bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific insertion and deletion of insertion sequence 1-like DNA element causes the reversible expression of the virulence capsular antigen Vi of Citrobacter freundii in Escherichia coli.
Authors: Ou J T , Baron L S , Rubin F A , Kopecko D J
Published: May 2006

Citrobacter freundii strain WR7004 reversibly expresses the virulence capsular antigen Vi, whose production is controlled by two distinct chromosomal loci, viaA and viaB. The rate of oscillation between the Vi-producing (Vi+) state and the Vi-nonproducing (Vi-) state in strain WR7004 ranges from 2 X 10(-4) to 7 X 10(-3) transitions per bacterium per generation. A similarly high conversion rate from Vi+ to Vi- occurs in Escherichia coli HB101 harboring pWR127, a plasmid that contains the 18-kilobase-pair (kb) viaB region cloned from WR7004. However, the Vi- state in HB101 harboring pWR127 was so stable that transition to the Vi+ state was not detected (less than 10(-10) per bacterium per generation). When pWR127 DNA derived from Vi- strains of HB101 harboring pWR127 was transformed in HB101 recipient, a small number of Vi+ transformants were seen among the transformants, which were predominantly Vi-. The viaB region consists of two EcoRI digestion fragments, A (8.6 kb) and B (9.4 kb). A discrete 700- to 800-base-pair DNA element was found to be inserted in the EcoRI B fragment of the viaB region of pWR127 when derived from Vi- strains. However, no such DNA element was found in the pWR127 DNA isolated from Vi+ strains. This discrete DNA element inserts into a recombinational hot spot 1.1 kb from the end of the EcoRI B fragment and behaves as an insertion sequence 1 (IS1)-like element. The insertion of this IS1-like element in the viaB region thus disrupts expression of the Vi antigen. Restoration of Vi expression results when this element is excised.
Thy-1-mediated regulation of a low-threshold transient calcium current in cultured sensory neurons.
Authors: Saleh M , Lang R J , Bartlett P F
Published: May 2006

The physiological effect of ligand binding to the Thy-1 molecule expressed on the neuronal cell surface was examined. Sensory neurons obtained from dorsal root ganglia of 2-day-old CBA/CAH mice (Thy-1.2) were cultured in vitro for 8 days, and then patch-clamp recordings were obtained of calcium currents. It was found that binding of an anti-Thy-1.2 monoclonal antibody to neurons expressing the Thy-1.2 moiety increased the amplitude of a low-threshold transient calcium current. Monoclonal antibodies directed against the Thy-1.1 molecule or a neuronal surface ganglioside (A2B5) had no effect on this calcium current in Thy-1.2-bearing cells, nor did they interfere with subsequent activation by the anti-Thy-1.2 monoclonal antibody. These results demonstrate that ligand binding to the Thy-1 molecule transduces a physiological signal in sensory neurons by increasing a voltage-activated calcium current.
Ion transport by the Na-Ca exchange in isolated rod outer segments.
Authors: Lagnado L , Cervetto L , McNaughton P A
Published: May 2006

The inward membrane current generated by the coupled exchange of external sodium for internal calcium has been investigated in isolated rod outer segments. The exchange rate is sensitive to voltage, with a reduction by a factor of e occurring for a 70-mV depolarization in normal Ringer's solution. The voltage sensitivity is not a constant property of the exchange, as it is reduced by an increase in external Na+ or by the removal of external Ca2+, Mg2+, or K+. Changes in membrane potential do not appear to affect the affinity of the exchange mechanism for internal Ca2+, but hyperpolarization increases the affinity for external Na+. When the external Na+ concentration is raised sufficiently to saturate the exchange mechanism, the voltage sensitivity is no longer apparent. We propose that the voltage dependence of the exchange is due to the external Na+-binding site being sensitive to membrane potential, perhaps because it is located within the membrane electric field.
Number of visits to a state in a random walk, before absorption, and related topics.
Authors: Hill T L
Published: May 2006

Equations are derived for the probability of n visits to a given state during the course of a random walk on a finite diagram that starts from a specified state and ends with absorption. By deriving the mean number of visits in two different ways, certain conjectures or theorems are encountered that connect properties of different but related diagrams in an interesting way. Other subjects included are (i) number of one-way transitions between two states before absorption; (ii) time dependence of the rate of cycle completions before absorption; and (iii) the relation of this work to the "return process" of Karlin and Taylor.
In vitro expression in eukaryotic cells of a prion protein gene cloned from scrapie-infected mouse brain.
Authors: Caughey B , Race R E , Vogel M , Buchmeier M J , Chesebro B
Published: May 2006

It has been proposed that the causative agent of scrapie represents a class of infectious particle that is devoid of nucleic acid and that an altered form of the endogenous prion protein (PrP) is the agent. However, it has been difficult to exclude the possibility that PrP purified from scrapie tissues might be contaminated with a more conventional viral agent. To obtain PrP uncontaminated by scrapie-infected tissues, PrP cDNA cloned from a scrapie-infected mouse brain was expressed in mouse C127 cells in vitro. mRNA and protein encoded by the cloned PrP gene were identified. The expressed PrP polypeptides appeared to be glycosylated and were released from the cell surface into the medium. Homogenates of the cells expressing the cloned PrP gene were inoculated into susceptible mice but failed to induce clinical signs of scrapie. Thus, either PrP is not the transmissible agent of scrapie or the expressed PrP requires additional modification to be infectious.
Identification and purification of a human immunoglobulin-enhancer-binding protein (NF-kappa B) that activates transcription from a human immunodeficiency virus type 1 promoter in vitro.
Authors: Kawakami K , Scheidereit C , Roeder R G
Published: May 2006

