bluuebunny/crossref_metadata_2025 huggingface dataset:
Pyrophosphate:protein phosphotransferase: A membrane-bound enzyme of endoplasmic reticulum
Authors: Lam Kit S. , Kasper Charles B.
Published: May 2006

Recently, we demonstrated that highly purified rat liver microsomal membrane was capable of selectively phosphorylating two intrinsic membrane polypeptides (
M
r
145,000 and
M
r
130,000) and that the course of the reaction was kinetically divided into two distinct stages [Lam, K. S. & Kasper, C. B. (1980)
J. Biol. Chem.
255, 259-266]. Evidence was also presented that strongly suggested that a phosphoryl donor other than ATP was involved in the second stage of phosphorylation. In the present study, we demonstrate that incubation of microsomal membrane with [γ-
32
P]ATP produces a prominent
32
P-labeled compound detectable by thin-layer chromatography on polyethyleneimine-impregnated cellulose. DEAE-cellulose fractionation of detergent-solubilized microsomal membrane generated a protein fraction that could convert in excess of 90% of the [γ-
32
P]ATP into this newly
32
P-labeled unknown compound (I∼
P
) without the formation of significant levels of
32
P
i
. When [α-
32
P]ATP was used, I∼
P
was unlabeled. Enzymically synthesized I∼
P
was purified and determined to be pyrophosphate by using
31
P NMR spectroscopy. [
32
P]Pyrophosphate, synthesized chemically or enzymically, was capable of selectively phosphorylating the
M
r
145,000 and
M
r
130,000 polypeptides. Time course studies utilizing pyrophosphate as the phosphate source showed only one phase of phosphorylation that was strongly inhibited by micromolar levels of ATP as well as by NaF (5 mM). These studies further establish that pyrophosphate is the phosphoryl donor involved in the second stage of phosphorylation.
On reagents that convert cytochrome oxidase from an inactive to an active coupling state.
Authors: Green D E , Fry M
Published: May 2006

Cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) of beef heart mitochondria, prepared by a standard method and brought to the highest purity level, is essentially inactive when tested in the aerobic assay involving oxidation of reduced cytochrome c by molecular oxygen. Three reagents (lysolecithin, Tween 20, and exogenous phospholipids) can convert cytochrome oxidase from an inactive to an active coupling state. These conversions are reversible: i.e., removal of the inducing agent leads to loss of activity. The evidence for the intrinsic coupling capability is that cytochrome oxidase in the active state invariably generates a proton gradient during respiration, and such gradient formation is demonstrable even when cytochrome oxidase is not inserted into a liposome.
Ecological and evolutionary significance of mycorrhizal symbioses in vascular plants (A Review)
Authors: Malloch D. W. , Pirozynski K. A. , Raven P. H.
Published: May 2006

Mycorrhizae, the symbioses between fungi and plant roots, are nearly universal in terrestrial plants and can be classified into two major types: endomycorrhizae and ectomycorrhizae. About four-fifths of all land plants form endomycorrhizae, whereas several groups of trees and shrubs, notably Pinaceae, some Cupressaceae, Fagaceae, Betulaceae, Salicaceae, Dipterocarpaceae, and most Myrtaceae form ectomycorrhizae. Among legumes, Papilionoideae and Mimosoideae have endomycorrhizae and usually form bacterial nodules. The members of the third subfamily, Caesalpinioideae, rarely form nodules, and one of the included groups, the two large, pantropical, closely related tribes Amherstieae and Detarieae, regularly form ectomycorrhizae. Nodules and ectomycorrhizae may well be alternative means of supplying organic nitrogen to the plants that form them.

Those plants having endomycorrhizae usually occur in forests of high species richness, whereas those with ectomycorrhizae usually occur in forests of low species richness. The roots of ectomycorrhizal trees, however, support a large species richness of fungal symbionts, probably amounting to more than 5000 species worldwide, whereas those of endomycorrhizal trees have low fungal species richness, with only about 30 species of fungi known to be involved worldwide. Ectomycorrhizal forests are generally temperate or occur on infertile soils in the tropics. They apparently have expanded in a series of ecologically important events through the course of time from the Middle Cretaceous onward at the expense of endomycorrhizal forests.
Resistance of herpes simplex virus to acycloguanosine: role of viral thymidine kinase and DNA polymerase loci.
Authors: Schnipper L E , Crumpacker C S
Published: May 2006

Acycloguanosine [9-(2-hydroxyethoxymethyl)guanine; acyclo-Guo] is a potent inhibitor of herpes simplex viruses (HSV); it is selectively phosphorylated in virus-infected cells. In order to define those viral functions that may mediate resistance to acyclo-Guo, the drug sensitivities of temperature-sensitive (ts) and phosphonoacetic acetic acid (PAA)-resistant mutants of HSV-1 and HSV-2 have been determined. Two distinct viral genetic loci are independently associated with acyclo-Guo resistance. Mutations resulting in diminished thymidine kinase activity are associated with resistance to inhibition by acyclo-Guo. Several PAA-resistant viruses that express wild-type levels of thymidine kinase activity are also resistant to acyclo-Guo. This suggests the importance of the viral DNA polymerase region in mediating acyclo-Guo resistance and is consistent with a close relationship between the PAAr mutation site and the AGGr locus. When wild-type HSV-1 is serially propagated under the selective pressure of acyclo-Guo, rapid emergence of resistant virus occurs, accompanied by the simultaneous appearance of thymidine kinase-deficient progeny.
Denervation increases turnover rate of junctional acetylcholine receptors.
Authors: Loring R H , Salpeter M M
Published: May 2006

