bluuebunny/crossref_metadata_2025 huggingface dataset:
Trans-activating rev protein of the human immunodeficiency virus 1 interacts directly and specifically with its target RNA.
Authors: Daefler S , Klotman M E , Wong-Staal F
Published: May 2006

The 20-kDa phosphorylated rev protein from human immunodeficiency virus 1 has been shown to transactivate posttranscriptionally the expression of viral structural proteins by selective stabilization and nuclear export of unspliced and incompletely spliced viral mRNA. We could demonstrate in gel-mobility and immunoprecipitation assays that the recombinant rev protein purified from a baculovirus expression system forms a distinct and specific complex with its target RNA (rev-responsive element), a 234-nucleotide sequence within the envelope coding region of human immunodeficiency virus 1. No complex formation could be observed using RNAs with similar secondary structure nor with other human immunodeficiency virus 1 recombinant proteins. Deletion analysis mapped this specific binding to the first 90 nucleotides of this rev-responsive element, which contains a U2 small nuclear RNA homologous region. We propose that the specific binding of rev to its target RNA sequence plays an essential part in releasing an incompletely spliced viral mRNA containing this target sequence to the cytoplasm.
Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya.
Authors: Woese C R , Kandler O , Wheelis M L
Published: May 2006

Molecular structures and sequences are generally more revealing of evolutionary relationships than are classical phenotypes (particularly so among microorganisms). Consequently, the basis for the definition of taxa has progressively shifted from the organismal to the cellular to the molecular level. Molecular comparisons show that life on this planet divides into three primary groupings, commonly known as the eubacteria, the archaebacteria, and the eukaryotes. The three are very dissimilar, the differences that separate them being of a more profound nature than the differences that separate typical kingdoms, such as animals and plants. Unfortunately, neither of the conventionally accepted views of the natural relationships among living systems--i.e., the five-kingdom taxonomy or the eukaryote-prokaryote dichotomy--reflects this primary tripartite division of the living world. To remedy this situation we propose that a formal system of organisms be established in which above the level of kingdom there exists a new taxon called a "domain." Life on this planet would then be seen as comprising three domains, the Bacteria, the Archaea, and the Eucarya, each containing two or more kingdoms. (The Eucarya, for example, contain Animalia, Plantae, Fungi, and a number of others yet to be defined). Although taxonomic structure within the Bacteria and Eucarya is not treated herein, Archaea is formally subdivided into the two kingdoms Euryarchaeota (encompassing the methanogens and their phenotypically diverse relatives) and Crenarchaeota (comprising the relatively tight clustering of extremely thermophilic archaebacteria, whose general phenotype appears to resemble most the ancestral phenotype of the Archaea.
Characterization of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41.
Authors: Freed E O , Myers D J , Risser R
Published: May 2006

The human immunodeficiency virus transmembrane glycoprotein gp41 has at its amino terminus a strongly hydrophobic stretch of 28 amino acids flanked by a highly conserved series of polar amino acids. To investigate the role in syncytium formation of the hydrophobic amino terminus of gp41 and the polar border of this hydrophobic region, we introduced eight single-amino acid substitutions and one double-amino acid substitution in the amino-terminal 31 amino acids of gp41. The mutant envelope glycoproteins were expressed from two distinct human immunodeficiency virus type 1 envelope glycoprotein expression vectors; the effects of the mutations on syncytium formation, envelope glycoprotein transport, secretion, and CD4 receptor-binding were analyzed. Results showed that polar substitutions throughout the hydrophobic amino terminus of gp41 greatly reduced or blocked syncytium formation mediated by the human immunodeficiency virus type 1 envelope glycoproteins, as did nonconservative mutations in the polar border of the hydrophobic amino terminus. Mutations at gp41 amino acids 15, 26, and 29 also significantly increased the extent of gp120 secretion into the extracellular medium. None of the mutations detectably affected envelope glycoprotein processing or envelope glycoprotein binding to CD4.
Cooperative interactions between transcription factors Sp1 and OTF-1.
Authors: Janson L , Pettersson U
Published: May 2006

We have examined whether the functional synergism between transcription factors Sp1 and OTF-1 involves cooperativity in binding. To demonstrate cooperativity, synthetic enhancers were constructed in which Sp1-binding sites were combined with various OTF-1-binding sites that differed in their binding affinities. The ability of these constructions to activate transcription from the human U2 small nuclear RNA promoter was measured. The results showed that an Sp1-binding site stimulated transcription 2-fold when combined with a high-affinity binding site for OTF-1. When combined with a low-affinity OTF-1-binding site, in contrast, a 20-fold stimulation of transcription was observed. The stimulatory effect of Sp1 was moreover influenced by the distance between the Sp1- and OTF-1-binding sites and the functional cooperation was mirrored by the cooperative formation of OTF-1- and Sp1-specific protein-DNA complexes in vitro. We conclude from these results that the functional cooperation between OTF-1 and Sp1 involves physical interactions between the two transcription factors resulting in cooperative binding. The results thus reveal a mechanism by which Sp1 can modulate transcription.
Functional expression of murine multidrug resistance in Xenopus laevis oocytes.
Authors: Castillo G , Vera J C , Yang C P , Horwitz S B , Rosen O M
Published: May 2006