The enhancer-binding factor NF-kappa B, which is found only in cells that transcribe immunoglobulin light chain genes, has been purified from nuclear extracts of Namalwa cells (human Burkitt lymphoma cells) by sequence-specific DNA affinity chromatography. The purified NF-kappa B has been identified as a 51-kDa polypeptide by UV-crosslinking analysis. "Footprint" and methylation-interference analyses have shown that purified NF-kappa B has a binding activity specific for the kappa light chain enhancer sequence. The purified factor activated in vitro transcription of the human immunodeficiency virus type I promoter by binding to an upstream NF-kappa B-binding site.
Differential regulation of S6 phosphorylation by insulin and epidermal growth factor in Swiss mouse 3T3 cells: insulin activation of type 1 phosphatase.
Authors: Olivier A R , Ballou L M , Thomas G
Published: May 2006

Insulin and epidermal growth factor (EGF) induce distinct kinetics of S6 kinase activation and S6 phosphorylation in Swiss 3T3 cells. Both events are differentially regulated by specific phosphatases. The major S6 phosphatase in cell extracts was identified as a type 1 enzyme by its chromatographic properties, its sensitivity to inhibitor 2, and its substrate specificity. This enzyme is different from the major S6 kinase phosphatase, which is a type 2A enzyme. Insulin at physiological concentrations causes up to a 2-fold activation of a type 1 S6 phosphatase, whereas at higher concentrations this effect is significantly diminished. EGF alone has little effect on this enzyme, and with both agents together the total phosphatase activity remains basal. The results are consistent with the phosphorylation state of S6 observed in vivo and suggest a role of phosphatase type 1 in the regulation of protein synthesis.
Expression of receptors for cholecystokinin and other Ca2+-mobilizing hormones in Xenopus oocytes.
Authors: Williams J A , McChesney D J , Calayag M C , Lingappa V R , Logsdon C D
Published: May 2006

The expression of receptors for cholecystokinin (CCK) and other similar acting Ca2+-mobilizing hormones was studied in Xenopus laevis oocytes. Poly(A)+ RNA was prepared from pancreatic AR42J cells, which normally express receptors for CCK and bombesin and the RNA injected into oocytes. The presence of these pancreatic receptors on the oocytes was then demonstrated by hormone-induced mobilization of 45Ca2+. CCK receptors were present 1 day (maximum, 2 days) after injection of RNA and were generally proportional to the amount of poly(A)+ RNA injected (1-50 ng). Oocyte CCK receptors retained selectivity for CCK analogs (CCK8 greater than unsulfated CCK8 greater than CCK4) and were blocked by the specific CCK receptor antagonist CR 1409. When poly(A)+ RNA was subjected to size fractionation on sucrose gradients, activity-inducing CCK receptors showed a single peak centered at 3 kilobases. The generality of this oocyte system for expressing Ca2+-mobilizing hormone receptors was further shown by expression of a response to bombesin after injection of AR42J cell RNA and a response to vasopressin and angiotensin II when poly(A)+ RNA from rat liver was injected. No response to CCK was demonstrable after injection of liver RNA, demonstrating the specificity of this assay.
Active site-directed inhibition of Ca2+/calmodulin-dependent protein kinase type II by a bifunctional calmodulin-binding peptide.
Authors: Kelly P T , Weinberger R P , Waxham M N
Published: May 2006

The activation of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM-KII) by Ca2+/CaM results in autophosphorylation and the generation of Ca2+/CaM-independent enzyme activity. We postulated that CaM binding and subsequent autophosphorylation alters the conformation of CaM-KII and exposes its substrate-binding and catalytic site(s). Previous peptide mapping studies on CaM-KII demonstrated the close proximity of CaM-binding and autophosphorylation domains. Analyses of the deduced amino acid sequences encoding CaM-KII have allowed the identification of its CaM-binding domain and have revealed two consensus phosphorylation sites that flank this regulatory domain. We report herein the distinct properties of two synthetic peptides modeled after the CaM-binding domain of CaM-KII. The first peptide binds CaM in a Ca2+-dependent manner and is an antagonist of CaM-KII activation (IC50 approximately equal to 75 nM). It does not, however, inhibit CaM-KII activity. A second peptide containing the same CaM-binding domain plus a putative autophosphorylation sequence at its N terminus displayed bifunctional regulatory properties. In addition to being a CaM antagonist, the latter was a potent inhibitor of Ca2+/CaM-independent kinase activity (IC50 approximately equal to 2 microM). We suggest that this bifunctional peptide represents an active site-directed inhibitory element of CaM-KII. The separation of CaM antagonist and active site-directed inhibitory properties of this peptide distinguishes CaM-KII from other CaM-dependent enzymes in which bifunctional regulatory properties appear to reside in the same peptide domain. These results indicate that the definition of site-directed inhibitory peptides should, in some cases, be expanded to include bona fide phosphorylation sites.
Toward a better understanding of protein folding pathways.
Authors: Creighton T E
Published: May 2006