The turnover rates of junctional acetylcholine receptors were measured in innervated and denervated mouse sternomastoid neuromuscular junctions by 125I-labeled alpha-bungarotoxin binding. First, we determined that the density of labeled toxin initially bound to the neuromuscular junction was essentially unchanged up to 16 days after denervation. Innervated muscles and muscles that had been denervated 8 days previously were then saturated with labeled toxin, and the specific label at the endplate regions was compared by gamma counting 7 days later. At that time, the residual junctional label seen in innervated muscle was 3.2 times greater than in denervated muscle. Electron microscope autoradiography further showed that, after saturation with unlabeled toxin, new binding sites appeared rapidly at the specialized receptive region of the postsynaptic membrane with an apparent half-time of turnover of 2-3 days. At innervated junctions, the half-time of turnover was about 10 days. These data show that the mechanisms that control receptor turnover rates are different from those that control high-density receptor clustering. The slow turnover rate of junctional receptors appears to be more directly dependent on the presence of the nerve than is the clustering of junctional receptors.
Identification of prosomatostatin in pancreatic islets.
Authors: Patzelt C , Tager H S , Carroll R J , Steiner D F
Published: May 2006

A 12.5-kilodalton protein not related to insulin or glucagon was detected in pulse-chase-labeled rat islets of Langerhans. Although this protein reacted poorly with various somatostatin antisera, analysis of two-dimensional peptide maps showed that it contains all of the tryptic fragments of somatostatin, which is located at its COOH terminus. Proteolytic conversion of the putative prosomatostatin, which took place parallel to the processing of proinsulin and proglucagon in pulse-chase experiments, coincided with the appearance of newly synthesized somatostatin and proceeded without the apparent involvement of major intermediate forms.
Photolyzed rhodopsin catalyzes the exchange of GTP for bound GDP in retinal rod outer segments.
Authors: Kwok-Keung Fung B , Stryer L
Published: May 2006

We have studied the binding of guanyl nucleotides to retinal rod outer segment membranes to determine how light activates a cyclic GMP phosphodiesterase and a GTPase. We found that rod outer segment membranes contain tightly bound radioactive GDP after incubation in the dark with [3H]GDP or [alpha-32P]GTP. Reconstituted membranes containing only rhodopsin and phospholipid bind almost no GDP. More than 80% of the radioactive GDP bound to rod outer segment membranes could be released by subsequent illumination. At low light levels, the rate and extent of GDP release were markedly enhanced by the presence of GTP or p[NH]ppG, a nonhydrolyzable analog of GTP. The kinetics of binding of p[NH]ppG paralleled the kinetics of release of bound GDP, indicating that p[NH]ppG was exchanged for bound GDP. The maximal amount of bound p[NH]ppG was 1 per 30 rhodopsins when photolyzed membranes were incubated with 10 micro M nucleotide. Under these conditions, p[NH]ppG binding was half-maximal when only 1 in 90,000 rhodopsins was photolyzed. This corresponds to the catalyzed exchange of 500 p[NH]ppG for bound GDP per photolyzed rhodopsin. We propose a light-activated GTP-GDP amplification cycle involving a guanyl nucleotide binding protein with GTPase activity (E). The essence of this cycle is that photolyzed rhodopsin catalyzes the formation of E . GTP from E . GDP (the major species in the dark) by nucleotide exchange. The formation of several hundred E . GTP per photolyzed rhodopsin may be the first stage of amplification in visual excitation.
Contrasts between oxygenic and anoxygenic photoreduction of ferredoxin: Incompatibilities with prevailing concepts of photosynthetic electron transport
Authors: Arnon Daniel I. , Tsujimoto Harry Y. , Tang George M.-S.
Published: May 2006

An investigation by paramagnetic resonance spectroscopy of the photoreduction of ferredoxin, oxygenically by water and anoxygenically by a direct electron donor to photosystem I, led to the unexpected findings that different reductive mechanisms may be involved. Ferredoxin photoreduced by water was not reoxidized in the light under aerobic conditions and, under anaerobic conditions, it was remarkably resistant to reoxidation in the dark. By contrast, ferredoxin photoreduced by a donor to photosystem I was readily reoxidized in the light by air and, under anaerobic conditions, by exposure to darkness. Furthermore, when electron transport linking photosystems I and II was inhibited by a plastoquinone antagonist, ferredoxin was photoreduced by water with no evidence for an accompanying photoreduction of the more electronegative bound iron-sulfur centers in chloroplasts. These findings are at variance with the now prevalent concepts of photosynthetic electron transport.
Effect of uncouplers on radiosensitivity and mutagenicity in x-irradiated mammalian cells.
Authors: Laval F
Published: May 2006

The number of x-irradiated mammalian cells surviving is markedly increased when the cells are incubated with an uncoupler of oxidative phosphorylation prior to or immediately after irradiation. This increase is greater in plateau-phase cells than in exponentially growing cells. The increase in survival is related to the potency of the uncouplers, which do not modify the effective x-ray dose. The influence of uncouplers on survival is related to an increase of repair and semiconservative DNA synthesis. The mutation frequency (8-azaguanine-resistant mutants) is significantly higher in irradiated cells treated with uncouplers than in untreated cells. These results suggest the existence of an error-prone repair process in mammalian cells.
Calmodulin localization in mammalian spermatozoa.
Authors: Jones H P , Lenz R W , Palevitz B A , Cormier M J
Published: May 2006

