bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of the mutagenicity of bay-region diol epoxides of polycyclic aromatic hydrocarbons by naturally occurring plant phenols: Exceptional activity of ellagic acid
Authors: Wood Alexander W. , Huang Mou-Tuan , Chang Richard L. , Newmark Harold L. , Lehr Roland E. , Yagi Haruhiko , Sayer Jane M. , Jerina Donald M. , Conney Allan H.
Published: May 2006

Ferulic, caffeic, chlorogenic, and ellagic acids, four naturally occurring plant phenols, inhibit the mutagenicity and cytotoxicity of (±)-7β,8α-dihydroxy-9α, 10α-epoxy-7,8,9,10-tetrahydrobenzo[
a
]pyrene (B[
a
]P 7,8-diol-9,10-epoxide-2), the only known ultimate carcinogenic metabolite of benzo[
a
]pyrene. The mutagenicity of 0.05 nmol of B[
a
]P 7,8-diol-9,10-epoxide-2 in strain TA100 of
Salmonella typhimurium
is inhibited 50% by incubation of the bacteria and the diol epoxide with 150 nmol of ferulic acid, 75 nmol of caffeic acid, 50 nmol of chlorogenic acid or, most strikingly, 1 nmol of ellagic acid in the 0.5-ml incubation mixture. A 3-nmol dose of ellagic acid inhibits mutation induction by 90%. Ellagic acid is also a potent antagonist of B[
a
]P 7,8-diol-9,10-epoxide-2 in Chinese hamster V79 cells. Mutations to 8-azaguanine resistance induced by 0.2 μM diol epoxide are reduced by 50% when tissue culture media also contains 2 μM ellagic acid. Similar to results obtained with the bacteria, ferulic, caffeic, and chlorogenic acids are approximately two orders of magnitude less active than ellagic acid in the mammalian cell assay. The antimutagenic effects of the plant phenols result from their direct interaction with B[
a
]P 7,8-diol-9,10-epoxide-2, because a concentration-dependent increase in the rate of diol epoxide disappearance in cell-free solutions of 1:9 dioxane/water, pH 7.0, is observed with all four phenols. In parallel with the mutagenicity studies, ellagic acid is 80-300 times more effective than the other phenols in accelerating the disappearance of B[
a
]P 7,8-diol-9,10-epoxide-2. Ellagic acid at 10 μM increases the disappearance of B[
a
]P 7,8-diol-9,10-epoxide-2 by approximately 20-fold relative to the spontaneous and hydronium ion-catalyzed hydrolysis of the diol epoxide at pH 7.0. Ellagic acid is a highly potent inhibitor of the mutagenic activity of bay-region diol epoxides of benzo[
a
]pyrene, dibenzo[
a,h
]pyrene, and dibenzo[
a,i
]pyrene, but higher concentrations of ellagic acid are needed to inhibit the mutagenic activity of the chemically less reactive bay-region diol epoxides of benz[
a
]anthracene, chrysene, and benzo[
c
]phenanthrene. These studies demonstrate that ellagic acid is a potent antagonist of the adverse biological effects of the ultimate carcinogenic metabolites of several polycyclic aromatic hydrocarbons and suggest that this naturally occurring plant phenol, normally ingested by humans, may inhibit the carcinogenicity of polycyclic aromatic hydrocarbons.
Frozen tissue sections as an experimental system to reveal specific binding sites for the regulatory subunit of type II cAMP-dependent protein kinase in neurons.
Authors: Miller P , Walter U , Theurkauf W E , Vallee R B , De Camilli P
Published: May 2006

Specific binding sites for the regulatory subunit of type II cAMP-dependent protein kinase (RII) were revealed in neurons by an immunohistochemical approach. Fixed frozen sections of several regions of the rat central nervous system were incubated in the presence of bovine RII. Bound bovine RII was subsequently detected by an immunofluorescence procedure using antibodies that recognize bovine but not rat RII. The results indicate that RII binds with high affinity to neurons. Binding is prominent in dendrites and almost undetectable in axons and axon terminals. The morphological distribution of the RII binding sites is almost identical to that of microtubule-associated protein 2 (MAP 2) immunoreactivity. Preadsorption of RII with a MAP preparation highly enriched in MAP 2 completely abolished binding of RII to tissue sections, suggesting that the binding is mediated by MAP 2. Our results indicate that frozen sections of fixed tissues are a suitable experimental system for study of specific interactions of cellular macromolecules at a morphological level.
Mutations of glucocerebrosidase: discrimination of neurologic and non-neurologic phenotypes of Gaucher disease.
Authors: Ginns E I , Brady R O , Pirruccello S , Moore C , Sorrell S , Furbish F S , Murray G J , Tager J , Barranger J A
Published: May 2006