Experimental observations of how unfolded proteins refold to their native three-dimensional structures contrast with many popular theories of protein folding mechanisms. The available experimental evidence (ignoring slow cis-trans peptide bond isomerization) is largely consistent with the following general scheme: under folding conditions, unfolded protein molecules rapidly equilibrate between different conformations prior to complete refolding. This rapid prefolding equilibrium favors certain compact conformations that have somewhat lower free energies than the other unfolded conformations. Some of the favored conformations are important for productive folding. The rate-limiting step occurs late in the pathway and involves a high-energy, distorted form of the native conformation; there appears to be a single transition state through which essentially all molecules refold. Consequently, proteins are not assembled via a large number of independent pathways, nor is folding initiated by a nucleation event in the unfolded protein followed by rapid growth of the folded structure. The known folding pathways involving disulfide bond formation follow the same general principles. An exceptional folding mechanism for reduced ribonuclease A proposed by Scheraga et al. (Scheraga, H.A., Konishi, Y., Rothwarf, D.M. & Mui, P.W. (1987) Proc. Natl. Acad. Sci. USA 84, 5740-5744) is shown to result from experimental shortcomings, an incorrect kinetic analysis, and a failure to consider the kinetics of unfolding.
Adhesion domain of human T11 (CD2) is encoded by a single exon.
Authors: Richardson N E , Chang H C , Brown N R , Hussey R E , Sayre P H , Reinherz E L
Published: May 2006

The 50-kDa T11 (CD2) T-lymphocyte surface glycoprotein facilitates physical adhesion between T-lineage cells and their cognate cellular counterparts (cytotoxic T-lymphocytes-target cells, helper T lymphocytes-antigen-presenting cells, or thymocytes-thymic epithelium) as well as signaling through the antigen-specific T3-Ti receptor complex. To examine the relationship between the structure and function of the T11 molecule, we have utilized a baculoviral expression system to produce milligram quantities of the hydrophilic extracellular T11 segment. Enzyme cleavage, microsequencing, and HPLC analyses of the expressed protein in conjunction with genomic cloning information show that the domain involved in cellular adhesion is encoded by a single 321-base-pair exon.
Human immunodeficiency virus type 1 challenge of chimpanzees immunized with recombinant envelope glycoprotein gp120.
Authors: Berman P W , Groopman J E , Gregory T , Clapham P R , Weiss R A , Ferriani R , Riddle L , Shimasaki C , Lucas C , Lasky L A
Published: May 2006

The major envelope glycoprotein, gp120, of human immunodeficiency virus type 1 (HIV-1) was purified from a Chinese hamster ovary cell line transfected with a truncated form of the HIV-1 env gene. The recombinant glycoprotein (rgp120) was formulated with aluminum hydroxide adjuvant and was used to immunize chimpanzees. The recombinant preparation was effective in eliciting cellular and humoral immunity as well as immunologic memory. Anti-rgp 120 antibodies reacted with authentic viral gp120 in immunological blot assays and were able to neutralize HIV-1 infectivity in vitro. Sera from the rgp120-immunized animals were able to neutralize HIV-1 pseudotypes of vesicular stomatitis virus prepared from the IIIB isolate, from which the gene encoding rgp120 was derived, as well as two heterologous isolates, ARV-2 and RF. The immune response elicited against the rgp120 was not effective in preventing viral infection after intravenous challenge with HIV-1. The implications of these results on HIV-1 vaccine development are discussed.
Homozygous deletion of the alpha- and beta 1-interferon genes in human leukemia and derived cell lines.
Authors: Diaz M O , Ziemin S , Le Beau M M , Pitha P , Smith S D , Chilcote R R , Rowley J D
Published: May 2006

The loss of bands p21-22 from one chromosome 9 homologue as a consequence of a deletion of the short arm [del(9p)], unbalanced translocation, or monosomy 9 is frequently observed in the malignant cells of patients with lymphoid neoplasias, including acute lymphoblastic leukemia and non-Hodgkin lymphoma. The alpha- and beta 1-interferon genes have been assigned to this chromosome region (9p21-22). We now present evidence of the homozygous deletion of the interferon genes in neoplastic hematopoietic cell lines and primary leukemia cells in the presence or absence of chromosomal deletions that are detectable at the level of the light microscope. In these cell lines, the deletion of the interferon genes is accompanied by a deficiency of 5'-methylthioadenosine phosphorylase (EC 2.4.2.28), an enzyme of purine metabolism. These homozygous deletions may be associated with the loss of a tumor-suppressor gene that is involved in the development of these neoplasias. The relevant genes may be either the interferon genes themselves or a gene that has a tumor-suppressor function and is closely linked to them.
Catalysis of a stereospecific bimolecular amide synthesis by an antibody.
Authors: Benkovic S J , Napper A D , Lerner R A
Published: May 2006

We report a nonhydrolytic bimolecular aminolysis reaction catalyzed by an antibody. The stereospecific formation of an amide from racemic lactone plus an aromatic amine is described by a random equilibrium bireactant kinetic sequence. The observed turnover rate may be approximated from the measured difference between the binding of reactants and the transition state analog.
Compact isospectral sets of plane domains
Authors: Osgood B. , Phillips R. , Sarnak P.
Published: May 2006

Any isospectral family of two-dimensional Euclidean domains is shown to be compact in the
C
∞
topology. Previously Melrose, using heat invariants, was able to establish the
C
∞
compactness of the curvature of the boundary curves. The additional ingredient used in this paper to obtain the compactness of the domains is the behavior of the determinant of the Laplacian near the boundary of the moduli space.
"Subversive" substrates for the enzyme trypanothione disulfide reductase: alternative approach to chemotherapy of Chagas disease.
Authors: Henderson G B , Ulrich P , Fairlamb A H , Rosenberg I , Pereira M , Sela M , Cerami A
Published: May 2006