The location of calmodulin in rabbit and guinea pig spermatozoa was determined by indirect immunofluorescence techniques. Spermatozoa that had not undergone the acrosome reaction exhibited four distinct regions of calmodulin-specific immunofluorescence: around the acrosome, in a band across the lower third of the head, and in two localized areas at the base and tip of the flagellum. In contrast, after the acrosome reaction, although other features of calmodulin distribution remained the same, the fluorescence associated with the anterior half of the head was notably absent. Instead, fluorescence was associated with the membranes that had separated from the sperm head. These findings suggest a potential role for calmodulin in the Ca2+-dependent control of sperm activation, in sperm-egg fusion, and in microtubule disassembly processes in the flagellum.
Gene-directed mutagenesis in bacteriophage T7 provided by polyalkylating RNAs complementary to selected DNA sites.
Authors: Salganik R I , Dianov G L , Ovchinnikova L P , Voronina E N , Kokoza E B , Mazin A V
Published: May 2006

Bacteriophage T7 early transcripts were used as carriers of alkylating groups to affect complementary T7 DNA sites for inducing mutations in preselected genes. A heterofunctional polyalkylating agent N,N,N'-tri-(beta-chloroethyl)-N'-(p-formylphenyl)propylene diamine-1,3 was attached to 3-5% of the transcript nucleotides. The controlled alkylating groups carried on RNA were activated after RNA . DNA hybridization. The modified transcripts were shown to hybridize only with the complementary H strand and to form covalently bound R loops in the appropriate T7 sites. The T7 DNA molecules locally alkylated by the modified transcripts of gene 1.3 coding for T7 ligase were packaged into T7 proteins and used to infect Escherichia coli B. As judged by plating efficiency and the ligase-deficient E. coli BL2 strain, 4 of 140 plaques obtained after infection contained mutants defective in gene 1.3 in amounts of 0.7-1.5%. The T7 DNA locally alkylated by the modified transcripts of gene 0.3 and 1.1 were used for the transfection of E. coli C1757. Analysis of 24 plaques produced by transfection indicated that 3 contained mutants defective in gene 0.3 in amounts of 2-10%. The mutants had also a second unidentified mutation. Complementation analysis data suggest that the second mutation is due to a defect in gene 1.1. The results obtained demonstrate the efficiency of the approach developed for gene-directed mutagenesis.
Natural cytotoxic cells against solid tumors in mice: blocking of cytotoxicity by D-mannose.
Authors: Stutman O , Dien P , Wisun R E , Lattime E C
Published: May 2006

Natural cytotoxic (NC) and natural killer (NK) cells have been defined by their ability to lyse certain solid or lymphoid tumor targets in vitro, without prior sensitization. Our present studies describe an attempt to characterize the structures involved in the effector-target recognition leading to tumor cell lysis. Addition of the monosaccharide D-mannose to the NC cell assay significantly blocked cytotoxicity of the fibrosarcoma Meth A target by the effector cells at 50 mM and lower concentrations. D-Galactose showed blocking activity in one of five experiments, only at 50 mM. L-Fucose, D-glucose, and N-acetyl-D-glucosamine did not affect NC cell cytotoxicity at similar concentrations. All of the sugars tested inhibited NK cell lysis of the lymphoma YAC-I target. None of the sugars affected killing of the appropriate target by allosensitized cytotoxic T lymphocytes. The blocking of NC-mediated cytotoxicity was not due to a direct toxic action of the sugars on the effector cells. These findings suggest that, in the NC system, recognition involves lectin-like structures with a specificity for D-mannose (or D-galactose, or both), whereas, in the NK system, such lectin-like structures are less restricted. Such structures appear not to be involved in the specific cytotoxicity mediated by T cells.
Characterization of a monoclonal antibody that defines an immunoregulatory T cell subset for immunoglobulin synthesis in humans.
Authors: Haynes B F , Mann D L , Hemler M E , Schroer J A , Shelhamer J H , Eisenbarth G S , Strominger J L , Thomas C A , Mostowski H S , Fauci A S
Published: May 2006

This study characterizes a monoclonal antibody (3A1), and partially characterizes the cell surface antigen and the functional peripheral blood T cell subset that it defines. The 3A1 antigen is present on the surface of several human T cell lines (HSB-2, CEM, MOLT-4, and others) in various amounts but is absent from the T cell line YT4E and all human B cell lines tested. Immunoprecipitation of an HSB-2 extract with 3A1 yielded one specific band with a molecular weight of approximately 40,000 in the presence of reducing agent. With directly fluoresceinated 3A1 antibody, fluorescence-activated cell sorter analysis showed that 85% of peripheral blood E-rosette-positive T cells were positive for the 3A1 antigen. After E-rosette-positive cells had been separated into 3A1+ and 3A1- cell suspensions, the 3A1+ cells helped autologous peripheral blood B cell suspensions toward pokeweed mitogen-driven proliferation and intracytoplasmic Ig production, whereas 3A1-T cells did not. Further, addition of 3A1- cells from some but not all normal subjects to cocultures of 3A1+ cells and B cells actively suppressed intracytoplasmic Ig production. However, the 3A1+T cell subset could be activated by concanavalin A to maximally suppress B cell Ig synthesis in vitro. Thus, the 3A1 antibody defines a major functional subject of peripheral blood T cells and should provide a useful marker for the study of human T cell function.
Potential leukemic cells among bone marrow cells of young AKR/J mice.
Authors: Haran-Ghera N
Published: May 2006