Multiple molecular forms of beta-glucocerebrosidase that permit discrimination between neurologic and non-neurologic phenotypes of Gaucher disease have been identified radioimmunologically in fibroblasts and human brain tissue. In normal human fibroblasts these forms have been shown by NaDodSO4/polyacrylamide gel electrophoresis to have apparent Mr of 63,000 (form A1), 61,000 (form A2), and 56,000 (form B). The Mr 63,000 form may be a precursor of the Mr 56,000 form. Non-neurologic Gaucher disease (type 1) fibroblasts and normal brain tissue are characteristic in that they contain only one major immunoreactive protein, the Mr 56,000 form. In contrast, fibroblast extracts and brain tissue from neurologic Gaucher disease phenotypes contain only the higher molecular weight forms A1 and A2. These data and the low residual activity of the enzyme in all the variants of Gaucher disease suggest that the mutations of beta-glucocerebrosidase are allelic and involve the active site.
Isolation of cDNA clones for the human complement protein factor B, a class III major histocompatibility complex gene product.
Authors: Woods D E , Markham A F , Ricker A T , Goldberger G , Colten H R
Published: May 2006

cDNA clones corresponding to a major histocompatibility class III antigen, the complement protein factor B, have been isolated from a human adult liver cDNA library. The clones, ranging in size from 1.0 to 2.3 kilobases, were identified by direct hybridization with two synthetic oligonucleotide mixtures. Two regions of the factor B amino acid sequence, each with minimal ambiguity in codon assignment, were chosen for synthesis of the oligonucleotides. The sequences of two clones have been partially determined. They contain coding information for the amino acid sequence of the Bb fragment of factor B and the entire 3' -untranslated region.
Stable expression in mouse cells of nuclear neoantigen after transfer of a 3.4-megadalton cloned fragment of Epstein-Barr virus DNA.
Authors: Summers W P , Grogan E A , Shedd D , Robert M , Liu C R , Miller G
Published: May 2006

All cells that harbor the Epstein-Barr virus (EBV) genome contain a neoantigen in the nucleus (EBNA). By transfection we located a segment of the genome that encodes or induces an antigen serologically related to EBNA. The responsible genes are found in the 3.4-megaldalton BamHI fragment K of EBV DNA, specifically in the left 1.9 megadaltons represented by HindIII fragment I1. Mouse LTK- cells were cotransformed with recombinant plasmids, containing the herpes simplex virus thymidine kinase gene and either EcoRI fragment B or BamHI fragment of K of EBV DNA. The TK+ cells surviving in selective medium were cloned. About 50% of the clones expressed the neoantigen in every nucleus. These mouse cells were used as antigens in immunofluorescence tests. Antibody to the nuclear antigen was found in 30 human sera known to contain antibody to EBNA; it was not detected in 18 sera that did not have antibody to EBNA. Mouse cells expressing EBNA as the result of acquisition of cloned EBV DNA fragments should prove useful in the characterization of the structure of this antigen and as reagents for the diagnosis of EBV infections.
Regulation of dopamine- and adenosine-dependent adenylate cyclase systems of chicken embryo retina cells in culture.
Authors: de Mello M C , Ventura A L , Paes de Carvalho R , Klein W L , de Mello F G
Published: May 2006

We have obtained evidence that receptor-stimulated adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] is regulated physiologically in both embryonic and mature neurons. In a series of experiments using cultured retina cells from chicken embryos, we found that dopamine-sensitive adenylate cyclase activity spontaneously desensitized as cultures differentiated. The cellular response to dopamine reached a maximum after 5 days in culture and then decreased to 40% during the next 5 days. This spontaneous desensitization appeared to be caused by functional dopaminergic transmission because it could be blocked by the dopamine antagonist haloperidol. The ability of added dopamine at 100 microM to cause near-complete desensitization is consistent with this conclusion. Pharmacologically induced desensitization required 31 hr for maximal effect and was half-maximal at 1-10 microM dopamine. Analogous desensitization of the adenosine-dependent adenylate cyclase system also was noted. When dopamine was removed from the medium of chronically treated cultures, cells resensitized to subsequent stimulation at a very slow rate. Resensitization likely depended on replacement of dopamine receptors because chronic dopamine treatment caused the disappearance of binding sites for the ligand [3H]spiroperidol. In a second series of experiments, using hatched animals, we found that similar regulation of dopamine receptor binding sites and activity could be elicited by manipulation of environmental light, a treatment thought to influence dopaminergic transmission. Retinas from animals in constant light had less specific [3H]spiroperidol binding (35 fmol/mg of protein) than did retinas from animals in constant darkness (66 fmol/mg of protein) and made less cAMP in response to added dopamine. Our results indicate that regulation of the dopamine receptor system begins early in development and continues to function in mature synapses.
Visual evoked potentials: evidence for lateral interactions.
Authors: Zemon V , Ratliff F
Published: May 2006

Electrical potentials evoked in the human brain by visual stimulation can easily be recorded by using electrodes attached to the scalp. It is difficult, however, to relate these visual evoked potentials (VEPs) to specific neural processes: scalp electrodes, far removed from the brain, sum potentials from large areas of cortex. We improved identification and localization of lateral interactions by differentially modulating small neighboring parts of a "windmill-dartboard" stimulus pattern-a central disc surrounded by three contiguous annuli, all radially divided into light and dark segments. With temporal contrast reversal of all segments in the pattern, the major component of the VEP is at the second harmonic of the frequency of modulation--as expected. Temporal contrast reversal of the segments in the central disc and second annulus, with contrast of segments held constant in the first and third annuli, unexpectedly amplifies the VEP at the fundamental frequency of modulation and attenuates it at the second harmonic. Slight spatial separation of static and dynamic zones reduces both the amplification of the fundamental and the attenuation of the second harmonic. Thus, both phenomena appear to result from strong lateral interactions over relatively short distances. Nevertheless, different neural mechanisms must be involved; fundamental and second-harmonic components of the VEP are different functions of spatial separation and relative contrast of the segments in contiguous static and dynamic zones.
Complete amino acid sequence and glycosylation sites of glycoprotein gp71A of Friend murine leukemia virus.
Authors: Chen R
Published: May 2006