The development of multidrug resistance (MDR) is associated with the overproduction of a plasma membrane glycoprotein, P glycoprotein. Here we report the functional expression of a member of the murine mdr family of proteins and show that Xenopus oocytes injected with RNA encoding the mouse mdr1b P glycoprotein develop a MDR-like phenotype. Immunological analysis indicated that oocytes injected with the mdr1b RNA synthesized a protein with the size and immunological characteristics of the mouse mdr1b P glycoprotein. These oocytes exhibited a decreased accumulation of [3H]vinblastine and showed an increased capacity to extrude the drug compared to control oocytes not expressing the P glycoprotein. In addition, competition experiments indicated that verapamil, vincristine, daunomycin, and quinidine, but not colchicine, can overcome the rapid drug efflux conferred by the expression of the mouse P glycoprotein.
Reversible import of apocytochrome c into mitochondria.
Authors: Hakvoort T B , Sprinkle J R , Margoliash E
Published: May 2006

35S-labeled Drosophila melanogaster apocytochrome c was made by in vitro transcription/translation of the gene and purified to the monomeric, fully reduced form. It was found that in the presence of a wheat germ extract factor there was a high-affinity phase of the uptake into mouse liver mitochondria at 10-300 pM apocytochrome c, and a lower-affinity phase through 4000 pM. Without the factor, the high-affinity phase was absent. The stimulatory effect of the factor could not be elicited with various reductants, such as NADH, FMN, and ferrous protoheme IX. Conversely, when mitochondria loaded with apocytochrome c were resuspended in fresh medium, the protein readily reequilibrated. Successive washings depleted greater than 95% of the associated apoprotein but removed no holoprotein. Proteases (proteinase K or trypsin) added to a suspension of mitochondria loaded with apoprotein digested an amount of apoprotein similar to that which would have been dissociated during the same time, as measured by successive washings in the absence of protease. Mitochondria loaded with apoprotein and similarly treated with protease continued exporting the apoprotein, even after the protease was inhibited and removed, suggesting that most of the apoprotein associated with the organelle was in a protease-resistant compartment. Apocytochrome c mutants in which serines or alanines replaced cysteines 14 and 17, which bind the prosthetic group, behaved like the cysteine-containing protein, indicating that the covalent attachment of the heme is unrelated to the translocation of the apoprotein.
Cell cycle synchronization: reversible induction of G2 synchrony in cultured rodent and human diploid fibroblasts.
Authors: Tobey R A , Oishi N , Crissman H A
Published: May 2006

In accord with a set of prespecified principles of cell synchrony induction, a three-step procedure was developed to arrest cells reversibly in the G2 phase of the cell cycle. Cultures of Chinese hamster ovary (CHO) cells were presynchronized in early S phase by sequential treatment with isoleucine deficiency and hydroxyurea blockades; then they were switched to medium supplemented with either of two agents that inhibit DNA topoisomerase II activity by different mechanisms, Hoechst 33342 at 7.5 micrograms/ml for 12 hr or VM-26 at 0.5 micrograms/ml for 8 hr. Up to 95% of the cells accumulated in G2 phase under those conditions. After switch of Hoechst 33342-treated cells to drug-free medium, the cells divided as a highly synchronized cohort of cells within 3 hr. Up to 85% of the cells in a culture of human diploid dermal fibroblasts (HSF-55 cells) could be accumulated in G2 phase by placing cells presynchronized in early-S phase in medium containing Hoechst 33342 at 0.1 micrograms/ml for 10 hr. Reversal of G2 arrest in the HSF-55 cultures resulted in cells dividing synchronously over 3.5 hr. By varying the concentration of Hoechst 33342 and the duration of the treatment period, it was possible to alter the position within G2 phase at which cells accumulated. This synchronization protocol should greatly facilitate study of G2/M biochemical events in mammalian cells, in particular, those associated with cdc2 gene regulation of the onset of mitosis.
Beta 2-adrenergic receptors are colocalized and coregulated with "whisker barrels" in rat somatosensory cortex.
Authors: Vos P , Kaufmann D , Hand P J , Wolfe B B
Published: May 2006

Autoradiography has been used to visualize independently the subtypes of beta-adrenergic receptors in rat somatosensory cortex. Beta 2-Adrenergic receptors, but not beta 1-adrenergic receptors colocalize with "whisker barrels" in this tissue. Thus, each whisker sends a specific multisynaptic pathway to the somatosensory cortex that can be histochemically visualized and only one subtype of beta-adrenergic receptor is specifically associated with this cortical representation. Additionally, neonatal lesion of any or all of the whisker follicles results in loss of the corresponding barrel(s) as shown by histochemical markers. This loss is paralleled by a similar loss in the organization of beta 2-adrenergic receptors in the somatosensory cortex. Other results indicate that these beta 2-adrenergic receptors are not involved in moment-to-moment signal transmission in this pathway and, additionally, are not involved in a gross way in the development of whisker-barrel array.
Oxidative damage to brain proteins, loss of glutamine synthetase activity, and production of free radicals during ischemia/reperfusion-induced injury to gerbil brain.
Authors: Oliver C N , Starke-Reed P E , Stadtman E R , Liu G J , Carney J M , Floyd R A
Published: May 2006