Potentially leukemic cells have been identified among bone marrow cells of AKR/J mice from the age of 14 days onward. Transfer of AKR/J bone marrow into irradiated hybrid mice (AKR/J X DBA/2)F1 caused a high leukemia incidence (50-100%) of AKR origin, very often within a short latent period. Similar transfer of AKR/J marrow into irradiated AKR/J recipients did not enhance spontaneous tumor development. In contrast to the leukemic AKR cells that express the T-cell surface component Thy-1.1, the potential leukemic cells among bone marrow cells of young AKR mice were shown to lack the expression of this antigen. The development of preleukemic AKR marrow into overt leukemia in hybrid mice was dependent on the presence of an intact thymus and exposure of the recipients to x-rays shortly before marrow transfer. Evidently, preleukemic AKR bone marrow undergoes sequential changes, affected by host factors, leading ultimately to development of overt leukemia.
Multiple components of blood group A and B antigens in human erythrocyte membranes and their difference between A1 and A2 status.
Authors: Fujii H , Yoshida A
Published: May 2006

Human type O erythrocyte membranes were converted to type A1 by purified human A1-enzyme, to type A2 by purified human A2-enzyme, and to type B by purified human B-enzyme in the presence of radioactive sugar donors (i.e., UDP-N-acetylgalactosamine for A-enzyme and UDP-galactose for B-enzyme, respectively). Type A2 erythrocyte membranes were also converted to type A1 by purified A1-enzyme A1-enzyme. The labeled blood group antigens (A1, A2, and B) thus produced were analyzed by sodium dodecyl sulfate gel electrophoresis and by isoelectric focusing. It was demonstrated that blood group antigens on erythrocyte membranes are mostly (greater than 80%) glycolipid, not glycoprotein. The blood group glycolipids were presumably conjugated with other membrane materials and formed three major components in type A1 and type B erythrocyte membranes. However, two of the three components were absent in type O erythrocyte membranes converted to type A2. These results indicate the heterogeneity of blood group components in erythrocytes membranes and the qualitative difference between A1 and A2 antigens on these membranes.
Binding of O2 and CO to hemes and hemoproteins.
Authors: Traylor T G , Berzinis A P
Published: May 2006

Enthalpies and entropies have been determined for the reversible binding of O2 and CO to chelated protoheme, a compound having a covalently attached imidazole bound to the iron. The values, based upon 1 atm standard state, are delta HO2 = -14.0 kcal (1 kcal = 4.18 kJ)/mol, delta SO2 = -35 eu, delta HCO = -17.5 kcal/mol, delta SCO = -34 eu, delta H identical to O2 = 21 kcal/mol (dissociation), and delta H identical to CO = 25 kcal/mol (dissociation). The similarity of these values to those of high-affinity hemoproteins such as isolated hemoglobin chains or R-state hemoglobin (delta HO2 = -13.5, delta HCO = -17.5) show that this model compound accurately mimics the dynamic behavior of these hemoproteins, in contrast to the behavior of other, more elaborate, model compounds. The enthalpy of the replacement of O2 by CO, delta HM, is 3.5 kcal/mol, abut the same as that of R-state hemoglobin. This result obtained with the model compound which most resembles the hemoglobin active site, indicates that distal side steric effects in these hemoproteins neither decrease CO affinity nor differentiate between the binding of CO anmd O2. Consequences of these findings in the binding of O2 and CO to hemoproteins are discussed.
Theoretical model for the cooperative equilibrium binding of myosin subfragment 1 to the actin-troponin-tropomyosin complex.
Authors: Hill T L , Eisenberg E , Greene L
Published: May 2006

Recent experimental data on the equilibrium binding of myosin subfragment 1 (S-1) to regulated actin filaments in the presence and in the absence of Ca(2+) are analyzed by using a linear Ising model. In the model, each tropomyosin-troponin unit (including seven sites on the actin filament) can be in one of two possible states, which have different intrinsic free energies and different binding constants for S-1. Bound S-1 molecules do not interact with each other. There are nearest-neighbor (pair) interactions between these units that depend on the state of each member of the pair and on the number of Ca(2+) bound to one member of the pair. There are two sources of positive cooperativity in this system: the fact that seven actin sites change state together as part of a single unit; and the existence of attractive nearest-neighbor interactions between units. Parameters in the model are evaluated by fitting the data, both in the presence and in the absence of Ca(2+). Several extensions of this model are discussed.
Structure of an actively exchanging complex between carboxypeptidase A and a substrate analogue.
Authors: Rees D C , Honzatko R B , Lipscomb W N
Published: May 2006

An x-ray diffraction study at 2.8 A resolution has yielded the structure of a complex between bovine carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and (-)-2-benzyl-3-p-methoxybenzoylpropionic acid. This substrate is an analogue of N-(p-methoxy)-benzoylphenylalanine, in which the amide NH is replaced by CN2. T. Sugimoto and E T. Kaiser (1979) J. Am. Chem. Soc. 101, 39469--3951] have shown that this complex catalyzes stereospecific exchange of that proton of the CH2 group which is in the R configuration. Our structure of this complex suports the model proposed by Sugimoto and Kaiser and is very similar to the productive peptide binding mode suggested by Lipscomb et al. [Lipscomb, W. N., Hartsuck, J. A., Reeke, G. N., Quiocho, F. A., Bethge, P. A., Ludwig, M. L., Steitz, T. A., Muirhead, H. & Coppola. J. C. (1968) Brookhaven Symp. Biol. 21, 24--90]. The proposed roles of glutamic acid 270 in the proton exchange and the interaction of zinc with the carbonyl group of the substrate are consistent with the observed structure.
Silent delta-globin gene in Old World monkeys.
Authors: Martin S L , Zimmer E A , Kan Y W , Wilson A C
Published: May 2006