The trypanosomatid flavoprotein disulfide reductase, trypanothione reductase, is shown to catalyze one-electron reduction of suitably substituted naphthoquinone and nitrofuran derivatives. A number of such compounds have been chemically synthesized, and a structure-activity relationship has been established; the enzyme is most active with compounds that contain basic functional groups in side-chain residues. The reduced products are readily reoxidized by molecular oxygen and thus undergo classical enzyme-catalyzed redox cycling. In addition to their ability to act as substrates for trypanothione reductase, the compounds are also shown to effectively inhibit enzymatic reduction of the enzyme's physiological substrate, trypanothione disulfide. Under aerobic conditions, trypanothione reductase is not inactivated by these redox-cycling substrates, whereas under anaerobic conditions the nitrofuran compounds cause irreversible inactivation of the enzyme. When tested for biological activity against Trypanosoma cruzi trypomastigotes, many of the test compounds were trypanocidal, and this activity correlated with their relative ability to act as substrates for trypanothione reductase. The activity of the enzyme with these redox-cycling derivatives constitutes a subversion of its normal antioxidant role within the cell. For this reason these compounds may be termed "subversive" substrates for trypanothione reductase.
Increased tyrosine kinase activity associated with the protein encoded by the activated neu oncogene.
Authors: Bargmann C I , Weinberg R A
Published: May 2006

A single mutation altering the transmembrane domain of the receptor-like p185 protein encoded by the rat neu gene converts the normal neu gene into a potent oncogene. The biochemical consequences of this mutation were studied by examining phosphorylation of the normal and transforming p185 molecules in membrane preparations. Here we show that the transforming p185 is phosphorylated to a much higher extent in vitro than its normal counterpart. This preferential phosphorylation has the properties that would be expected of p185 autophosphorylation: it takes place on tyrosine and requires intact p185 kinase activity. The normal p185 protein does not demonstrate increased phosphorylation even when it coexists in a transformed cell with the transforming p185 protein. These data show that transforming p185 is specifically associated with an active tyrosine kinase activity and suggest that this activity is intrinsic to the transforming protein. Thus, the transmembrane domain of p185 appears to directly regulate its kinase activity.
Relevance of the reaction of a manganese(III) chelate with hydroxide ion to photosynthesis: Reaction of hydroxide ion with 5,10,15,20-tetrakis(2,4,6-trimethylphenyl)porphinatomanganese(III) in ligating and nonligating solvents
Authors: Nakagaki Paul C. , Calderwood Thomas S. , Bruice Thomas C.
Published: May 2006

The reaction of HO
-
with 5,10,15,20-tetrakis(2,4,6-trimethylphenyl)porphinatomanganese(III) chloride [(TMP)Mn
III
(Cl)] in ligating solvents (CH
3
CN, dimethyl sulfoxide, pyridine) results in formation of (TMP)Mn
II
(≈10
6
M
-1
·s
-r
), which in a slower reaction is converted to a product whose structure is suggested to be that of a porphyrin manganese(III) peroxo dimer. Admittance of O
2
at any time during these reactions leads to formation of the manganese(III) peroxide (TMP)Mn
III
(O
2
)
-
. In nonligating solvents [CH
2
Cl
2
, (CH
3
)
2
CO], the reaction of HO
-
with (TMP)Mn
III
(Cl) yields (TMP)Mn
IV
(OH)
2
.
Sequence, tissue distribution, and chromosomal localization of mRNA encoding a human glucose transporter-like protein.
Authors: Fukumoto H , Seino S , Imura H , Seino Y , Eddy R L , Fukushima Y , Byers M G , Shows T B , Bell G I
Published: May 2006

cDNA clones encoding a glucose transporter-like protein have been isolated from adult human liver and kidney cDNA libraries by cross-hybridization with the human HepG2/erythrocyte glucose transporter cDNA. Analysis of the sequence of this 524-amino acid glucose transporter-like protein indicates that it has 55.5% identity with the HepG2/erythrocyte glucose transporter as well as a similar structural organization. Studies of the tissue distribution of the mRNA coding for this glucose transporter-like protein in adult human tissues indicate that the highest amounts are present in liver with lower amounts in kidney and small intestine. The amounts of glucose transporter-like mRNA in other tissues, including colon, stomach, cerebrum, skeletal muscle, and adipose tissue, were below the level of sensitivity of our assay. The single-copy gene encoding this glucose transporter-like protein has been localized to the q26.1----q26.3 region of chromosome 3.
Expression of the human erythrocyte glucose transporter in Escherichia coli.
Authors: Sarkar H K , Thorens B , Lodish H F , Kaback H R
Published: May 2006

The gene encoding the human erythrocyte glucose transporter, cloned from HepG2 hepatoma cells, was expressed in Escherichia coli by introducing a prokaryote-type ribosome binding site, subcloning the gene into the T7 promoter/T7 polymerase expression system, and transforming a strain that is defective in glucose transport. Cells bearing plasmids with the transporter gene take up 2-deoxy-D-glucose and D-glucose, unlike cells bearing plasmids without the transporter gene. Moreover, 2-deoxy-D-glucose uptake is inhibited by unlabeled D-glucose, cytochalasin B, or mercuric chloride but not by L-glucose. The glucose transport protein is inserted into the membrane of E. coli, as evidenced by immunoblotting experiments with two site-directed polyclonal antibodies, one directed against the COOH terminus of the glucose transporter and the other directed against a synthetic peptide containing amino acid residues 225-238. As detected with both antibodies, the protein migrates with apparent molecular mass of 34 kDa in sodium dodecyl sulfate/12% polyacrylamide, a size similar to that of the unglycosylated glucose-transport protein synthesized in vitro.
Alveolar type II cells express a high-affinity receptor for pulmonary surfactant protein A.
Authors: Kuroki Y , Mason R J , Voelker D R
Published: May 2006