Free radical-mediated oxidative damage has been implicated in tissue injury resulting from ischemia/reperfusion events. Global cortical ischemia/reperfusion injury to Mongolian gerbil brains was produced by transient occlusion of both common carotid arteries. Protein oxidation, as measured by protein carbonyl content, increased significantly during the reperfusion phase that followed 10 min of ischemia. The activity of glutamine synthetase, an enzyme known to be inactivated by metal-catalyzed oxidation reactions, decreased to 65% of control levels after 2 hr of reperfusion that followed 10 min of ischemia. We also report that the free radical spin trap N-tert-butyl-alpha-phenylnitrone [300 mg/kg (body weight)] administered 60 min before ischemia/reperfusion is initiated, partially prevents protein oxidation and protects from loss of glutamine synthetase activity. In addition, we report a N-tert-butyl-alpha-phenylnitrone-dependent nitroxide radical obtained in the lipid fraction of the ischemia/reperfusion-lesioned brains, but there was very little radical present in the comparable sham-operated control brains. These data strengthen the previous observation utilizing in vivo-trapping methods, that free radical flux is increased during the reperfusion phase of the ischemia-lesioned gerbil brain. The loss of glutamine synthetase would be expected to increase the levels of brain L-glutamate. Thus, the oxidative inactivation of glutamine synthetase may be a critical factor in the neurotoxicity produced after cerebral ischemia/reperfusion injury.
Initial electron-transfer in the reaction center from Rhodobacter sphaeroides.
Authors: Holzapfel W , Finkele U , Kaiser W , Oesterhelt D , Scheer H , Stilz H U , Zinth W
Published: May 2006

The initial electron transfer steps in the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides have been investigated by femtosecond time-resolved spectroscopy. The experimental data taken at various wavelengths demonstrate the existence of at least four intermediate states within the first nanosecond. The difference spectra of the intermediates and transient photodichroism data are fully consistent with a sequential four-step model of the primary electron transfer: Light absorption by the special pair P leads to the state P*. From the excited primary donor P*, the electron is transferred within 3.5 +/- 0.4 ps to the accessory bacteriochlorophyll B. State P+B- decays with a time constant of 0.9 +/- 0.3 ps passing the electron to the bacteriopheophytin H. Finally, the electron is transferred from H- to the quinone QA within 220 +/- 40 ps.
Disruption of microfilament organization in living nonmuscle cells by microinjection of plasma vitamin D-binding protein or DNase I.
Authors: Sanger J M , Dabiri G , Mittal B , Kowalski M A , Haddad J G , Sanger J W
Published: May 2006

Plasma vitamin D-binding protein (DBP), which binds to monomeric actin, causes the breakdown of stress fibers when it is microinjected into nonmuscle cells. Disruption of the stress fiber network is also accompanied by shape changes in the cell that resemble those seen after cytochalasin treatment. When DBP was coinjected with fluorescently labeled alpha-actinin, no fluorescent stress fibers or attachment plaques were visible 30 min after injection. Twelve hours later the cells regained their flattened shape and their stress fibers. Fluorescently labeled DBP causes the same reversible changes in cell shape as the unlabeled protein. Upon injection, the labeled DBP diffuses throughout the cytoplasm, becoming localized by 12 hr in a punctate pattern, presumably due to lysozomal sequestration. Similar injections of DBP into skeletal myotubes and cardiac myocytes did not lead to shape changes or breakdown of nascent and/or fully formed myofibrils, even though DBP has a 2-fold higher binding affinity for muscle actin over that of the nonmuscle isoactins. Similar differential effects in nonmuscle cells were also observed after the microinjection of DNase I, another protein capable of binding monomer actin. The effects of these microinjected monomer actin-binding proteins imply that an accessible pool of monomer actin is needed to maintain stress fiber integrity in nonmuscle cells but not the integrity of the nascent or fully formed myofibrils in muscle cells.
Monoclonal antibody R2D5 reveals midsagittal radial glial system in postnatally developing and adult brainstem.
Authors: Mori K , Ikeda J , Hayaishi O
Published: May 2006

Radial glial cells and their processes play critical roles in organizing the spatial arrangement of the nervous system in the embryonic brain. It has been thought that following completion of their roles in the embryo, most of the radial glial processes disappear before or shortly after birth. Here we use R2D5, a monoclonal antibody to a soluble cytosolic protein, to demonstrate that a specific system of midsagittal radial glial cells persists in postnatal and adult brain. In the brainstem of postnatally developing and adult rabbits and cats, the R2D5-positive processes of radial glial cells were observed to be arranged in a precisely parallel array at the midsagittal seam. These radial glial processes formed a continuous palisade separating the right and left brainstem. In early postnatal animals, R2D5-positive radial processes were found to reach the pial surface and to cover the entire midsagittal seam of the brainstem. These processes embraced dendrites and somata of neurons in almost all of the midsagittal nuclei, including the raphe nuclei, suggesting that the radial glial cells may interact with the midsagittal groups of neurons. In addition, the palisade of R2D5-positive radial processes formed loose openings for crossing axonal bundles at the midline decussations of fiber tracts. In more mature brains, somata of R2D5-positive radial glial cells that had migrated ventrally were observed within the palisades, and in adult cats, most of the R2D5-positive radial processes were found to have retracted from the ventral parts of the midsagittal seam. The spatial arrangement of R2D5-positive processes suggests that they may have persistent functional roles as an interface between ventricular humoral signals and midsagittal groups of neurons in the postnatally developing brainstem and in the adult brainstem. The structure of the midline glial system suggests also that it plays a role in organizing the spatial arrangement of decussating axons during development.
Stark spectroscopy of the Rhodobacter sphaeroides reaction center heterodimer mutant.
Authors: Hammes S L , Mazzola L , Boxer S G , Gaul D F , Schenck C C
Published: May 2006