The delta polypeptide chain is present in the adult hemoglobin of all higher primates except Old World monkeys. Because Old World monkeys have evolved from higher primate ancestors, it can be concluded that the ability to synthesize this polypeptide has been lost relatively recently. It is shown here that the gene for delta globin exists in two of these species, the rhesus monkey (Macaca mulatta) and the baboon (Papio papio). Restriction endonuclease fragments of monkey genomic DNA bearing the delta- and beta-globin genes were detected after hybridization of human globin cDNA probes to filter-bound primate DNAs according to the Southern method. A restriction map prepared for rhesus DNA was identical in overall organization to the map of the human region. This indicates that large deletions or additions of DNA are not responsible for the Old World monkeys' lack of delta globin.
Convexity properties of products of random nonnegative matrices
Authors: Cohen Joel E.
Published: May 2006

Consider a sequence of
N
×
N
random nonnegative matrices in which each element depends on a vector
u
of parameters. The
n
th partial product is the random matrix formed by multiplying, from right to left, the first
n
of these random matrices in order. Under certain conditions, the elements of the
n
th partial product grow asymptotically exponentially as
n
increases, and the logarithms of the discrete long-run growth rates are convex functions of
u
. These conditions are met by some models in statistical mechanics and demography. Consequently, the Helmholtz free energy is concave and the population growth rate is convex in these models.
The “excess entropy” around nonequilibrium steady states, (δ
2
S
)

ss

, is not a Liapunov function
Authors: Fox Ronald Forrest
Published: May 2006

In a response to my recent paper [Fox, R. F. (1979)
Proc. Natl. Acad. Sci. USA
76, 2114-2117], Nicolis and Prigogine [Nicolis, G. & Prigogine, I. (1979)
Proc. Natl. Acad. Sci. USA
76, 6060-6061] reasserted that the “excess entropy” around nonequilibrium steady states, (δ
2
S
)

ss

, is a Liapunov function. A simple, explicit counterexample which invalidates this claim is presented. The existence of an alternative theory possessing a proper Liapunov function for steady states is reviewed.
Cucurbitacins as kairomones for diabroticite beetles
Authors: Metcalf Robert L. , Metcalf Robert A. , Rhodes A. M.
Published: May 2006

The characteristic bitter substances of the Cucurbitaceae act as kairomones for a large group of diabroticite beetles (Chrysomelidae, Galerucinae, Luperini), promoting host selection and compulsive feeding behavior. These beetles (e.g.,
Diabrotica undecimpunctata howardi
) respond to as little as 1 ng of cucurbitacin (Cuc) B on thin-layer plates by arrest and compulsive feeding. Six species of diabroticite beetles were about 10 times more responsive to Cuc B than to Cuc E and less responsive to Cuc D, I, and L. Chloroform extracts of 18 species of
Cucurbita
were developed on thin-layer chromatograms and exposed to diabroticite beetles. The feeding patterns showed pronounced beetle responses to three general Cuc distribution patterns: Cuc B and D as in
Cucurbita andreana
and
C. ecuadorensis
; Cuc E and I as in
C. okeechobeensis
and
C. martinezii
; and Cuc E glycoside in
C. texana
. All the diabroticites responded in exactly the same feeding patterns. The results demonstrate a coevolutionary association between the Cucurbitaceae and the Luperini, during which the intensely bitter and toxic Cucs that arose to repel herbivores and protect the plants from attack became specific kairomone feeding stimulants for the beetles.
Dolichyldiphosphoryloligosaccharide—protein oligosaccharyltransferase: Solubilization, purification, and properties
Authors: Das Rathindra C. , Heath Edward C.
Published: May 2006

Dolichyldiphosphoryloligosaccharide—protein oligosaccharyltransferase was solubilized from hen oviduct rough endoplasmic reticulum by extraction with 0.2% Nonidet P40. Oligosaccharyltransferase activity was assayed in an incubation mixture containing Glc

n

-Man

x

-GlcNAc
2
-diphosphoryldolichol as an oligosaccharyl donor and the
125
I-labeled tryptic peptide consisting of residues 29-58 from bovine α-lactalbumin as acceptor. The transferase was purified approximately 2000-fold by fractionation on a bovine α-lactalbumin-Sepharose column; the active material bound quantitatively to the gel and was eluted by removal of divalent cation from the wash buffer. The product of the transferase activity,
125
I-glycopeptide, was determined as concanavalin A-agarose-adsorbed radioactivity by a filter disc assay method.
125
I-Labeled concanavalin A-agarose-bound product was characterized as a glycopeptide as follows: (
i
) gel filtration behavior on Sephadex G-50; (
ii
) elution from concanavalin A-agarose with 1% α-methyl mannoside; (
iii
) absence of affinity for ricin-Sepharose and loss of affinity for concanavalin A-agarose after treatment with endo-β-
N
-acetylglucosaminidase
H
; (
iv
) enzymatic synthesis of identical product upon using [
3
H]oligosaccharyldiphosphoryldolichol and unlabeled peptide acceptor; and (
v
) digestion of
3
H-labeled peptide with Pronase, resulting in the formation of lower molecular weight glycopeptide. Oligosaccharyltransferase activity exhibited an absolute requirement for divalent cations (3 mM Mn
2+
; Mg
2+
was 30% as effective), complete dependence on exogenously supplied peptide acceptor (1.33 μg/ml) and oligosaccharyldiphosphoryldolichol (approximately 10 nmol/ml), and an optimum pH between 7 and 7.5.
Nucleotide sequence of the primary origin of bacteriophage T7 DNA replication: relationship to adjacent genes and regulatory elements.
Authors: Saito H , Tabor S , Tamanoi F , Richardson C C
Published: May 2006