The complete amino acid sequence of glycoprotein gp71A of Friend murine leukemia virus (F-MuLV) is presented. The protein moiety of gp71A was digested with Staphylococcus aureus (SV8) protease, trypsin, and thermolysin. The sequences of the peptides were determined by the micro dansyl Edman procedure. gp71A is composed of 445 amino acid residues and contains eight oligosaccharide side chains, which are attached exclusively to asparagine by N-glycosyl bonds primarily in the COOH-terminal half of the polypeptide. gp71A is rich in proline (49 residues), tryptophan (16 residues), and cysteine (19 residues). Proline has the highest molar content (11%) of all amino acids. The prolines cluster in two segments. The most interesting one stretches between residue 233 and residue 283 and contains 18 prolines within 51 amino acids. This proline-rich domain most likely forms a flexible polyproline helix. The comparison of gp70 of Moloney murine leukemia virus (Mo-MuLV gp70) with F-MuLV gp71A revealed that 70 amino acids have been exchanged and 9 residues have been deleted from Mo-MuLV gp70. The most striking alterations have taken place within the large polyproline segment (residues 247 to 281). In this part of the molecule 7 amino acids have been deleted in Mo-MuLV and 18 residues have been replaced. This evidence supports the proposal of Shinnick et al. [Shinnick, T. M., Lerner, R. A. & Sutcliffe, J. G. (1981) Nature (London) 293, 543-548] that this area is a "hot spot" for recombination.
18
O isotope shift in
15
N NMR analysis of biological N-oxidations: H
2
O-NO
2
-
exchange in the ammonia-oxidizing bacterium
Nitrosomonas
Authors: Andersson Kristoffer K. , Philson Stephen B. , Hooper Alan B.
Published: May 2006

The
18
O/
16
O shifts in
15
N NMR were determined for nitrite (0.13 ppm or 4.2 Hz at 7.05 T) and nitrate (0.056 ppm or 1.7 Hz at 7.05 T) at neutral pH. The technique, which allows clear differentiation between
16
O and
18
O derivatives of
15
N, was used to assess the source of oxygens in nitrite produced by oxidation of ammonia in
Nitrosomonas
. The two oxygens of nitrite produced by cell-catalyzed oxidation of ammonia or hydroxylamine had the
16
O/
18
O isotope composition of water.
Nitrosomonas
is shown to catalyze the rapid exchange of oxygen between nitrite and water. The exchange reaction required the concomitant oxidation of ammonia. The amount of nitrite exchanged could exceed the amount of ammonia oxidized by a factor of 3. This exchange explains previous difficulties in the determination of the source of nitrite oxygen in ammonia oxidation. When cells oxidized [
15
N]ammonia in the presence of a great excess of exogenous [
14
N]nitrite, 20% of one oxygen in the resulting [
15
N]nitrite was derived from dioxygen. Dioxygen is apparently the source of at least one oxygen in nitrite produced by
Nitrosomonas
.
Three distinct stages of B-cell defects in common varied immunodeficiency.
Authors: Saiki O , Ralph P , Cunningham-Rundles C , Good R A
Published: May 2006

B-lymphocyte function of 15 patients with primary common varied immunodeficiency or related disease were examined. All patients had low serum levels of IgM, IgG, and IgA, but 12 of 15 patients had nearly normal numbers of peripheral blood B lymphocytes. Mononuclear cells and B cells from peripheral blood were assayed for B-cell mitogenic responses to anti-Ig mu chain antibodies or to Staphylococcus aureus strain Cowan I (referred to as Cowan I), and for differentiation to Ig-secreting cells of IgM, IgG, and IgA classes in the presence of Cowan I and pokeweed mitogen or T-cell factor. The patients all showed profound B-cell defects in one or more of the assays and could be divided into three approximately equal groups based on their responses. The first group showed normal proliferation in response to the two B-cell mitogens and near normal numbers of IgM-secreting cells but no IgG- or IgA-secreting cells. B cells in the second group showed proliferative responses to Cowan I or anti-mu, but no differentiation to Ig-secreting cells. The third group had no B-cell proliferative responses or differentiation in our assays. In several patients from each group, (i) helper T cells were functional in Ig-secreting-cell responses with purified normal B cells, (ii) patient T cells did not significantly suppress formation of Ig-secreting cells by normal cells in coculture, and (iii) removal of T cells with addition of T-cell-replacing factor, or partial removal of monocytes, did not alleviate any of the defects. These studies show that primary B-cell defects in common varied immunodeficiency occur at several levels, probably representing blocks at different stages of differentiation.
Similarity of secretory protein I from parathyroid gland to chromogranin A from adrenal medulla.
Authors: Cohn D V , Zangerle R , Fischer-Colbrie R , Chu L L , Elting J J , Hamilton J W , Winkler H
Published: May 2006

We have compared the amino acid and carbohydrate compositions, partial amino acid sequences, immunological crossreactivity, and physical properties of secretory protein I of the parathyroid gland and chromogranin A of adrenal gland. This comparison indicates that these proteins are similar molecules. Because secretory protein I is present in secretory granules containing parathormone and is cosecreted with the hormone, and because chromogranin A is contained within chromaffin granules and, likewise, is secreted with the catecholamines, the present observations raise the possibility that this class of protein plays a general role in hormone secretion or storage mechanisms.
Gene order and gene-polypeptide relationships of the proton-translocating ATPase operon (unc) of Escherichia coli.
Authors: Gunsalus R P , Brusilow W S , Simoni R D
Published: May 2006