Primary cultures of rat alveolar type II cells bind radiolabeled pulmonary surfactant protein A (SP-A) with high affinity. The binding of 125I-labeled SP-A is time- and temperature-dependent and is not accompanied by significant degradation. The binding process is saturable at low concentrations of SP-A (5 micrograms/ml), and unlabeled SP-A readily competes with labeled SP-A for cellular binding sites. Subsequent to binding, two pools of cell-associated 125I-labeled SP-A can be identified based upon sensitivity to trypsin at 0 degrees C. It is likely that the trypsin-sensitive pool comprises 125I-labeled SP-A bound to the cell surface and the trypsin-insensitive pool comprises the internalized protein. Scatchard analysis of cell surface binding of SP-A at 0.1-10 micrograms/ml shows positive cooperativity at concentrations between 0.1 and 1 micrograms/ml. Hill plots give nH = 1.34 +/- 0.08 with an apparent dissociation constant K'd = 1.02 +/- 0.32 micrograms/ml (which is 0.64 +/- 0.19 nM if the native molecular mass of oligomeric SP-A is assumed to be 1.6 MDa). The binding of SP-A to type II cells shows an absolute requirement for Ca2+. The putative receptor for SP-A is unaffected by treatment of type II cells with a variety of proteases and N-Glycanase (EC 3.5.1.52). Alveolar macrophages also exhibit high-affinity binding of SP-A, but rat lung fibroblasts and the alveolar epithelial cell line L2 exhibit only nonspecific binding.
Continuous, on-line DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores.
Authors: Brumbaugh J A , Middendorf L R , Grone D L , Ruth J L
Published: May 2006

A method for sequencing DNA by using a difluoresceinated primer and laser excitation is described. Dideoxy protocols have been determined that provide sequences for 600 bases starting with base 1 with less than 1% error in a single load. Electrophoresis is at 20 W and the bands are detected 24 cm from the bottom of the loading well with a scanning fluorescence detector. Bands are imaged on a TV screen in two dimensions. The sequences can be read from the TV screen manually or semiautomatically by using a simple software program. The system allows more bases to be read with a lower error rate than any other reported automated sequencing method.
Inhibitors of glycoprotein processing alter T-cell proliferative responses to antigen and to interleukin 2.
Authors: Wall K A , Pierce J D , Elbein A D
Published: May 2006

Most of the cell-surface molecules involved in T-cell immune responses are N-linked glycoproteins. We have investigated the effects of inhibitors of glycoprotein processing on specific T-cell functions, with the dual aims of examining the functional role of carbohydrate and of testing the usefulness of such compounds as immunomodulators. Treatment of a cloned murine helper T-cell line with these inhibitors differentially affects the proliferative response of the cell, depending upon the nature of the stimulus. Treatment with the plant alkaloid swainsonine, which inhibits the processing mannosidase II and causes the accumulation of glycoproteins bearing hybrid-type oligosaccharide structures, enhances the proliferative response of the T-cell clone to antigen and to the mitogen concanavalin A. Treatment with another plant alkaloid, castanospermine, which inhibits glucosidase I and causes the accumulation of glucose-containing high-mannose structures, has the opposite effect and inhibits the proliferative response of the T cell to antigen. Cell-surface oligosaccharide alteration does not affect antigen recognition, as judged by the lack of effect of either drug on interleukin 2 production following antigen stimulation. Cells treated with either alkaloid proliferate poorly to exogenous interleukin 2 and may have defective interleukin 2 receptor function. Swainsonine-treated cells apparently have compensatory alterations that can overcome the reduced responsiveness to interleukin 2. Antibody-binding studies indicate that normal quantities of many cell-surface molecules, including the T-cell receptor for antigen, are expressed by the treated cells.
Primary structure of cotranscribed genes encoding the Rieske Fe-S and cytochrome f proteins of the cyanobacterium Nostoc PCC 7906.
Authors: Kallas T , Spiller S , Malkin R
Published: May 2006