The 682-base-pair nucleotide sequence between positions 14.45 and 16.15 on the bacteriophage T7 DNA molecule has been determined. We can identify not only the sequence of the primary origin of DNA replication but also the termination of gene 1, all of genes 1.1 and 1.2, the start of gene 1.3, and a number of regulatory sequences. The endpoints of four deletion mutations that extend into this region have been determined. These mutations are inferred to have arisen by recombination between short homologous sequences, three of which ar T7 RNA polymerase promoters. The base changes of four point mutations in gene 1.2 have been identified. The sequence essential for initiation at the primary origin is located between the left endpoints of the two deletions D2 and D303. Sequence analysis of these mutants assigns the primary origin to a 129-base-pair segment between positions 14.73 and 15.05. This intergenic segment is A+T-rich (75%) and contains a single T7 gene 4 protein recognition site; it is preceded by two tandem T7 RNA polymerase promoters. A model for initiation of T7 DNA replication is presented.
The tetramer d(CpGpCpG) crystallizes as a left-handed double helix.
Authors: Crawford J L , Kolpak F J , Wang A H , Quigley G J , van Boom J H , van der Marel G , Rich A
Published: May 2006

The structure of the tetramer d(CpGpCpG) has been solved by x-ray analysis in two different crystal forms with and without spermine cations. The molecules crystallize in hexagonal unit cells and they form a left-handed double helix of Z-DNA similar to that previously reported for the hexamer d(CpGpCpGpCpG). In the crystal lattice the molecules stack together to form a virtually continuous left-handed double helix in which every fourth phosphate group is missing. The stacking of bases upon each other is similar to that seen in the hexamer. However, the base pairs have a slightly different orientation in that the cytosine residues are slightly removed from the axis of the molecule compared to the position they occupy in the hexamer. The structures are similar in two crystal forms with and without spermine cations.
In vitro biosynthesis of fish islet preprosomatostatin: evidence of processing and segregation of a high molecular weight precursor.
Authors: Shields D
Published: May 2006

Evidence is presented for a precursor to somatostatin that is 10-12 times larger than the authentic secreted hormone. mRNA from angler fish (Lophius americanus) islets of Langerhans was translated in the wheat germ cell-free system and the products were identified by immunoprecipitation with specific antibodies to somatostatin followed by sodium dodecyl sulfate gel electrophoresis. One 18,000-dalton polypeptide was specifically immunoprecipitable. Competition experiments showed that authentic somatostatin competed with the 18,000-dalton molecule for antibody binding. When dog pancreas microsomal membranes were present during translation, an additional polypeptide of 16,000 daltons was also immunoprecipitable. Comparison of their tryptic peptides demonstrated that the 16,000-dalton polypeptide was derived from the 18,000-dalton one. Tryptic peptide analysis of somatostatin and the 18,000-dalton precursor demonstrated that the 18,000-dalton polypeptide contains the authentic somatostatin amino acid sequence and suggests that it is located at the carboxyl terminus of the precursor molecule and is preceded by a basic amino acid.
Receptor-mediated endocytosis of antibody-opsonized liposomes by tumor cells.
Authors: Leserman L D , Weinstein J N , Blumenthal R , Terry W D
Published: May 2006

Specific receptor-mediated delivery of the contents of small, sonicated liposomes was studied with three murine tumor cell types: an IgG Fc receptor-negative nonphagocytic line (EL4); an Fc receptor-positive phagocytic line (P388D1); and an Fc receptor-positive nonphagocytic line (P388). The liposomes (formed from phosphatidylcholines, cholesterol, and dinitrophenyl-substituted phosphatidylethanolamine) contained carboxyfluorescein as a fluorescent marker and methotrexate as a pharmacologic agent. Binding and internalization of the liposomes were observed by fluorescence microscopy and measured by flow microfluorometry. The hapten-derivatized lipid was used as a binding point on the liposome for the antibody-combining site of the immunoglobulin. In the presence of IgG anti-dinitrophenyl, but not F(ab')2 or IgA anti-dinitrophenyl, liposomes bound to the Fc receptor-bearing cells. The liposomes underwent endocytosis by the P388D1 cells and, to a lesser extent, by the P388 cells. As measured by depression of [3H]deoxyuridine incorporation, methotrexate in IgG-opsonized liposomes had a much greater pharmacologic effect on the P388D1 cells than did the same amount in unopsonized liposomes or in free solution. This observation indicates that an appropriately chosen drug, incorporated in liposomes, can exert its effect on a cytoplasmic target after endocytosis. P388 cells showed a moderate effect of the drug in liposomes. Neither P388 nor P388D1 cells bound or ingested unopsonized liposomes, and the Fc receptor-negative EL4 line neither bound nor ingested opsonized liposomes. The data demonstrate specific interaction of opsonized liposomes with the cells' IgG Fc receptor.
DNA-protein interactions in the Drosophila melanogaster mitochondrial genome as deduced from trimethylpsoralen crosslinking patterns.
Authors: Potter D A , Fostel J M , Berninger M , Pardue M L , Cech T R
Published: May 2006