We have constructed an extensive set of plasmids that carry the genes specifying the eight polypeptides of the proton-translocating ATPase of Escherichia coli. Using detailed restriction analysis and in vitro protein synthesis directed by these plasmids, we have established the order of the eight unc genes to be BEFHAGDC and the corresponding polypeptides to be a, c, b, delta, alpha, gamma, beta, and epsilon. These analyses include determining the location of the gene coding for the delta subunit of the F1 portion of the complex. We call this gene uncH. We have now established the gene order and gene-polypeptide relationships of the unc operon. This approach should be of use for study of other multigene bacterial operons, especially those with genes coding for polypeptides with unknown or unmeasurable catalytic activity.
Regulation of fructose-6-phosphate 2-kinase by phosphorylation and dephosphorylation: possible mechanism for coordinated control of glycolysis and glycogenolysis.
Authors: Furuya E , Yokoyama M , Uyeda K
Published: May 2006

The kinetic properties and the control mechanism of fructose-6-phosphate 2-kinase (ATP: D-fructose-6-phosphate 2-phosphotransferase) were investigated. The molecular weight of the enzyme is approximately 100,000 as determined by gel filtration. The plot of initial velocity versus ATP concentration is hyperbolic with a Km of 1.2 mM. However, the plot of enzyme activity as a function of fructose-6-phosphate is sigmoidal. The apparent K0.5 for fructose-6-phosphate is 20 microM. Fructose-6-phosphate 2-kinase is inactivated by the catalytic subunit of cyclic AMP-dependent protein kinase, and the inactivation is closely correlated with phosphorylation. The enzyme is also inactivated by phosphorylase kinase in the presence of Ca2+ and calmodulin. The phosphorylated fructose-6-phosphate 2-kinase, which is inactive, is activated by phosphorylase phosphatase and alkaline phosphatase. The possible physiological significance of these observations in the coordinated control of glycogen metabolism and glycolysis is discussed.
Ultrastructure of the first component of human complement: electron microscopy of the crosslinked complex.
Authors: Strang C J , Siegel R C , Phillips M L , Poon P H , Schumaker V N
Published: May 2006

Electron micrographs are shown of the first component of human complement (C1) which has been crosslinked with a water-soluble carbodiimide to prevent dissociation into its C1q and C1r2C1s2 subunits. Two projections of the crosslinked molecule are seen in the electron micrographs, which are called "top" and "profile." In both views, the C1q heads are visible. From the top, the C1r2C1s2 tetrameric subunits appears to be located centrally on the C1q and folded to form a compact mass obscuring most of the arms and central bundle. In profile, the tetramer appears to be located in the region of the arms between the C1q heads and the central bundle. Both the heads and the rod-like central bundle appear to be free of C1r2C1s2 in these profile projections. Sometimes it is possible to count more than six domains in the region of the C1q heads, as though a portion of the tetramer had unfolded to protrude among the heads.
IgA interaction with the asialoglycoprotein receptor.
Authors: Stockert R J , Kressner M S , Collins J C , Sternlieb I , Morell A G
Published: May 2006

IgA present in normal human serum reacts with the hepatic receptor specific for asialoglycoproteins as demonstrated by inhibition of receptor-mediated erythroagglutination. Inhibition is reversibly abolished by the oxidation of the galactose or N-acetylgalactosamine residues of IgA with galactose oxidase. The site of receptor recognition appears to be the O-glycosidically linked oligosaccharides present on the hinge region of the IgAI subtype of IgA. The demonstration of a specific binding, in vitro, of IgA by the hepatic receptor suggests that the uptake of polymeric IgA by the liver in vivo may be mediated by this reaction.
In vitro assembly of the nonglycosylated membrane protein (M) of Sendai virus.
Authors: Heggeness M H , Smith P R , Choppin P W
Published: May 2006

The nonglycosylated membrane protein (M) of Sendai virus was purified from virions and conditions were found under which the protein assembled in vitro into three types of ordered structures: narrow tubes, wide tubes, and sheets. These structures were examined by high resolution electron microscopy by using negative staining and metal shadowing techniques. The tubes and sheets are formed from strands 7.2 nm wide that are composed of annular subunits. The wide tubes appear to be formed by the rolling of a sheet into a cylinder in which the 7.2-nm strands are inclined with a pitch of 26-33 degrees and have a left-handed orientation. In addition to the strong reflections corresponding to the 7.2-nm spacings generated by the strands, optical diffraction patterns also showed weak reflections that could be indexed on a lattice corresponding to real-space lattice constants of 7.6 nm and 5.3 nm, with an included angle of 71 degrees. The dimensions and arrangements of these structures formed in vitro are strikingly similar to those of ordered arrays of particles found by others to be associated with the inner surface of the plasma membrane of infected cells. The results support the concept that ordered arrays of M protein, similar to those assembled in vitro, are involved in the assembly of the virus particle by budding from the cell membrane and that they provide specific recognition sites for the viral nucleocapsid at the cytoplasmic surface of the plasma membrane.
Cloning and sequence determination of the gene for the human immunoglobulin epsilon chain expressed in a myeloma cell line.
Authors: Kenten J H , Molgaard H V , Houghton M , Derbyshire R B , Viney J , Bell L O , Gould H J
Published: May 2006

