: May 2006

The influence of external noise on the steady-state properties of a chemical system with very simple kinetics was studied. We dealt with an isomerization reaction, in a closed system, coupled to a light absorption and radiationless deexcitation process involving only one of the isomers. The theoretical analysis established that external noise deeply modified the macroscopic properties of the system and even induced transition phenomena that are totally unexpected from the usual deterministic description.
Delimitation of a promoter for RNA polymerase III by means of a functional test.
Authors: Telford J L , Kressmann A , Koski R A , Grosschedl R , Müller F , Clarkson S G , Birnstiel M L
Published: May 2006

A Xenopus laevis DNA segment containing the structural gene for tRNA1Met and 22 base pairs at the 5' side of the gene is active in tRNA production. The DNA segment was ligated to sea urchin histone DNA and was also inserted into plasmid pCR1. Both recombinant DNAs were shown to produce mature tRNA1Met at a high rate when injected into centrifuged Xenopus oocytes.
Fibronectin and the multiple interaction model for platelet-collagen adhesion.
Authors: Santoro S A , Cunningham L W
Published: May 2006

A rapid, sensitive, and reproducible assay to determine the adhesion of platelets to collagen has been developed. Collagen fibers and adherent platelets are retained on polycarbonate membrane filters. Chemical modification of collagen by acetylation and of platelets by treatment with chymotrypsin markedly reduces adhesion. The role of fibronectin in the collagen-platelet interaction has been examined. Treatment of platelets with purified antibody or Fab' fragments to fibronectin only slightly reduces adhesion. Preincubation of platelets with high concentrations of gelatin reduces adhesion by only 22% but fails to inhibit aggregation. Thus, fibronectin has only a limited role in the adhesion of platelets to collagen and is either not involved in the adhesion that leads to aggregation or is only one of several adhesion mechanisms, any of which alone can initiate aggregation.
Stimulation of sugar uptake and glycolysis in chicken embryo fibroblasts by the major glycoprotein from avian myeloblastosis virus.
Authors: Papamatheakis J D , Marciani D J
Published: May 2006

Addition of purified major glycoprotein from avian myeloblastosis virus to growing or quiescent chicken embryo fibroblasts rapidly stimulates the rate of hexose transport and increases the lactic acid production. These stimulatory effects are dependent on the time of exposure and the dose of viral glycoprotein. In contrast, the glycoprotein only marginally affects hexose transport in chicken cells transformed by Rous sarcoma virus. Some effects of the glycoprotein on serum-starved quiescent cells were similar to those observed upon re-addition of serum; however, the viral glycoprotein did not stimulate DNA synthesis. Quiescent cells stimulated by saturating levels of serum showed little further stimulation of hexose uptake upon exposure to viral glycoprotein for 3 hr. This behavior suggests that the glycoprotein may be acting on a system that is also a target for serum action.
Clonal evolution of myeloma cells leads to quantitative changes in immunoglobulin secretion and surface antigen expression.
Authors: Leibson P J , Loken M R , Panem S , Schreiber H
Published: May 2006

We report that a cloned population of tumor cells can rapidly produce variants that differ in their quantitative expression of surface proteins and in their rate of immunoglobulin secretion. A fresh clonal isolate of S107 myeloma cells possessing large amounts of surface IgA was continuously passaged in vitro for 2 years. During this period, fluorescence-activated cell sorter analysis indicated the development of subpopulations possessing decreased amounts of surface IgA. Cells from these variant subpopulations were isolated by first using the cell sorter to enrich for cells with decreased amounts of surface IgA and then cloning the selected population in soft agar. The 50 sublines that were isolated showed heritable differences in their levels of surface IgA and H-2 antigens and in their rates of myeloma protein secretion. Sublines having either large amounts, intermediate amounts, or absence of surface IgA also had corresponding large amounts, intermediate amounts, or absence of myeloma protein secretion. In contrast, a decrease or loss of surface Ig did not correlate with a decrease or loss of viral envelope glycoprotein gp71 and H-2 antigens. The variants did not resemble the phenotypes of less-differentiated normal lymphocyte populations of the B-cell lineage. The isolation and characterization of these variants allows us to explore the mechanisms and pathways of tumor cell differentiation as well as to study the regulation and function of cell surface proteins.
Fetal and adult albumins are indistinguishable by immunological and physicochemical criteria
Authors: Gitzelmann-Cumarasamy Nilou , Gitzelmann Richard , Wilson Kenneth J. , Kuenzle Clive C.
Published: May 2006