The effect of an electric field has been measured on the absorption spectrum (Stark effect) of the heterodimer mutant (M)H202L of Rhodobacter sphaeroides reaction centers, where the primary electron donor consists of one bacteriochlorophyll alpha and one bacteriopheophytin alpha. The electronic absorption spectrum of the heterodimer mutant from 820-950 nm is relatively featureless in a poly(vinyl alcohol) film, but it exhibits some structure in a glycerol/water glass at 77 K. A feature is seen in the Stark effect spectrum of the heterodimer at 77 K centered at 927 and 936 nm in poly(vinyl alcohol) and a glycerol/water glass, respectively. This feature has approximately the same shape and width as the Stark effect for the primary electron donor of the wild type, which consists of a pair of bacteriochlorophyll alpha molecules. The angle zeta A between the transition moment at the frequency of absorption and the difference dipole delta muA is 36 +/- 2 degrees in the wild type and 32 +/- 2 degrees for that feature in the heterodimer. A range of values for [delta muA] = (13-17)/f Debye units (where f is the local field correction) is obtained for the 936-nm feature in glycerol/water, depending on analysis method. This feature is interpreted as arising from a transition to the lower exciton state of the heterodimer, which is more strongly mixed with a low-lying charge transfer transition than in the wild type.
Mitotic role for the Cdc28 protein kinase of Saccharomyces cerevisiae.
Authors: Reed S I , Wittenberg C
Published: May 2006

The Cdc28 protein kinase functions in the G1 to S phase transition of the cell cycle of the budding yeast Saccharomyces cerevisiae. This is in contrast with observations of the homologous protein kinase from a variety of metazoans, where activity and function are associated with the G2 to M phase transition. We present evidence that the Cdc28 protein kinase is also required for mitosis and that this function is executed in the G2 interval of the cell cycle. We show, in addition, that the protein kinase is highly active during this phase of the cell cycle. The dual role of the Cdc28 protein kinase in the S. cerevisiae cell cycle thus parallels that demonstrated for the cdc2 protein kinase of the fission yeast Schizosaccharomyces pombe.
Amino acid sequence requirements for the association of apocytochrome c with mitochondria.
Authors: Sprinkle J R , Hakvoort T B , Koshy T I , Miller D D , Margoliash E
Published: May 2006

To examine the amino acid sequence requirements for the biphasic association of Drosophila melanogaster apocytochrome c with mouse liver mitochondria in vitro, recombinant constructs of the protein were prepared. Removal of the C-terminal sequence to residue 58 had little influence, but truncation to residue 50 decreased the association to low levels and removal to residue 36 was even more effective. However, a mutant missing the segment between residues 35 and 66 was fully functional, but, when the C-terminal segment from residue 36 was replaced with a noncytochrome c sequence, the high-affinity phase of the association was lost. A mutant in which residues 90, 91, 92, 96, and 100 were replaced by lysine, leucine, proline, proline, and proline, respectively, to prevent the possible formation of the C-terminal alpha-helix and another mutant in which the C-terminal segment from residue 90 to residue 120 was a noncytochrome c sequence had normal association. In contrast, replacing lysine-5, -7, and -8 by glutamine, glutamic acid, and asparagine, respectively, resulted in loss of the high-affinity phase. The same mutations in the apoprotein lacking the segment between residues 35 and 66 caused, in addition, a decrease of the low-affinity phase association. Thus, the N-terminal region is most critical for apocytochrome c association, but alternative segments of the central and/or C-terminal region can be utilized, where noncytochrome c sequences are ineffective. These results emphasize the wide disparity between the structural requirements for association with mitochondria and for the production of a functional holoprotein.
Endogenous biosynthesis of arachidonic acid epoxides in humans: increased formation in pregnancy-induced hypertension.
Authors: Catella F , Lawson J A , Fitzgerald D J , FitzGerald G A
Published: May 2006

Arachidonic acid is metabolized by means of P450 isoenzyme(s) to form epoxyeicosatrienoic acids (EETs) and their corresponding dihydroxy derivatives (DHETs). In the present study, we established the presence in human urine of 8,9-, 11,12-, and 14,15-EETs and their corresponding DHETs by developing quantitative assays and using negative ion, chemical ionization GC/MS and octadeuterated internal standards. Urinary excretion of 8,9- and 11,12-DHET increased in healthy pregnant women compared with nonpregnant female volunteers. By contrast, excretion of 11,12-DHET and 14,15-DHET, but not the 8,9-DHET regioisomer, increased even further in patients with pregnancy-induced hypertension. Intravenous administration of [3H]14,15-EET to three dogs markedly increased its DHET in plasma. The terminal half-life ranged from 7.9-12.3 min and the volume of distribution (3.5-5.3 liters) suggested limited distribution outside the plasma compartment. Negligible radioactivity was detected in urine; this fact infers that under physiological circumstances, urinary DHETs largely derive from the kidney. That P450 metabolites of arachidonic acid are formed in humans supports the hypothesis that these metabolites contribute to the physiological response to normal pregnancy and the pathophysiology of pregnancy-induced hypertension.
Inhibition of T-cell proliferation by a MYB antisense oligomer is accompanied by selective down-regulation of DNA polymerase alpha expression.
Authors: Venturelli D , Travali S , Calabretta B
Published: May 2006