Messenger RNA has been isolated from cells of the human myeloma line 266BL which synthesizes IgE of the myeloma ND. A fraction enriched in mRNA for the epsilon heavy chain was copied into DNA and the DNA was cloned in Escherichia coli. A chemically synthesized oligonucleotide probe, based on the experimentally determined sequence of the specific message, was used to screen colonies. The largest epsilon chain cDNA cloned, 2.0 kilobases, was characterized by restriction endonuclease mapping and DNA sequence analysis. It appears to encode the complete amino acid sequence of the epsilon chain, including a signal peptide at the NH2 terminus as well as untranslated sequences at the 5' and 3' ends of the mRNA. The missing part of the previously published amino acid sequence of the ND epsilon chain was determined from the DNA sequence.
Monoclonal antibodies to the thyrotropin receptor: stimulating and blocking antibodies derived from the lymphocytes of patients with Graves disease.
Authors: Valente W A , Vitti P , Yavin Z , Yavin E , Rotella C M , Grollman E F , Toccafondi R S , Kohn L D
Published: May 2006

Human monoclonal antibodies have been generated from heterohybridomas obtained by fusing mouse myeloma cells with peripheral lymphocytes from patients with active Graves disease. This report characterizes four antibodies as presumptive thyrotropin receptor antibodies because they specifically inhibit thyrotropin binding and competitively inhibit thyrotropin-induced cAMP levels in human thyroid cells. Two of these antibodies, 208F7 and 206H3, are representative of autoimmune stimulators in Graves disease sera because they stimulate thyroid function in all assays, including the mouse bioassay; their ability to inhibit thyrotropin-induced cAMP increases in thyroid cells competitively is complemented by more than additive agonism at low (10 pM) thyrotropin concentrations. These stimulating antibodies interact more potently with human thyroid ganglioside preparations than with bovine thyroid or brain gangliosides; in contrast, they are poor inhibitors of 125I-labeled thyrotropin binding to liposomes containing the glycoprotein component of the human thyrotropin receptor. Antibodies 129H8 and 122G3 appear to be representative of inhibiting or "blocking" antibodies in Graves disease sera. Thus they have no intrinsic stimulatory action in assays of thyroid function but rather inhibit thyrotropin activity in the assays tested. These two antibodies do not react with human thyroid gangliosides but are strong inhibitors of thyrotropin binding to liposomes containing the high-affinity glycoprotein component from human, bovine, and rat thyroid membranes. The data unequivocally establish the pluritopic nature of the immunoglobulins in Graves disease and relate individual components or determinants of the thyrotropin receptor structure with specific autoimmune immunoglobulins.
Membrane potential and gentamicin uptake in Staphylococcus aureus.
Authors: Mates S M , Eisenberg E S , Mandel L J , Patel L , Kaback H R , Miller M H
Published: May 2006

At pH 5.0, the electrical potential (delta psi, interior negative) across the plasma membrane of Staphylococcus aureus exhibits a minimum of -85 to -90 mV; the pH gradient (delta pH, interior alkaline) across the membrane approximates a maximum of about -100 mV. Under these conditions, uptake of the aminoglycoside gentamicin is negligible, and viability of the organism is not impaired by the antibiotic. In contrast, at pH 7.5, at which delta psi is about -130 mV and delta pH is 0, gentamicin uptake is observed and the drug markedly decreases viability. Dramatically, when the ionophore nigericin is added at pH 5.0, gentamicin uptake is induced, there is a striking decrease in viability, and the effect is associated with an increase in delta psi at the expense of delta pH. Consistently, valinomycin, which dissipates delta psi in the presence of potassium, abolishes gentamicin uptake and killing. In addition, from pH 5.0 to pH 7.5, there is a direct relationship between the magnitude of delta psi and both gentamicin uptake and its bactericidal effect. However, a threshold delta psi of -75 to -90 mV is apparently necessary to initiate uptake and killing. These observations provide a strong indication that delta psi plays a critical role in the uptake and antibacterial action of gentamicin and suggest that nigericin-like ionophores may be clinically useful in synergy with aminoglycosides.
Immunocytochemical localization of sodium channel distributions in the excitable membranes of Electrophorus electricus.
Authors: Ellisman M H , Levinson S R
Published: May 2006

The tetrodotoxin binding protein, a major component of the Na+ channel, has been purified from the electric organ of the South American eel Electrophorus electricus. Antibodies to this protein were raised in rabbits and their specificity was demonstrated by a highly sensitive radioimmunoassay and by immunoprecipitation procedures. These antibodies were used to examine the distribution of the binding protein in the eel electroplax membranes and along myelinated nerve axons. The distribution of the antigen was determined by using the peroxidase-antiperoxidase technique at both the light and electron microscopic levels. In the electrocytes of the electric organ, only the innervated face showed staining in experimental material. The stained regions of electroplax plasmalemma included the caveolae of the innervated surface while caveolae of the non-innervated surface did not stain. Thus, the innervated surface including caveolae exclusively contains the Na+ channels. Along myelinated axons, staining was limited to the nodal zone of the node of Ranvier. The paranodal and internodal zones did not stain for the binding protein. Limited diffusion of primary IgG and subsequent reactants into the paranodal and internodal sites was eliminated as a possible source of focal staining at nodes because mechanically demyelinated preparations also exhibited focal nodal staining. Thus, this tetrodotoxin binding protein component of the Na+ channel is located solely within the nodal zone of the node of Ranvier.
Tubulin bound to colchicine forms polymers different from microtubules.
Authors: Andreu J M , Timasheff S N
Published: May 2006

The purified tubulin-colchicine complex undergoes in vitro polymerization under the same conditions that promote the assembly of microtubules from purified tubulin. The need for a critical concentration, the apparent free energy change of the reaction, and the effects of divalent cations and nucleotide binding indicate interactions similar to those involved in microtubule formation. The large polymers formed are not microtubules, suggesting that the mode of action of the antimitotic drug may be the production of an incorrect bonding geometry between tubulin molecules.
Nucleotide sequence requirements for specific initiation of transcription by RNA polymerase I.
Authors: Grummt I
Published: May 2006