The thylakoid membrane cytochrome b6-f complex (plastoquinol:oxidized-plastocyanin oxidoreductase, EC 1.10.99.1) catalyzes electron-transfer and proton-translocation reactions essential for oxygenic photosynthesis. We have isolated and determined the nucleotide sequences of the petC and petA genes encoding the Rieske Fe-S and cytochrome f polypeptides from the filamentous cyanobacterium Nostoc PCC 7906. These genes occur as single genomic copies, are tightly linked, and, as indicated by hybridization of gene-specific probes to Nostoc RNA, are cotranscribed as a 2.0-kilobase message. The Rieske Fe-S/cytochrome f gene pair thus represents an example of clustering and cotranscription in cyanobacteria of functionally related genes that, in photosynthetic eukaryotes, reside on separate nuclear and plastid genomes. These data are consistent with the progressive degeneration of the modern chloroplast genome from the ancestral, cyanobacterial-like genome of an endosymbiont. The Rieske Fe-S and the mature cytochrome f apoproteins are encoded by 537 and 867 nucleotides and have molecular masses of 19.2 and 31.2 kDa, respectively. They show 59% and 60% protein sequence identity, respectively, relative to spinach. Forty-four amino acids (4.7 kDa) resembling a prokaryotic signal sequence precede apocytochrome f. In contrast, the Rieske Fe-S protein appears to be translated without a presequence. The 183 bases separating the Rieske Fe-S and preapocytochrome f genes contain two families of 7- to 9-base tandem repeats, and some part of this sequence is highly reiterated in the genome. The C terminus of the Rieske Fe-S protein contains cysteine and histidine residues (probable ligands for the Fe2S2 center) in two peptides, Cys-Thr-His-Leu-Gly-Cys-Val and Cys-Pro-Cys-His-Gly-Ser, which have been conserved in spinach and in the five available Rieske Fe-S sequences from the mitochondrial-type cytochrome b-c1 complexes. Cytochrome f shows the heme binding residues Cys-Xaa-Xaa-Cys-His near its N terminus. Single, long hydrophobic stretches occur near the N and C termini, respectively, of the Rieske Fe-S and cytochrome f proteins and may form membrane-spanning helices.
Comparison between DNA melting thermodynamics and DNA polymerase fidelity.
Authors: Petruska J , Goodman M F , Boosalis M S , Sowers L C , Cheong C , Tinoco I
Published: May 2006

The relation between DNA polymerase fidelity and base pairing stability is investigated by using DNA primer-template duplexes that contain a common 9-base template sequence but have either correct (A.T) or incorrect (G.T, C.T, T.T) base pairs at the primer 3' terminus. Thermal melting and enzyme kinetic measurements are compared for each kind of terminus. Analysis of melting temperatures finds that differences between the free energy changes upon dissociation (delta delta Go) are only 0.2, 0.3, and 0.4 kcal.mol-1 (1 cal = 4.18 J) for terminal A.T compared to G.T, C.T, and T.T mispairs, respectively, at 37 degrees C. We show that enthalpy changes are directly correlated with entropy changes for normal and abnormal base pairs in DNA in aqueous solution and that delta delta Go values are small because of near cancellation of corresponding enthalpy and entropy components. The kinetics of elongating primer termini are measured with purified Drosophila DNA polymerase alpha. The matched A.T terminus is found to be extended approximately 200 times faster than a G.T mismatch and 1400 and 2500 times faster than C.T and T.T mismatches, respectively. Enzymatic discrimination against elongating mismatched termini is based mainly on Km rather than Vmax differences. From Km at 37 degrees C, we find delta delta Go values of 2.6-3.7 kcal.mol-1, about an order of magnitude greater than indicated by melting data. A similar measurement of nucleotide insertion kinetics has previously found rates of forming A.T base pairs to be 500 times greater than G.T mispairs and 20,000 times greater than C.T and T.T mispairs. Here also, Km differences are mainly responsible for discrimination and indicate even larger delta delta Go values (4.3-4.9 kcal.mol-1). Thus, free energy differences between correct and incorrect base pairs in the active site cleft of polymerase appear to be greater than 10 times as large as in aqueous medium. We explore the idea that a binding cleft that snugly fits correct base pairs and excludes water at the active site may amplify base-pair free energy differences by reducing entropy differences and increasing enthalpy differences sufficiently to account for nucleotide insertion and extension fidelity.
Effects of linkage on rates of molecular evolution.
Authors: Birky C W , Walsh J B
Published: May 2006

When an advantageous mutation is fixed in a population by selection, a closely linked selectively neutral or mildly detrimental mutation may "hitchhike" to fixation along with it. It has been suggested that hitchhiking might increase the rate of molecular evolution. Computer simulations and a mathematical argument show that complete linkage to either advantageous or deleterious mutations does not affect the substitution of selectively neutral mutations. However, the simulations show that linkage to selected background mutations decreases the rate of fixation of advantageous mutations and increases the rate of fixation of detrimental mutations. This is true whether the linked background mutations are advantageous or detrimental, and it verifies and extends previous observations that linkage tends to reduce the effects of selection on evolution. These results can be interpreted in terms of the Hill-Robertson effect: a locus linked to another locus under selection experiences a reduction in effective population size. The interpretation of differences in evolutionary rates between different genomes or different regions of a genome may be confounded by the effects of strong linkage and selection. Recombination is expected to reduce the overall rate of molecular evolution while enhancing the rate of adaptive evolution.
Selection of the mRNA translation initiation region by Escherichia coli ribosomes.
Authors: Calogero R A , Pon C L , Canonaco M A , Gualerzi C O
Published: May 2006