We recently found that inhibition of MYB protein synthesis in human peripheral blood mononuclear cells (PBMC) exposed to human c-myb (designated MYB) antisense oligodeoxynucleotides prevents entry into S phase and cell proliferation. To determine the mechanism(s) by which down-regulation of human c-myb protein (MYB) synthesis interferes with DNA synthesis, we analyzed mRNA levels of DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), transcripts of two genes required for DNA synthesis, in normal and leukemic T lymphocytes exposed to MYB antisense oligodeoxynucleotides. Expression of DNA polymerase alpha was inhibited both in normal T lymphocytes progressing from G0 to S phase and in exponentially growing CCRF-CEM leukemic cells, whereas expression of PCNA was inhibited only in mitogen-stimulated PBMC and remained essentially unaffected in the leukemia T-cell line. The functional link between expression of MYB and DNA polymerase alpha mRNAs was further demonstrated by analyzing DNA polymerase alpha mRNA levels in a temperature-sensitive (ts) fibroblast cell line (TK-ts13; TK is thymidine kinase) constitutively expressing human MYB mRNA driven by the simian virus 40 (SV40) promoter. In the MYB-expressing TK-ts13 cells, DNA polymerase alpha mRNA levels were unaffected following shift to the nonpermissive temperature of 39.6 degrees C, whereas in the parental line, DNA polymerase alpha mRNA levels were readily down-regulated. These findings indicate that the expression of MYB is related to that of DNA polymerase alpha in cells expressing MYB at high levels and suggest that there is a functional link between c-myb and DNA polymerase alpha mRNA expression during cell cycle progression of normal T lymphocytes.
De novo methylation of the MyoD1 CpG island during the establishment of immortal cell lines.
Authors: Jones P A , Wolkowicz M J , Rideout W M , Gonzales F A , Marziasz C M , Coetzee G A , Tapscott S J
Published: May 2006

CpG dinucleotides are unevenly distributed in the vertebrate genome. Bulk DNA is depleted of CpGs and most of the cytosines in the dinucleotide in this fraction are methylated. On the other hand, CpG islands, which are often associated with genes, are unmethylated at testable sites in all normal tissues with the exception of genes on the inactive X chromosome. We used Hpa II/Msp I analysis and ligation-mediated polymerase chain reaction to examine the methylation of the MyoD1 CpG island in adult mouse tissues, early cultures of mouse embryo cells, and immortal fibroblastic cell lines. The island was almost devoid of methylation at CCGG sites in adult mouse tissues and in low-passage mouse embryo fibroblasts. In marked contrast, the island was methylated in 10T 1/2 cells and in six other immortal cell lines showing that methylation of this CpG island had occurred during escape from senescence. The island became even more methylated in chemically transformed derivatives of 10T 1/2 cells. Thus, CpG islands not methylated in normal tissues may become modified to an abnormally high degree during immortalization and transformation.
Acetylcholine receptor-alpha-bungarotoxin interactions: determination of the region-to-region contacts by peptide-peptide interactions and molecular modeling of the receptor cavity.
Authors: Ruan K H , Spurlino J , Quiocho F A , Atassi M Z
Published: May 2006

In previous studies from this laboratory, the binding regions of alpha-neurotoxins on human and Torpedo acetylcholine (AcCho) receptors (AcChoRs) and the binding regions for the receptor on the toxin were characterized with synthetic peptides of the respective molecules. In the present work, peptides representing the active regions of one molecule are each allowed to bind to each of the active-region peptides of the other molecule. Thus, the interaction of three alpha-bungarotoxin (alpha-BTX) synthetic loop peptides with four synthetic peptides representing the toxin-binding regions on human AcChoR permitted the determination of the region-region interactions between alpha-BTX and the human receptor. Based on the known three-dimensional structure of the toxin, the active peptides of the receptor were then assembled to their appropriate toxin-contact regions by computer model building and energy minimization. This allowed the three-dimensional construction of the toxin-binding cavity on human AcChoR. The cavity appears to be conical, 30.5 A in depth, involving several receptor regions that make contact with the alpha-BTX loop regions. One AcChoR region (within residues 125-136) involved in the binding to alpha-BTX also resides in a known AcCho-binding site, thus demonstrating in three dimensions a critical site involved in both AcCho activation and alpha-BTX blocking. The validity of this approach was first established for three of four peptides corresponding to regions on the beta chain of human hemoglobin involved in binding to the alpha chain. Thus, studying the interaction between peptides representing the binding regions of two protein molecules may provide an approach in molecular recognition by which the binding site on one protein can be described if the three-dimensional structure of the other protein is known.
Expression of melatonin receptors in arteries involved in thermoregulation.
Authors: Viswanathan M , Laitinen J T , Saavedra J M
Published: May 2006