The existence of a functionally immature fetal albumin has been postulated to explain the reduced ability of newborn plasma to bind bilirubin and various drugs. In support of this, cord and adult albumin, isolated by a simple salting-out technique, were reported to differ in electrophoretic and chromatographic properties and in their resistance to alkali and proteolytic enzymes. However, the interpretation of these findings has since been questioned. To resolve this controversy, we have purified to homogeneity human serum albumins from pooled umbilical cord and adult donor plasma. The two albumins were compared and found to be indistinguishable by polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, as well as by immunoelectrophoresis and double immunodiffusion using specific antibodies against both albumins. Furthermore, the amino acid compositions, the aminoterminal sequence (Asp-Ala-His-Lys-Ser-Glu-Val-Ala-), the carboxy terminus (Leu), and the peptide fingerprints were identical in the two albumins. No significant differences were found by circular dichroism in the ultraviolet (200-350 nm). Binding studies with bilirubin showed association constants of 3.7 ± 0.7 × 10
7
M
-1
for cord and 2.9 ± 0.3 × 10
7
M
-1
for adult albumin, respectively. The circular dichroic spectra of 1:1 bilirubin·albumin complexes showed considerable variation between the batches but were not significantly different. The only difference was found in the fluorescence spectra of the bilirubin·albumin complexes, in which complexes with adult albumin showed only 75% of the relative fluorescence exhibited with cord albumin. The combined results nevertheless strongly indicate that fetal and adult albumins are very similar, if not identical.
Pattern recognition in genetic sequences
Authors: Sellers Peter H.
Published: May 2006

This paper announces an algorithm for finding pattern similarities between two given finite sequences. Two portions, one from each sequence, are similar if they are close in the metric space of evolutionary distances. In its most general form the algorithm allows a complete list to be made of all pairs of intervals, one from each of the two given sequences, such that each pair displays a maximum local degree of similarity; if the lengths of the sequences are
m
and
n
, then the algorithm requires on the order of
mn
steps. This result lends itself to detecting similarities by computer between pairs of biological sequences, such as proteins and nucleic acids.
Sequence heterogeneity at the 5' termini of late simian virus 40 19S and 16S mRNAs.
Authors: Canaani D , Kahana C , Mukamel A , Groner Y
Published: May 2006

The 5'-cap-containing leader sequence of the most abundant 19S and 16S mRNAs of simian virus 40 (SV40) was previously mapped between 0.67 and 0.76 map units. We now find that the two late mRNA species contain multiple 5' ends. Eight different RNase T2-resistant cap structures were identified:m7GpppmAmpU (47%); m7GpppmAmpUmpU (19%); m7GpppmAmpC (16%); m7GpppmAmpCmpA (5%); m7GpppmAmpG (6%); m7GpppGmpC (3%); m7GpppmAmGmpA (2%); m7GpppGmpCmpG (2%). Capped T1 oligonucleotides of 19S and 16S mRNAs have been isolated by two different procedures: (i) chromatography on a DEAE-cellulose column followed by paper electrophoresis and (ii) two-dimensional electrophoresis/homochromatography. Cap structures of the isolated 5' oligonucleotides were identified. Each of the major caps was found to be associated with a few differential 5' oligonucleotides, implying a vast heterogeneity at the termini of SV40 late mRNAs. The results suggest that on SV40 DNA, RNA polymerase II has a reportoire of initiation points. In most of the cases, initiation takes place with adenosine triphosphate followed by a pyrimidine. Alternatively, transcription may start at one specific point but a unique mechanism of processing generates heterogeneous populations of termini with a common 5' adenosine triphosphate.
Real-time solvent exchange studies of the imino and amino protons of yeast phenylalanine transfer RNA by Fourier transform NMR.
Authors: Johnston P D , Figueroa N , Redfield A G
Published: May 2006