Two genes specifying model mRNAs of minimal size and coding capacity, with or without the Shine-Dalgarno (SD) sequence, were assembled, cloned, and transcribed in high yields. These mRNAs, as well as synthetic polynucleotides, phage MS2 RNA, and a deoxyoctanucleotide complementary to the 3' end of 16S rRNA were used to study the mechanism of translation initiation in vitro. Escherichia coli 30S ribosomal subunits interact with all these nucleic acids, albeit with different affinities; the affinity for the mRNA with the SD sequence (Ka approximately 2 x 10(7) M-1) is more than an order of magnitude higher than that for the mRNA lacking this sequence. The initiation factors are equally required, regardless of the presence of the SD sequence, for 30S and 70S initiation complex formation and for mRNA translation, but the initiation factors do not affect the SD interaction or the binding of the mRNAs to the ribosomes. The SD interaction is also mechanistically irrelevant for 30S initiation complex formation and is not essential for translation in vitro or for the selection of the mRNA reading frame. It is suggested that the function of the SD interaction is to ensure a high concentration of the initiation triplet near the ribosomal peptidyl-tRNA binding site, whereas the selection of the translational start is achieved kinetically, under the influence of the initiation factors, during decoding of the initiator tRNA.
HLA-DQ rather than HLA-DR region might be involved in dominant nonsusceptibility to diabetes.
Authors: Sterkers G , Zeliszewski D , Chaussée A M , Deschamps I , Font M P , Freidel C , Hors J , Betuel H , Dausset J , Levy J P
Published: May 2006

Since HLA-DRw15 (a subdivision of the HLA-DR2 specificity previously called DR2 long) is associated with dominant nonsusceptibility to insulin-dependent diabetes mellitus (IDDM), while HLA-DRw16 (another subdivision of HLA-DR2, previously called DR2 short) is positively associated with the disease, we looked for particular characteristics of HLA products encoded by the DR2 haplotypes of IDDM patients. The results show the following: (i) HLA-DQ molecules of HLA-DRw15-positive IDDM patients are different from those of HLA-DRw15-positive controls, suggesting that the HLA-DQ gene of DRw15 haplotypes is involved in a protective effect. (ii) HLA-DR and -DQ products of DRw16-positive IDDM are functionally indistinguishable from those of HLA-DRw16-positive controls. Furthermore, our data provide evidence that the residue at position 57 on the DQ beta chain could play a crucial biological role in antigen presentation to T cells as far as the DRw16 haplotype is concerned. This observation fits with the recent observation of correlation between DQ beta allelic polymorphism at position 57 and both susceptibility and resistance to IDDM.
Efficient expression of retroviral vector-transduced human low density lipoprotein (LDL) receptor in LDL receptor-deficient rabbit fibroblasts in vitro.
Authors: Miyanohara A , Sharkey M F , Witztum J L , Steinberg D , Friedmann T
Published: May 2006

Familial hypercholesterolemia is caused by a genetic deficiency of the low density lipoprotein (LDL) receptor. The Watanabe heritable hyperlipidemic (WHHL) rabbit, which is also defective in LDL receptor activity, provides an excellent animal model of homozygous familial hypercholesterolemia. As a step toward development of effective gene therapy for familial hypercholesterolemia, we have constructed a transmissible retroviral vector containing a full-length human cDNA for the LDL receptor. WHHL fibroblasts infected in vitro expressed the human receptor efficiently, as indicated by RNA and ligand blotting studies. Infected fibroblasts bound and degraded a monoclonal antibody specific for the human LDL receptor (IgGC7) in a manner comparable to that seen with normal human fibroblasts. Human LDL was also degraded by infected WHHL cells and promoted cholesterol esterification to the same degree as seen in normal human fibroblasts. Although technical problems remain to be solved, these studies show that, in principle, gene therapy may be possible for familial hypercholesterolemia patients.
A large family of bacterial activator proteins.
Authors: Henikoff S , Haughn G W , Calvo J M , Wallace J C
Published: May 2006

At least nine different bacterial proteins belong to the LysR family. The gene sequence for one of these proteins is presented here. Six others (Escherichia coli LysR, IlvY, CysB; Salmonella typhimurium MetR; Rhizobium NodD; and Enterobacter cloacae AmpR) are known to activate other genes. Based on sequence alignments, each member of this family is predicted to have a helix-turn-helix DNA binding motif near its amino terminus. The combined evidence indicates that all nine proteins are related by common ancestry, are similarly folded, and are not detectably related to other known bacterial regulatory proteins. The DNA database searching procedure and other methods used in this study should be useful in detecting other groups of related proteins.
Distinctive charge configurations in proteins of the Epstein-Barr virus and possible functions.
Authors: Blaisdell B E , Karlin S
Published: May 2006

The protein products of several open reading frames (ORFs) of the Epstein-Barr virus (EBV) are remarkable in their distribution of charged residues. The nuclear antigen proteins EBNA1-EBNA4 of the EBV latent state contain separate significant clusters of charge of each sign. They (excepting EBNA4) also feature distinctive periodic charge patterns [e.g., (+, O)8, (O, -, -)7] and significant tandem repeats. None of the other ORFs (about 80) of the genome possess the conjunction of these properties. Only the protein encoded from BMLF1, the first immediate early transactivator protein, contains significant multiple charge clusters and periodic charge patterns. All proteins that contain significant repeats also contain at least one significant charge cluster of a single sign. These include EBNA5 and LYDMA produced during latency and BZLF1, whose expression terminates latency and initiates productive growth. It is reasonable to conclude that these aggregate significant charge configurations and repeats are important functionally for the latent existence and for the initiation of the lytic cycle and may be characteristic of these conditions. We discuss how large multimeric protein structures bound together by clusters of unlike charge may provide a mechanism for regulation of the expression of these proteins.
Cell adhesion induces expression of growth-associated genes in suspension-arrested fibroblasts.
Authors: Dike L E , Farmer S R
Published: May 2006