The nucleotide sequence(s) specifying RNA polymerase I initiation has been investigated by studying the transcription of deleted and nondeleted mouse ribosomal RNA gene (rDNA) templates in vitro. The deletion of 5'-flanking sequences upstream from position -- 39 did not affect transcriptional activity, but removal of sequences between positions -- 39 and -- 34 resulted in a 90% decrease of rDNA transcription. The template activity was completely eliminated by the further deletion of nucleotides -- 33 to -- 13. It is concluded that sequences between -- 34 and -- 12, upstream from the transcribed region, represent an essential control region for the initiation of transcription in vitro. Therefore, this region may be functionally analogous to the T-A-T-A box of RNA polymerase II promoters. In addition to this control region, sequences located further upstream (between positions -- 45 and -- 169) may also exert some function in efficient transcription initiation as revealed by competition experiments between wildtype and mutant rDNA templates.
Birhythmicity, chaos, and other patterns of temporal self-organization in a multiply regulated biochemical system.
Authors: Decroly O , Goldbeter A
Published: May 2006

We analyze on a model biochemical system the effect of a coupling between two instability-generating mechanisms. The system considered is that of two allosteric enzymes coupled in series and activated by their respective products. In addition to simple periodic oscillations, the system can exhibit a variety of new modes of dynamic behavior; coexistence between two stable periodic regimes (birhythmicity), random oscillations (chaos), and coexistence of a stable periodic regime with a stable steady state (hard excitation) or with chaos. The relationship between these patterns of temporal self-organization is analyzed as a function of the control parameters of the model. Chaos and birhythmicity appear to be rare events in comparison with simple periodic behavior. We discuss the relevance of these results with respect to the regularity of most biological rhythms.
Interferon-gamma-like molecule induces Ia antigens on cultured mast cell progenitors.
Authors: Wong G H , Clark-Lewis I , McKimm-Breschkin J L , Schrader J W
Published: May 2006

Persisting (P) cells (murine cells that resemble mast cells and grow continuously in vitro for prolonged periods in the presence of a specific growth factor) did not express detectable levels of Ia antigens (murine class II major histocompatibility antigens) when their growth was supported by partially purified P cell-stimulating factor. However, when these Ia-negative P cells were transferred to medium conditioned by concanavalin A-stimulated spleen cells, Ia antigens appeared within 24 hr. The increase in Ia antigens was due to induction of synthesis of Ia antigens by P cells and not to absorption of Ia antigens from the conditioned medium or selective growth of Ia-positive cells from a low number of Ia-positive cells in the original population. The Ia-inducing activity was also found in supernatants from antigen-stimulated cloned T-cell lines, but not from certain T-cell hybridomas or the T lymphoma EL-4. The presence of Ia-inducing activity correlated with the presence of interferon-gamma (IFN-gamma). The gel filtration profiles of IFN-gamma activity and Ia-inducing activity were coincident and corresponded to an apparent molecular weight of 40,000-45,000. Both the IFN-gamma and Ia-inducing activity were destroyed by treatment at pH 2. These results indicate that IFN-gamma or a closely related molecule induces Ia antigens on P cells and suggest that regulation of Ia antigen expression may be an important aspect of the effects of IFN-gamma on the immune and hemopoietic systems.
Bacteriophage T4-induced anticodon-loop nuclease detected in a host strain restrictive to RNA ligase mutants.
Authors: David M , Borasio G D , Kaufmann G
Published: May 2006

The fate of host tRNAs during T4 bacteriophage infection was investigated with Escherichia coli CTr5x, the only known host strain that is restrictive to RNA ligase and polynucleotide kinase mutants. Three CTr5x tRNA species were cleaved during infection. One was leucine tRNA1, which was cleaved in the extra arm, as reported elsewhere for E. coli B infected with bacteriophage T2 or T4. The other two were specific to E. coli CTr5x and were not cleaved in various other hosts. One of the cleaved CTr5x-specific tRNAs had an anticodon sequence of the E. coli B "major" isoleucine tRNA but otherwise little sequence homology. Both CTr5x-specific tRNAs were cleaved by a distinct T4-induced endonuclease, other than that of leucine tRNA1, because the CTr5x-specific cleavages (i) were induced later in infection, (ii) persisted with a T4 mutant deficient in leucine tRNA1 endonuclease, and (iii) occurred in the anticodon loop. The specific manifestation of the anticodon-directed endonuclease activity in T4-infected E. coli CTr5x suggests roles for RNA ligase and polynucleotide kinase in processing of host tRNA species.
Nonspecific stabilization of stress-susceptible proteins by stress-resistant proteins: a model for the biological role of heat shock proteins.
Authors: Minton K W , Karmin P , Hahn G M , Minton A P
Published: May 2006

It is demonstrated experimentally that addition of proteins that are themselves resistant to denaturation by heat or ethanol can nonspecifically stabilize other proteins that are ordinarily highly susceptible to inactivation. It is proposed that the diffusion-limited rate with which unfolded protein molecules encounter each other and become irreversibly crosslinked is reduced in the presence of substantial concentrations of an unreactive globular protein. We suggest that one of the functions of heat shock proteins, which are synthesized in large amounts after exposure of cells to increased temperature and other forms of stress, may be to stabilize other proteins kinetically in a similarly nonspecific fashion.
Effect of acetylated and deacetylated 2-aminofluorene adducts on in vitro DNA synthesis.
Authors: Moore P D , Rabkin S D , Osborn A L , King C M , Strauss B S
Published: May 2006