Melatonin binding sites were localized and characterized in the vasculature of the rat by using the melatonin analogue 2-[125I]iodomelatonin (125I-melatonin) and quantitative in vitro autoradiography. The expression of these sites was restricted to the caudal artery and to the arteries that form the circle of Willis at the base of the brain. The arterial 125I-melatonin binding was stable, saturable, and reversible. Saturation studies revealed that the binding represented a single class of high-affinity binding sites with a dissociation constant (Kd) of 3.4 x 10(-11) M in the anterior cerebral artery and 1.05 x 10(-10) M in the caudal artery. The binding capacities (Bmax) in these arteries were 19 and 15 fmol/mg of protein, respectively. The relative order of potency of indoles for inhibition of 125I-melatonin binding at these sites was typical of a melatonin receptor: 2-iodomelatonin greater than melatonin greater than N-acetylserotonin much much greater than 5-hydroxytryptamine. Norepinephrine-induced contraction of the caudal artery in vitro was significantly prolonged and potentiated by melatonin in a concentration-dependent manner, suggesting that these arterial binding sites are functional melatonin receptors. Neither primary steps in smooth muscle contraction (inositol phospholipid hydrolysis) nor relaxation (adenylate cyclase activation) were affected by melatonin. Melatonin, through its action on the tone of these arteries, may cause circulatory adjustments in these arteries, which are believed to be involved in thermoregulation.
Probe mapping to facilitate transposon-based DNA sequencing.
Authors: Strausbaugh L D , Bourke M T , Sommer M T , Coon M E , Berg C M
Published: May 2006

A promising strategy for DNA sequencing exploits transposons to provide mobile sites for the binding of sequencing primers. For such a strategy to be maximally efficient, the location and orientation of the transposon must be readily determined and the insertion sites should be randomly distributed. We demonstrate an efficient probe-based method for the localization and orientation of transposon-borne primer sites, which is adaptable to large-scale sequencing strategies. This approach requires no prior restriction enzyme mapping or knowledge of the cloned sequence and eliminates the inefficiency inherent in totally random sequencing methods. To test the efficiency of probe mapping, 49 insertions of the transposon gamma delta (Tn1000) in a cloned fragment of Drosophila melanogaster DNA were mapped and oriented. In addition, oligonucleotide primers specific for unique subterminal gamma delta segments were used to prime dideoxynucleotide double-stranded sequencing. These data provided an opportunity to rigorously examine gamma delta insertion sites. The insertions were quite randomly distributed, even though the target DNA fragment had both A + T-rich and G + C-rich regions; in G + C-rich DNA, the insertions were found in A + T-rich "valleys." These data demonstrate that gamma delta is an excellent choice for supplying mobile primer binding sites to cloned DNA and that transposon-based probe mapping permits the sequences of large cloned segments to be determined without any subcloning.
Mechanism for cryptic splice site activation during pre-mRNA splicing.
Authors: Nelson K K , Green M R
Published: May 2006

The 5' splice site of a pre-mRNA is recognized by U1 small nuclear ribonucleoprotein particles (snRNP) through base pairing with the 5' end of U1 small nuclear RNA (snRNA). Single-base substitutions within a 9-nucleotide 5'-splice-site sequence can abolish or attenuate use of that site and, in higher eukaryotes, can also activate nearby "cryptic" 5' splice sites. Here we show that the effects of single-base substitutions within a 5' splice site can be completely or partially suppressed by cis mutations that improve the overall complementarity of the site to U1 snRNA. We further show that in the presence of the normal 5' splice site, a cryptic 5' splice site can be activated by increasing its complementarity to U1 snRNA. U1 snRNP binding experiments confirm that cryptic 5' splice sites are activated when their affinity for U1 snRNP approaches that of the authentic 5' splice site. Based upon these results, we propose a spliceosome competition model for 5'-splice-site selection and cryptic 5'-splice-site activation. We discuss our results with regard to the factors involved in 5'-splice-site recognition.
In vitro incorporation of selenium into tRNAs of Salmonella typhimurium.
Authors: Veres Z , Tsai L , Politino M , Stadtman T C
Published: May 2006

Broken-cell preparations of Salmonella typhimurium rapidly incorporated 75Se from 75SeO3(2-) into tRNA by an ATP-dependent process. Selenium incorporation in the presence of 50 microM 75SeO3(2-) (0.8-1 pmol per A260 unit) was enhanced by the selenocysteine precursor, O-acetyl-L-serine (to 3.7 pmol per A260 unit). This increase in incorporation was a function of O-acetyl-L-serine concentration. Neither O-acetyl-L-homoserine nor O-phospho-L-serine stimulated the incorporation of selenium into tRNA. The incorporation of 75Se from 75SeO3(2-) was decreased by adding L-selenocysteine but not by adding the D isomer. When homologous bulk tRNA was added to the broken-cell preparations, an increased rate of 75Se labeling was observed. The supernatant fraction of the broken-cell preparation contained all of the enzymes required for this process. Reversed-phase HPLC analysis of labeled bulk tRNA digested to nucleosides showed the presence of a labeled compound that coeluted with authentic 5-methylaminomethyl-2-selenouridine.
Octyl glucoside extracts GTP-binding regulatory proteins from rat brain "synaptoneurosomes" as large, polydisperse structures devoid of beta gamma complexes and sensitive to disaggregation by guanine nucleotides.
Authors: Nakamura S , Rodbell M
Published: May 2006