The location of proteins on the mitochondrial DNA (mtDNA) of Drosophila melanogaster was investigated by trimethylpsoralen photoreaction of embryos disrupted by gentle homogenization. After photoreaction, the mtDNA was isolated and the pattern of DNA crosslinking was determined by electron microscopy of the DNA under totally denaturing conditions. In contrast to nuclear DNA, which showed periodic crosslinks indicative of a nucleosome structure, most of each mtDNA molecule exhibited uniformly heavy crosslinking. A 10% region of the mtDNA was, however, protected from psoralen crosslinking in a distinctive manner: five uncrosslinked segments were closely clustered in the mtDNA. Four were 394 +/- 13 (SD) base pairs in size, while the fifth measured about 200 base pairs. These protected segments mapped within the A+T-rich region of the mtDNA, extending from the end of the A+T-rich region near the Bg1 II cleavage site to the center of the A+T-rich region. Protection of this part of the mtDNA from crosslinking was interpreted to be the result of association with proteins in the mitochondrion because mtDNA that was deproteinized before the photoreaction was uniformly crosslinked over its entire length. The origin of replication of the mtDNA is also located at the center of the A+T-rich region, which suggests that the protection from the psoralen photoreaction may be due to proteins involved in membrane attachment or replication.
Prostaglandin synthesis by macrophages requires a specific receptor-ligand interaction.
Authors: Rouzer C A , Scott W A , Kempe J , Cohn Z A
Published: May 2006

The ingestion of particles by macrophages leads to the prompt induction of prostaglandin (PG) synthesis. We have now dissected the endocytic process and examined the requirements of prostaglandin E (PGE) synthesis for particle attachment, membrane interiorization, and phagosome-lysosome fusion. Macrophages that were loaded with the polyanion dextran sulfate and exhibited a greater than 99% inhibition of phagosome-lysosome fusion produced normal amounts of PGE upon challenge with zymosan. Inhibition of membrane interiorization with cytochalasin D was similarly ineffective in blocking PGE synthesis. The addition of large numbers of unmodified polystyrene latex beads, which were readily ingested by macrophages, failed to stimulate PGE synthesis. However, when macrophages were challenged with latex beads coated with immune complexes, an increased synthesis of PGE resulted. No response occurred if the complex was prepared with the F(ab')2 fragment of IgG. Similar results occurred when nonphagocytizable Sephadex beads coated with immune complexes were employed. We conclude that particle binding to the Fc receptor of the macrophage plasma membrane is a sufficient stimulus for PGE synthesis.
Control of tumor growth in animals by infusion of an angiogenesis inhibitor.
Authors: Langer R , Conn H , Vacanti J , Haudenschild C , Folkman J
Published: May 2006

Angiogenesis and tumor growth were inhibited in two different animal models by regional infusion of a partially purified cartilage extract. In rabbits bearing corneal implants of V2 carcinoma and receiving the inhibitor, vascular growth rates were < 3% of those in control animals receiving either Ringer's solution or bovine trypsin inhibitor (Trasylol). Subconjunctival B16 melanoma implants in mice receiving the inhibitor weight < 2.5% of implants in mice receiving Ringer's solution, Trasylol, or albumin. Histologic study of major organs and standard blood tests revealed no toxic effects in any of the animals. The inhibitor did not retard the growth of either tumor cell type in tissue culture at concentrations as high as 1 mg/ml. These results suggest that the cartilage factor does not interfere with the growth of the tumor cell population directly but that it prevents tumor growth by inhibiting angiogenesis.
Synthesis of enkephalins by adrenal medullary chromaffin cells: reserpine increases incorporation of radiolabeled amino acids.
Authors: Wilson S P , Chang K J , Viveros O H
Published: May 2006

Adrenal medullary chromaffin cells maintained in a chemically defined, serum-free medium retain high levels of both catecholamines and opiate-like peptides. At least 25% of the opioid activity as measured by radioreceptor assay is present as [Met]- and [Leu]enkephalin. Incubation of chromaffin cells with [3H]tyrosine and [35S]methionine for 3 days results in incorporation of 3H and 35S into [Met]enkephalin and 3H incorporation into [Leu]enkephalin, demonstrating de novo enkephalin synthesis. Addition of 100 nM reserpine to the culture medium causes an 80% depletion of catecholamines and a 100% increase in opioid peptide content 3-4 days later. [Met]Enkephalin content is preferentially induced (290% above control) compared with [Leu]enkephalin (70%) Reserpine increases the fractional radiolabeling of [Met]- and [Leu]enkephalin in excess of control cultures, suggesting that the increase in opiate-like peptides results from an increased rate of synthesis rather than decreased degradation, decreased secretion, or increased processing of preformed precursor(s).
Photoaffinity label for the beta-adrenergic receptor: synthesis and effects on isoproterenol-stimulated adenylate cyclase.
Authors: Wrenn S M , Homcy C J
Published: May 2006

An azide derivative of the beta-adrenergic antagonist acebutolol has been synthesized and its effect examined on the isoproterenol-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] activity of rat reticulocytes. It behaved as an effective competitive antagonist (Kd = 2 X 10(-7) M) prior to photolysis. However, when the reticulocyte preparation pretreated with acebutolol azide was photolyzed, a noncompetitive inhibition of isoproterenol-stimulated adenylate cyclase was obtained. Photolysis of the azide derivative in buffer alone did not convert it to a product of higher affnity. Labeling of the beta-adrenergic receptor appeared to be irreversible; multiple washings could not reverse the inhibition produced during photolysis with the label whereas washing would completely reverse the antagonism produced by the same concentration of label prior to photolysis. The effect appears to be specific for the beta-adrenergic receptor because the inhibition could be blocked stereoselectively by propranolol and there was no inhibition of fluoride- or GMP-P(NH)P-stimulated adenylate cyclase. furthermore, no effect was observed on the glucagon-mediated stimulation of adenylate cyclase of liver membranes, whereas the catecholamine response in the same membranes was inhibited.
Abnormal behavior of interferon-induced enzymatic activities in an interferon-resistant cell line.
Authors: Verhaegen M , Divizia M , Vandenbussche P , Kuwata T , Content J
Published: May 2006