We have constructed primed phi X174 DNA templates containing either acetylated or deacetylated aminofluorene adducts at the C-8 position of guanine. T4 DNA polymerase terminates synthesis one nucleotide before the acetylated adducts but incorporates an additional nucleotide opposite the deacetylated guanylaminofluorene. These observations can be explained by the known preferred conformations of the acetylated and deacetylated guanosinylaminofluorene nucleosides--the former favoring the syn conformation (so that in DNA the guanine is displaced from the helix by the fluorene ring) and the latter preferring the anti conformation (which allows normal base pairing of the guanine with cytosine). A similar differentiation between the two adducts was found with Escherichia coli DNA polymerase I. In contrast, avian myeloblastosis virus (AMV) reverse transcriptase, which terminated with a nucleotide inserted opposite the acetylated adducts, was less able to do so at the deacetylated adducts. The nucleoside incorporated by AMV reverse transcriptase opposite the acetylated adduct was exclusively cytidine, which suggests regular base pairing with the reacted guanosine nucleoside in the anti conformation; however, synthesis was completely blocked and unable to continue beyond this point. The differences between the termination patterns of the prokaryotic enzymes and AMV reverse transcriptase indicates that specific properties of a replicating polymerase can influence the conformation of a reacted nucleoside in the DNA, thus altering its recognition and possibly its mutagenic activity.
Ultrastructural basis for chloroquine-induced increase in intracellular insulin in adipocytes: alteration of lysosomal function.
Authors: Smith R M , Jarett L
Published: May 2006

A quantitative morphological analysis of insulin uptake into adipocytes was undertaken to determine the structural basis for chloroquine-induced increases in intracellular insulin. Adipocytes were incubated with ferritin-labeled insulin in the presence or absence of 50 microM chloroquine at 37 degrees C for 2-90 min and the uptake of the hormone conjugate was determined quantitatively. Quantitative morphometry of cellular organelles also was performed. Chloroquine treatment of adipocytes incubated with 70 nM ferritin-labeled insulin resulted in: (i) a 120% increase in the number of lysosomes in the cytoplasm; (ii) a 75% increase in the average concentration of ferritin-labeled insulin in a lysosome; and (iii) a 25% increase in the percentage of lysosomes containing ferritin-labeled insulin. The cumulative result of these effects was a substantial increase in the amount of intact intracellular hormone within the lysosomes. These morphological data are consistent with biochemical data concerning chloroquine-induced accumulation of 125I-labeled insulin in adipocytes.
Surface antigenic change during differentiation of a parasitic protozoan, Leishmania mexicana: Identification by monoclonal antibodies.
Authors: Fong D , Chang K P
Published: May 2006

The fusion of SP2/0 myeloma cells with spleen cells from mice immunized with Leishmania mexicana amazonensis promastigotes produced hybridoma clones. Indirect immunofluorescent antibody assay with live leishmanias showed that the monoclonal antibody 6H12 recognized only the antigens bound to the surface of L. mexicana amazonensis promastigotes. It also showed that the antibody bound to neither amastigotes of this species nor to other Leishmania species--i.e., L. braziliensis braziliensis, L. tropica, and L. donovani. Monoclonal antibodies from three other clones (4D11, 4H9, and 6A11) were found to compete with 6H12 for binding to L. mexicana promastigotes. With lysates of [35S]methionine-labeled promastigotes, all four monoclonal antibodies precipitated the same triplet set of protein bands at the approximately equal to 68,000-dalton region, whereas another monoclonal antibody (6G5) precipitated a different band at approximately equal to 90,000 daltons. During differentiation of L. mexicana amazonensis from amastigotes to promastigotes, there was a 4- to 8-fold increase above the initial level in the binding of 6H12 monoclonal antibody to leishmanias, as detected by enzyme-linked immunosorbent assay and quantitative fluorometric assay, respectively. Thus, we have demonstrated the use of monoclonal antibodies as probes for antigens that change during leishmanial differentiation.
Genes controlling segmental specification in the Drosophila thorax.
Authors: Struhl G
Published: May 2006

The roles of three homeotic genes, Ubx+, Scr+, and Antp+, in the Drosophila thorax have been studied by determining the cellular phenotypes of mutations resulting in loss of gene function. The principal results are: (i) The Scr+ and Ubx+ genes are required in the prothorax and metathorax, respectively; in the absence of these genes, both segments develop like the mesothorax. (ii) The Antp+ gene is required in all three thoracic segments: in its absence, parts of the mesothorax are transformed into corresponding parts of the antenna, and similar transformations to antenna are found in the prothorax and metathorax if the Scr+ and Ubx+ genes also are absent. (iii) Loss of the Ubx+ gene early in embryogenesis, but not later, leads to the inappropriate activity of the Scr+ gene in the meso- and metathorax. Results i and ii argue strongly that segmental determination is specified in a combinatorial fashion in the head and thorax by the selective activities of the Scr+, Ubx+, Antp+, and putative head-determining genes. Result iii suggests that a product of the Ubx+ gene also plays an early, regulatory role in ensuring the correct spatial expression of the Scr+ gene during subsequent development.
Diverse origins of multiple ovarian teratomas in a single individual.
Authors: Carritt B , Parrington J M , Welch H M , Povey S
Published: May 2006