Real-time solvent exchange measurements using Fourier transform NMR at 270 MHz are presented. By means of the fast gel filtration column techniques originally developed for tritium exchange experiments, we were able to replace the solvent of a tRNA sample from an 1H2O to an 2H2O buffer and obtain a useful spectrum in 2-5 min. At 15 degrees C, there are 5 +/- 1 lowfield (-11 to -15 ppm relative to 2,2-dimethyl-2-silapentane-5-sulfonate) imino protons with exchange half times of minutes to hours. In addition, the m7G-46 C(8) proton and several amino protons are observed to exchange with similar rates. Analogous studies on unfractionated yeast tRNA suggest that such a class of slowly exchanging imino protons is present in several tRNAs, and that the activation energy for exchange is small [[approximatley 5 kcal/mol (21 kJ/mol)]. We speculate that these imino resonances arise from D-stem protons and that their slow exchange reflects stabilization by the numerous tertiary interactions involving this stem and the Mg2+ bound at the P-10 bend.
Contacts between Escherichia coli RNA polymerase and thymines in the lac UV5 promoter.
Authors: Simpson R B
Published: May 2006

I have identified those 5 positions of thymines in the lac UV5 promoter that lie close to bound Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). Although ultraviolet irradiation of DNA with 5-bromouracil substituted in place of thymine normally cleaves the DNA at the bromouracils, a protein bound to the DNA can perturb these cleavages at those locations at which the protein lies close to the bromine. In the lac promoter most of these contacts lie in three regions. Four contacts lie in the region where transcription initiates; four lie in the "Pribnow box," which is located about 10 base pairs upstream from the initiation site; and three more lie in the "-35 region," located about 35 base pairs upstream from the initiation site. The "Pribnow box" and the "-35 region" are regions whose sequences are partially conserved between promoters and in which most promoter mutations are located; thus, contacts in these two regions probably represent sites of sequence-specific recognition by RNA polymerase.
Subunit stoichiometry and molecular weight of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.
Authors: Angelides K J , Akiyama S K , Hammes G G
Published: May 2006

The molar ratio of the component enzymes of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was found to be 1.8:1.7:1[pyruvate decarboxylase (E1):dihydrolipoyl transacetylase (E2):dihydrolipoyl dehydrogenase (E3)]. This ratio was determined by measuring the Coomassie blue staining of the constituent enzymes after sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. The above ratio is the average of four separate experiments with two different enzyme preparations. The average molecular weights of the individual enzymes were found to be 96,000, 76,000, and 55,000 for E1, E2, and E3, respectively, by sodium dodecyl sulfate and sodium dodecyl sulfate/8 M urea polyacrylamide gel electrophoresis and by column chromatography in 6 M guanidine . HCl. The molecular weight of E2 was reduced to 33,000-36,000 after extensive reduction and alkylation with iodoacetamide. The molecular weights of the complex, E1, and E3 were found to be 4,800,000, 182,000, and 104,000, respectively, with low-angle laser light scattering. Both E1 and E3 are dimeric under the conditions employed. If octahedral symmetry is assumed for the E2 core, a polypeptide chain ratio of 24:24:12 (E1:E2:E3) is in good agreement with the measured molar ratio of component enzymes and the molecular weight of the pyruvate dehydrogenase complex.
Biofunctional evaluation of a hydrogen bond linking the ring and tail beta-turns of oxytocin.
Authors: Roy J , Roy U , Gazis D C , Schwartz I L
Published: May 2006

Deamino-[8-N-methylleucine]oxytocin and deamino-[8-alpha-hydroxyisocaproic acid]oxytocin were synthesized to study the importance of hydrogen bonding between the carboxamide carbonyl of asparagine and the peptide N-H of leucine in stabilizing the biologically active conformation of oxytocin. The analogs were synthesized by coupling deaminotocinoic acid with Pro-Leu(Me)-Gly-NH2 and Pro-HyIc-Gly-NH2, respectively. (HyIc is alpha-hydroxyisocaproic acid). Deamino-[8-N-methylleucine]oxytocin was found to possess 48 +/- 7 units of uterotonic activity, 33 +/- 5 units of avian vasodepressor activity, and 3.15 +/- 1.5 units of antidiuretic activity per mg; deamino-[8-alpha-hydroxyisocaproic acid]oxytocin possessed 134 +/- 12 units of uterotonic activity, 31 +/- 3 units of avian vasodepressor activity, 9.6 +/- 3.0 units of antidiuretic activity, and 0.26 +/- 0.02 unit of pressor activity per mg. Neither of the analogs possesses the peptide N-H at residue 8 required for the formation of a hydrogen bond with the asparagine carboxamide; however, both can assume the conformation needed to evoke the characteristic biological activities of oxytocin although in lower potency. It is concluded that such a hydrogen bond does not constitute a conformational constraint that is essential for hormone action.