A methylcellulose suspension system that prevents cell-surface contact with the substrate was used to study the role of cell adhesion in the regulation of proliferation. The nonadhesive conditions established by suspension culture cause BALB/c 3T3 (A31) cells to enter a G0 state of growth arrest within 48 hr as defined by an inhibition of DNA synthesis and a suppression of c-myc and histone mRNA expression. The adhesion of these suspension-arrested cells rapidly induces c-fos, c-myc, and actin gene expression. This stimulation did not depend on the presence of serum since the adhesion of suspension-arrested cells, in the absence of serum, also induced the expression of c-fos and c-myc mRNAs. In addition, adhesion onto fibronectin increased the number of cells able to respond to epidermal growth factor and insulin and progress into S phase. These results indicate that adhesion of suspension-arrested cells activates the G0/G1 transition independent of growth factors.
Detection of DNA "fingerprints" of cultivated rice by hybridization with a human minisatellite DNA probe.
Authors: Dallas J F
Published: May 2006

A human minisatellite DNA probe detects several restriction fragment length polymorphisms in cultivars of Asian and African rice. Certain fragments appear to be inherited in a Mendelian fashion and may represent unlinked loci. The hybridization patterns appear to be cultivar-specific and largely unchanged after the regeneration of plants from tissue culture. The results suggest that these regions of the rice genome may be used to generate cultivar-specific DNA fingerprints. The demonstration of similarity between a human minisatellite sequence and polymorphic regions in the rice genome suggests that such regions also occur in the genomes of many other plant species.
Anoxic contractile failure in rat heart myocytes is caused by failure of intracellular calcium release due to alteration of the action potential.
Authors: Stern M D , Silverman H S , Houser S R , Josephson R A , Capogrossi M C , Nichols C G , Lederer W J , Lakatta E G
Published: May 2006

Anoxia of the heart causes failure of contraction before any irreversible injury occurs; the mechanism by which anoxia blocks cardiac excitation-contraction coupling is unknown. Studies in whole muscle are confounded by heterogeneity; however, achieving the low oxygen tensions required to study anoxia in a single myocyte during electrophysiological recording has been a barrier in experimental design. Guided by calculations of oxygen transport, we developed a system to insulate myocytes in an open dish from oxygen by a laminar counterflowing argon column, permitting free access to the cell by microelectrodes while maintaining a PO2 less than 0.02 torr (1 torr = 133 Pa). In the absence of glucose, the amplitude of stimulated contraction of anoxic ventricular myocytes fell to zero over 2 min after a lag period attributable to the consumption of endogenous glycogen. The cytosolic calcium concentration transient, measured by indo-1 fluorescence, fell to zero simultaneously with contraction. After the twitch had failed, microinjection of caffeine around the cell still caused a large calcium release and contraction, indicating that sarcoplasmic reticular calcium stores were not depleted. Twitch failure was accompanied by shortening and then failure of the action potential; under voltage clamp, large outward currents, reversing at the resting potential, developed during contractile failure. After failure of action potential-mediated contraction, voltage-clamp depolarization, with a large command voltage to compensate for the series-resistance error due to outward currents, restored normal twitch contraction. We conclude that anoxic contractile failure in the rat myocyte is due to alteration of the action potential and the distal pathways of excitation-contraction coupling remain essentially intact.
Phorbol ester-induced synaptic facilitation is different than long-term potentiation.
Authors: Muller D , Turnbull J , Baudry M , Lynch G
Published: May 2006

The studies described here tested the hypothesis that the changes in synaptic efficacy produced by phorbol esters in hippocampal slices are equivalent to the long-term potentiation (LTP) induced by high-frequency stimulation. In contrast to the extremely stable synaptic potentiation induced by electrical stimulation, the facilitatory effects of phorbol 12,13-diacetate and phorbol 12,13-dibutyrate were transient: washout of the drugs restored normal responses in approximately 1-2 and 2-4 hr for phorbol diacetate and phorbol dibutyrate, respectively. It is noteworthy that the more liposoluble of the phorbol esters required longer washout periods. Robust LTP still occurred in response to high-frequency stimulation after washout of phorbol esters and to a lesser degree during their application. Treatment of slices with H-7, an inhibitor of protein kinase C, did not prevent LTP induction although it significantly affected neuronal excitability and produced effects opposite to those of phorbol esters. Finally, phorbol esters altered responses to repetitive stimulation in a way that could account for the reduced LTP elicited in their presence. These results indicate that the increases in synaptic responses caused by phorbol esters and high-frequency electrical stimulation are quite different and thus do not support the hypothesis that activation of protein kinase C, the presumed target of the phorbol esters, triggers LTP.
Vesicular localization of immunoreactive [Met5]enkephalin in the globus pallidus.
Authors: Coulter H D
Published: May 2006

The distribution of enkephalin immunoreactivity in the neuropil of globus pallidus was analyzed with a quick-freezing, postembedment-staining technique for light and electron microscopic immunocytochemistry. Fluorescence images of ultrathin sections on glass slides, obtained with a silicon-intensified-tube (type) video camera, revealed staining in the form of scattered fluorescent dots, each 200-400 nm in diameter. Colloidal gold staining under the electron microscope was associated with 80- to 100-nm vesicles of average electron density, widely dispersed in the neuropil, with usually one and no more than four vesicles in individual sectioned neuronal processes. Analysis of fluorescence images in paired serial sections of thicknesses varying from 25 to 150 nm proved that the 200- to 400-nm dots of fluorescence came from smaller structures, presumably the 80- to 100-nm vesicles. Enkephalinergic vesicles in the globus pallidus were nearly always found in what appeared to be axons of passage and were only infrequently associated with synaptic profiles.