Interferon induces two double-stranded RNA-dependent enzymatic activities: an oligoisoadenylate synthetase that converts ATP to ppp(A2'p)n5'A, and a protein phosphokinase. We have explored the level and inducibility of these two enzymes in a human cell line (HEC-1) totally insensitive to both the antiviral and the anticellular actions of interferon. The activities of both enzymes are high in untreated cells and only minor changes occur after treatment with interferon, even at high concentrations. Interferon-treated HEC-1 cells do not contain an inhibitor of the oligoisoadenylate synthetase activity. The products of this HEC-1 oligoisoadenylate synthetase consist mainly of dimers, trimers, and tetramers as found in other cell lines after interferon treatment. The synthetase level is unaffected by treating the cells with anti-interferon antiserum, indicating that the results cannot be explained by a spontaneous low production of interferon by these cells. Furthermore, virus multiplication is not inhibited, even after treatment with interferon. These observations suggest that either the two enzymatic activities do not suffice for the establishment of an antiviral state in vivo or that a regulatory control mechanism, lost in these cells and common for both enzymes, is required for the expression of the antiviral action of interferon. This might explain both the constitutivity of the two enzymes and the interferon resistance observed.
Structure of the potato inhibitor complex of carboxypeptidase A at 2.5-A resolution.
Authors: Rees D C , Lipscomb W N
Published: May 2006

The structure of the complex between the proteolytic enzyme carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and the 39-amino-acid carboxypeptidase A inhibitor from potatoes has been determined at 2.5-A resolution. A combination of multiple isomorphous replacement, molecular replacement, and noncrystallographic symmetry averaging techniques was used to solve the structure. The chain trace of the inhibitor and details of the binding interactions in the complex are described. A surprising aspect of the complex is that the carboxy-terminal peptide bond of the inhibitor has been hydrolyzed, and the carboxy-terminal glycine is trapped in the binding pocket of carboxypeptidase A. Consequently, the complex resembles a stage in the catalytic mechanism after hydrolysis of the peptide bond. The ring of tyrosine-248, which is known to undergo large conformational changes upon substrate binding, is in the "down" position and interacts with the inhibitor in the complex.
Energy transfer between photosystem II units in a connected package model of the photochemical apparatus of photosynthesis
Authors: Butler Warren L.
Published: May 2006

A model of the photochemical apparatus of photosynthesis, presented previously in a tripartite format, is used in a bipartite format to analyze energy transfer between photosystem II units. The model is used to develop analytical expressions for the photochemical properties of chloroplasts that include a term for the probability for energy transfer between photosystem II units. In particular, a normalized plot of the fluorescence of variable yield as a function of the closure of photosystem II reaction centers permits the evaluation of the energy transfer parameter. Data from a study by Melis [Melis, A. (1978)
FEBS Lett.
95, 202-206] are used to estimate that the probability for energy transfer between photosystem II units in the α component of photosystem II was approximately 0.57 when the photosystem II reaction centers were all open and 0.83 when the centers were closed. This connected package model appears to provide a better description of the system than the matrix model that was used previously.
Stoichiometry of system I and system II reaction centers and of plastoquinone in different photosynthetic membranes
Authors: Melis Anastasios , Brown Jeanette S.
Published: May 2006

The concentrations of photochemical centers and of plastoquinone were measured in several kinds of photosynthetic membranes by optical difference spectroscopy. Photosystem I reaction centers were measured from the light-induced absorbance change at 700 nm (oxidation of the primary electron donor, P700). Photosystem II reaction centers were estimated from the light-induced absorbance change at 325 nm (reduction of the primary electron acceptor, Q). Spinach chloroplasts and membrane fractions obtained by French press treatment, mature and developing pea chloroplasts, and blue-green algal membranes were investigated. No loss of primary photochemical activity occurred during fractionation of the chloroplasts. The results indicated a large variability in the ratio of system II to system I reaction centers (from 0.43 to 3.3) in different photosynthetic membranes. Oxygen-evolving plants may change the ratio of their photosystems in response to environmental light conditions. The amount of photoreducible plastoquinone was also measured at 263 nm. In spinach chloroplasts, seven to eight plastoquinone molecules were found per reaction center of system II. Most of the plastoquinone pool was associated with the grana. However, the ratio of chemically determined plastoquinone to chlorophyll was similar in the grana and stroma thylakoids.
Long-term culture of human bone marrow cells.
Authors: Gartner S , Kaplan H S
Published: May 2006

A method has been described for the long-term culture of human bone marrow cells in liquid medium. Hematopoiesis, as measured by the production of granulocytic-macrophage progenitor cells (CFUc), continued for at least 20 weeks and was dependent upon the presence of a marrow-derived adherent layer of cells. As in the case of murine marrow liquid cultures, the adherent layer consisted of mononuclear phagocytic cells, endothelial cells, and lipid-laden adipocytes, the latter being essential for long-term hematopoiesis. Optimal growth conditions included McCoy's medium supplemented with fetal bovine serum, horse serum, and hydrocortisone and incubation at 33 degrees C. Horse serum in conjunction with hydrocortisone appeared essential for the growth of adipocytes.