GTP-binding regulatory proteins are generally purified from cholate-extracted membranes in the form of heterotrimers (G proteins) consisting of a GTP-binding subunit (alpha protein) complexed with a tightly interacted heterodimer termed beta gamma. In this study we extracted the proteins from rat brain "synaptoneurosomes" using the neutral detergent 1-octyl beta-D-glucopyranoside (octyl glucoside). Using specific antibodies for detection by immunoblotting and sucrose gradients for analyzing hydrodynamic properties, we found that each species of alpha protein (alpha subunits of stimulatory, inhibitory, and brain GTP-binding proteins) exhibited a broad range (4 S to greater than 12 S) of polydisperse structures with peak values (5 S to 7 S) considerably greater than that of heterotrimeric G proteins. The beta subunit proteins, for example, appeared as a homogeneous peak at 4.4 S within which only a fraction of the total alpha proteins can be associated. Incubation of octyl glucose extracts at 30 degrees C rapidly sedimented the alpha proteins but not the beta proteins. Incubation at 30 degrees C with guanosine 5'[gamma-thio]triphosphate (10-100 microM) prevented rapid sedimentation. Hydrodynamic analysis revealed that all alpha proteins were converted to approximately 4 S structures by the actions of guanosine 5'-[gamma-thio]triphosphate without change in the hydrodynamic properties of the beta proteins. Extraction of the membranes with sodium cholate instead of octyl glucoside resulted in complete loss of the large, polydisperse structures of the alpha proteins; the S values were approximately 4 S, in the range for beta proteins. These findings suggest that the transducing GTP-binding proteins in synaptoneurosomes exist as polydisperse, possibly multimer, structures of various size that are stable in octyl glucoside but destroyed by cholate. The polydisperse structures are not associated with beta gamma complexes and are sensitive to the disaggregating effects of guanosine 5'-[gamma-thio]triphosphate.
Reduced expression of the liver/beta-cell glucose transporter isoform in glucose-insensitive pancreatic beta cells of diabetic rats.
Authors: Thorens B , Weir G C , Leahy J L , Lodish H F , Bonner-Weir S
Published: May 2006

Rats injected with a single dose of streptozocin at 2 days of age develop non-insulin-dependent diabetes 6 weeks later. The pancreatic beta islet cells of these diabetic rats display a loss of glucose-induced insulin secretion while maintaining sensitivity to other secretagogues such as arginine. We analyzed the level of expression of the liver/beta-cell glucose transporter isoform in diabetic islets by immunofluorescence staining of pancreas sections and by Western blotting of islet lysates. Islets from diabetic animals have a reduced expression of this beta-cell-specific glucose transporter isoform and the extent of reduction is correlated with the severity of hyperglycemia. In contrast, expression of this transporter isoform in liver is minimally modified by the diabetes. Thus a decreased expression of the liver/beta-cell glucose transporter isoform in beta cells is associated with the impaired glucose sensing characteristic of diabetic islets; our data suggest that this glucose transporter may be part of the beta-cell glucose sensor.
Prevention of spontaneous and radiation-induced tumors in rats by reduction of food intake.
Authors: Gross L , Dreyfuss Y
Published: May 2006

In our previous studies carried out on inbred Sprague-Dawley rats, we reported a striking increase in the incidence of tumors following total-body gamma-irradiation [150 rads (1.5 Gy) five times at weekly intervals]. Subsequently, we observed that two or three irradiations, and to a lesser extent even a single irradiation, were sufficient to induce an impressive increase in the incidence of tumors, particularly in females. A significant reduction of the incidence of radiation-induced tumors resulted when the rats were placed on calorically restricted diet. In experiments reported here, we increased slightly the amount of food given to animals on restricted diet. In the new study, among 102 irradiated females on full diet, 91 (89%) developed tumors, as compared with 29 out of 128 female rats (23%) also irradiated but maintained on restricted diet and 43 out of 89 (48%) untreated control females. None of 77 nonirradiated females on restricted diet developed tumors. Among 65 irradiated male rats, 29 (45%) developed tumors, as compared with 5 out of 74 (7%) rats also irradiated but maintained on restricted diet. Of the 49 males in the nonirradiated groups, 2 (4%) developed tumors. There was a significant weight reduction in both females and males maintained on restricted diet; animals on restricted diet lived longer than those on full diet.
Structural design and molecular evolution of a cytokine receptor superfamily.
Authors: Bazan J F
Published: May 2006

A family of cytokine receptors comprising molecules specific for a diverse group of hematopoietic factors and growth hormones has been principally defined by a striking homology of binding domains. This work proposes that the approximately 200-residue binding segment of the canonical cytokine receptor is composed of two discrete folding domains that share a significant sequence and structural resemblance. Analogous motifs are found in tandem approximately 100-amino acid domains in the extracellular segments of a receptor family formed by the interferon-alpha/beta and -gamma receptors and tissue factor, a membrane tether for a coagulation protease. Domains from the receptor supergroup reveal clear evolutionary links to fibronectin type III structures, approximately 90-amino acid modules that are typically found in cell surface molecules with adhesive functions. Predictive structural analysis of the shared receptor and fibronectin domains locates seven beta-strands in conserved regions of the chain; these strands are modeled to fold into antiparallel beta-sandwiches with a topology that is similar to immunoglobulin constant domains. These findings have strong implications for understanding the evolutionary emergence of an important class of regulatory molecules from primitive adhesive modules. In addition, the resulting double-barrel design of the receptors and the spatial clustering of conserved residues suggest a likely binding site for cytokine ligands.
Expression of tissue factor procoagulant activity: regulation by cytosolic calcium.
Authors: Bach R , Rifkin D B
Published: May 2006