Centromere heteromorphisms and enzyme markers were examined in cells cultured from seven benign ovarian teratomas arising in a single patient. Three of the teratomas were homozygous for all eight enzyme and centromere markers found to be heterozygous in the host. The other four tumors were heterozygous at the centromeres of chromosomes 1, 16, 17, and 18, as in the patient, but were homozygous for at least one of the enzyme markers. The linkage phases of the heterozygous enzyme markers phosphogluconate dehydrogenase and phosphoglucomutase 1 and the chromosome 1 centromere heteromorphism were established for the patient and for three of the heterozygous teratomas by analysis of Chinese hamster-human somatic cell hybrids. The linkage phase of these markers in homozygous and heterozygous tumors was in every case different from that in the host. The finding of heterozygous centromeres in ovarian teratomas excludes suppression of meiosis II as a mechanism for their origin, and we suggest rather that they arise by failure of meiosis I. The linkage phases in the fully homozygous tumors are most readily derived from that in the patient, we suggest, by endoreduplication of a haploid gamete. The varied origin of ovarian teratomas has important implications for the suitability of such material for centromere-based gene mapping.
A GAL10-CYC1 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site.
Authors: Guarente L , Yocum R R , Gifford P
Published: May 2006

We have identified the promoter region of the GAL10 gene (whose product is UDP-galactose epimerase) of Saccharomyces cerevisiae; this promoter mediates galactose induction of transcription in conjunction with the product of the GAL4 regulatory gene. This identification was achieved by excising a 365-base-pair fragment of GAL10 leader DNA with a GAL10 proximal endpoint greater than 100 base pairs upstream of the transcriptional start site and substituting it in place of the upstream activation site of the CYC1 (iso-1-cytochrome c) promoter [Guarente, L. & Ptashne, M. (1981) Proc. Natl. Acad. Sci. USA 78, 2199-2203]. The hybrid promoter is composed of DNA encoding CYC1 mRNA start sites and the GAL segment upstream of these sites. This promoter is regulated in a manner analogous to GAL10; i.e., it is induced by galactose and responds to mutations in the GAL4 and GAL80 regulatory loci. The activity of the hybrid promoter requires sequences in the region of the CYC1 mRNA start sites but does not require a precise spacing between these sequences and the GAL segment. The transposed GAL segment appears not to contain sequences that mediate glucose repression. Thus, the picture of the GAL10 promoter that emerges is one of an upstream activation site that responds to the GAL4 product plus galactose, and a region of transcription initiation that may contain sequences that mediate glucose repression. Experiments employing strains inducible (GAL80) or constitutive (gal80) for GAL10 expression indicate that an additional component of glucose repression is inducer exclusion.
Isolation of distinct cDNA clones encoding HLA-DR beta chains by use of an expression assay.
Authors: Long E O , Wake C T , Strubin M , Gross N , Accolla R S , Carrel S , Mach B
Published: May 2006

cDNA clones encoding different human Ia antigen beta chains were isolated by use of a complementation-expression assay in Xenopus oocytes. The assay was based on two previous findings. First, oocytes injected with mRNA from a human B-cell line express HLA-DR antigen. The three intracellular DR chains are assembled in oocytes and can be immunoprecipitated with anti-DR monoclonal antibodies. Second, we have isolated cDNA clones encoding DR alpha and intermediate chains. In order to identify beta-chain cDNA clones, mRNA was hybrid-selected with pools of cDNA clones, mixed with mRNA for the alpha and intermediate chains, and injected into oocytes. We isolated two distinct clones that could select DR beta-chain mRNA as demonstrated by assembly of the translation product with DR alpha chains and immunoprecipitation with DR-specific monoclonal antibodies. One clone is specific for a beta chain of the DR locus. The other clone, much weaker in its ability to select DR mRNA, encodes another Ia-like beta chain. Full-length cDNA clones corresponding to the DR and Ia-like beta chains were isolated and compared. Cross-hybridization was detectable in the coding regions but not in the 3' untranslated regions. Distinct RNAs homologous to the DR and the Ia-like beta-chain clones were present in B cells but were undetectable in three T-cell lines.
Response of vitamin B-6 deficiency and the carpal tunnel syndrome to pyridoxine.
Authors: Ellis J M , Folkers K , Levy M , Shizukuishi S , Lewandowski J , Nishii S , Schubert H A , Ulrich R
Published: May 2006

The specific activities and percentage deficiencies of the glutamic oxaloacetic transaminase of erythrocytes (EGOT) were determined for patients with carpal tunnel syndrome (CTS) diagnosed by clinical examination and electrical conduction data; the EGOT data revealed a severe deficiency of vitamin B-6. After double-blind treatment with pyridoxine and placebo, two physicians identified those receiving pyridoxine (clinically improved) and those receiving placebo (did not improve) without error, P less than 0.0078. Correcting a deficiency of the coenzyme at receptors of existing molecules of the apoenzyme appears to take place within days; correction of the deficiency in the number of molecules of the transaminase takes place over 10-12 weeks. The clinical response, appraised by the diminution of the symptoms of CTS, was correlated only with the restored levels of the transaminase which presumably results from a translational long-term increase in the number of molecules of EGOT by a mechanism activated by correcting a deficiency of pyridoxal 5'-phosphate. Apparent Km values of EGOT were identical for groups of patients with CTS and others without CTS but with identical specific activities, indicating that CTS is a primary deficiency of vitamin B-6 rather than one of a dependency state. Clinical improvement of the syndrome with pyridoxine therapy may frequently obviate hand surgery.