Intact bovine fibroblasts, pericytes, and kidney cells manifested significantly less tissue factor procoagulant activity than their disrupted counterparts. Addition of calcium ionophore A23187 rapidly and reversibly enhanced the cell-surface expression of tissue factor in intact cells up to the level achieved by disruption. Inhibitors of calmodulin blocked the ionophore-dependent enhancement of procoagulant activity. Similar kinetic parameters were obtained for factor X hydrolysis by tissue factor-factor VIIa on unperturbed pericytes and phosphatidylcholine vesicles. Increase in Vmax and decrease in apparent Km for this reaction were seen after either disruption or ionophore stimulation of the pericytes. Addition of phosphatidylserine to the reconstituted phospholipid vesicles also increased the Vmax and decreased the apparent Km for factor X hydrolysis. These data agree with the hypothesis that the expression of tissue factor procoagulant activity on cell surfaces is modulated by calcium-mediated changes in the asymmetric distribution of phosphatidylserine in plasma membrane.
Structure-function studies on Escherichia coli MetR protein, a putative prokaryotic leucine zipper protein.
Authors: Maxon M E , Wigboldus J , Brot N , Weissbach H
Published: May 2006

The Escherichia coli metR gene has been sequenced. The sequence predicts a protein of 317 amino acids and a calculated molecular weight of 35,628. This is about 15% larger than the protein from Salmonella typhimurium reported previously [Plamann, L.S. & Stauffer, G.V. (1987) J. Bacteriol. 169, 3932-3937]. The protein is a homodimer and contains a leucine zipper motif characteristic of many eukaryotic DNA-binding proteins. Replacement of two of the leucines in the leucine zipper region of the MetR protein, or substitution of proline for one of the leucines, results in loss of biological activity of the protein. In addition, truncation studies have identified a region on MetR that may be involved in the homocysteine activation of metE expression.
Structure of a protein superfiber: spider dragline silk.
Authors: Xu M , Lewis R V
Published: May 2006

Spider major ampullate (dragline) silk is an extracellular fibrous protein with unique characteristics of strength and elasticity. The silk fiber has been proposed to consist of pseudocrystalline regions of antiparallel beta-sheet interspersed with elastic amorphous segments. The repetitive sequence of a fibroin protein from major ampullate silk of the spider Nephila clavipes was determined from a partial cDNA clone. The repeating unit is a maximum of 34 amino acids long and is not rigidly conserved. The repeat unit is composed of three different segments: (i) a 6 amino acid segment that is conserved in sequence but has deletions of 3 or 6 amino acids in many of the repeats; (ii) a 13 amino acid segment dominated by a polyalanine sequence of 5-7 residues; (iii) a 15 amino acid, highly conserved segment. The latter is predominantly a Gly-Gly-Xaa repeat with Xaa being alanine, tyrosine, leucine, or glutamine. The codon usage for this DNA is highly selective, avoiding the use of cytosine or guanine in the third position. A model for the physical properties of fiber formation, strength, and elasticity, based on this repetitive protein sequence, is presented.
The invariant chain forms complexes with class II major histocompatibility complex molecules and antigenic peptides "in vivo".
Authors: Viguier M , Dornmair K , Clark B R , McConnell H M
Published: May 2006

The binding of a chicken ovalbumin peptide (residues 323-339), Ova-(323-339), to I-Ad molecules was investigated in vitro and in vivo. By using antigenic peptides labeled either with a hapten or with fluorescein, complexes formed in vitro between I-Ad and antigenic peptides were detected by Western blot analysis with an antibody recognizing the hapten 7-nitrobenzo-2-oxa-1,3-diazole and by scanning gels for fluorescence emitted by fluoresceinated peptide. Both techniques reveal that Ova-(323-339) binds not only to I-Ad alpha/beta heterodimers and separated alpha and beta chains but also to complexes of higher molecular mass. Additional analysis shows that one of these additional complexes contains I-Ad heterodimers, antigenic peptides, and also invariant chain. To explore the physiological role of these complexes, cells were incubated with haptenated peptide and the I-Ad-peptide complexes formed in vivo were purified by affinity chromatography using hapten-specific antibody. The complexes formed migrate with a significantly higher apparent molecular mass than the alpha/beta heterodimers. A band at 180 kDa contained the alpha/beta heterodimer, the antigenic peptide, and the invariant chain. These results show that in vivo high molecular mass complexes formed by the I-Ad heterodimer and the invariant chain bind antigenic peptides.
Global processing of visual stimuli in a neural network of coupled oscillators.
Authors: Sompolinsky H , Golomb D , Kleinfeld D
Published: May 2006

An oscillator neural network model is presented that is capable of processing local and global attributes of sensory input. Local features in the input are encoded in the average firing rate of the neurons while the relationships between these features can modulate the temporal structure of the neuronal output. Neurons that share the same receptive field interact via relatively strong feedback connections, while neurons with different fields interact via specific, relatively weak connections. This pattern of connectivity mimics that of primary visual cortex. The model is studied in the context of processing visual stimuli that are coded for orientation. We compare our theoretical results with recent experimental evidence on coherent oscillatory activity in the cat visual cortex. The computational capabilities of the model for performing discrimination and segmentation tasks are demonstrated.