bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinesin swivels to permit microtubule movement in any direction.
Authors: Hunt A J , Howard J
Published: May 2006

Kinesin is a motor protein that uses the energy derived from ATP hydrolysis to transport organelles along microtubules. By analyzing the thermal fluctuation of microtubules tethered to glass surfaces by single molecules of kinesin, we have measured the torsional flexibility of the motor protein. The torsional stiffness of kinesin, (117 +/- 19) x 10(-24) N.m.rad-1 (mean +/- SEM), is so low that one kT of energy (approximately 4.1 x 10(-21) J at room temperature) is sufficient to twist a kinesin molecule through more than 360 degrees from its resting orientation. Consistent with this flexibility, motility assays show that one or more kinesin molecules can move a microtubule equally well in any direction. These results explain how a motor on the surface of an organelle can rapidly bind to and capture a microtubule irrespective of the organelle's orientation. Furthermore, the flexibility ensures that several motors can efficiently work together even though they are randomly oriented on the surface of an organelle rather than being in precise arrays like the motors of muscle and cilia.
Functional kainate-selective glutamate receptors in cultured hippocampal neurons.
Authors: Lerma J , Paternain A V , Naranjo J R , Mellström B
Published: May 2006

Glutamate mediates fast synaptic transmission at the majority of excitatory synapses throughout the central nervous system by interacting with different types of receptor channels. Cloning of glutamate receptors has provided evidence for the existence of several structurally related subunit families, each composed of several members. It has been proposed that KA1 and KA2 and GluR-5, GluR-6, and GluR-7 families represent subunit classes of high-affinity kainate receptors and that in vivo different kainate receptor subtypes might be constructed from these subunits in heteromeric assembly. However, despite some indications from autoradiographic studies and binding data in brain membranes, no functional pure kainate receptors have so far been detected in brain cells. We have found that early after culturing, a high percentage of rat hippocampal neurons express functional, kainate-selective glutamate receptors. These kainate receptors show pronounced desensitization with fast onset and very slow recovery and are also activated by quisqualate and domoate, but not by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate. Our results provide evidence for the existence of functional glutamate receptors of the kainate type in nerve cells, which are likely to be native homomeric GluR-6 receptors.
Nck associates with the SH2 domain-docking protein IRS-1 in insulin-stimulated cells.
Authors: Lee C H , Li W , Nishimura R , Zhou M , Batzer A G , Myers M G , White M F , Schlessinger J , Skolnik E Y
Published: May 2006

Nck, an oncogenic protein composed of one SH2 and three SH3 domains, is a common target for various cell surface receptors. Nck is thought to function as an adaptor protein to couple cell surface receptors to downstream effector molecules that regulate cellular responses induced by receptor activation. In this report, we show that Nck forms a stable complex in vivo with IRS-1 in insulin-stimulated cells. The interaction between IRS-1 and Nck is mediated by the binding of the SH2 domain of Nck to tyrosine-phosphorylated IRS-1. Although Nck associates with IRS-1, Nck phosphorylation is not affected by insulin stimulation. Furthermore, in vitro and in vivo studies show that the SH2 domains of Nck, GRB2, and p85 bind distinct phosphotyrosine residues in IRS-1. After insulin stimulation all three signaling molecules can be found complexed to a single IRS-1 molecule. These findings provide further evidence that, in response to insulin stimulation, IRS-1 acts as an SH2 docking protein that coordinates the regulation of various different signaling pathways activated by the insulin receptor.
Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase.
Authors: Brown K A , Howell E E , Kraut J
Published: May 2006

X-ray crystal structures have been determined for a second-site revertant (Asp-27-->Ser, Phe-137-->Ser; D27S/F137S) and both component single-site mutants of Escherichia coli dihydrofolate reductase. The primary D27S mutation, located in the substrate binding pocket, greatly reduces catalytic activity as compared to the wild-type enzyme. The additional F137S mutation, which partially restores catalytic activity, is located on the surface of the molecule, well outside of the catalytic center and approximately 15 A from residue 27. Comparison of kinetic data for the single-site F137S mutant, specifically constructed as a control, and for the double-mutant enzymes indicates that the effects of the F137S and D27S mutations on catalysis are nonadditive. This result suggests that the second-site mutation might mediate its effects through a structural perturbation propagated along the polypeptide backbone. To investigate the mechanism by which the F137S substitution elevates the catalytic activity of D27S we have determined the structure of the D27S/F137S double mutant. We also present a rerefined structure for the original D27S mutant and a preliminary structural interpretation for the F137S single-site mutant. We find that while either single mutant shows little more than a simple side-chain substitution, the double mutant undergoes an extended structural perturbation, which is propagated between these two widely separated sites via the helix alpha B.
Unresponsiveness of primitive chronic myeloid leukemia cells to macrophage inflammatory protein 1 alpha, an inhibitor of primitive normal hematopoietic cells.
Authors: Eaves C J , Cashman J D , Wolpe S D , Eaves A C
Published: May 2006

Most primitive hematopoietic cells appear to be normally quiescent in vivo, whereas their leukemic counterparts in patients with chronic myeloid leukemia (CML) are maintained in a state of rapid turnover. This difference is also seen in the long-term culture system, where control of primitive hematopoietic progenitor proliferation is mediated by interactions of these cells with marrow-derived mesenchymal cells of the fibroblast lineage. We now show that exogenous addition of macrophage inflammatory protein 1 alpha (MIP-1 alpha) to normal long-term cultures can reversibly and specifically block the activation of "primitive" (high proliferative potential), but not "mature" (lower proliferative potential), progenitors in the adherent layer of these cultures. Moreover, addition of MIP-1 beta after primitive-progenitor activation can prevent the subsequent return of these cells to a quiescent state a few days later as shown previously in similar experiments using antibodies to transforming growth factor beta. This suggests that the level of MIP-1 alpha (or a related MIP-1 alpha agonist) produced in LTCs, like the level of transforming growth factor beta, may be necessary, but is not on its own sufficient, to mediate the inhibitory activity of the regulatory cells in the adherent layer. Addition of MIP-1 alpha to similar long-term cultures containing normal marrow adherent layers but supporting exclusively neoplastic (CML) hematopoiesis did not block the cycling of primitive neoplastic progenitors. A defect in the responsiveness of CML cells to MIP-1 alpha (or a similarly acting chemokine) would explain their deregulated proliferative behavior in this model and, by extrapolation to the in vivo setting, suggests a molecular mechanism whereby the leukemic clone may become amplified at the stem-cell level. In addition, these findings suggest approaches to the therapy of CML, using inhibitors such as MIP-1 alpha for the protection of primitive normal cells.
In vivo protection against endotoxin by plasma high density lipoprotein.
Authors: Levine D M , Parker T S , Donnelly T M , Walsh A , Rubin A L
Published: May 2006

Overwhelming bacterial infection is accompanied by fever, hypotension, disseminated intravascular coagulation, and multiple organ failure leading to death in 30-80% of cases. These classical symptoms of septic shock are caused by potent cytokines that are produced in response to endotoxin released from Gram-negative bacteria. Treatments with antibodies and receptor antagonists to block endotoxin or cytokine mediators have given mixed results in clinical trials. High density lipoprotein (HDL) is a natural component of plasma that is known to neutralize endotoxin in vitro. We report here that raising the plasma HDL concentration protects mice against endotoxin in vivo. Transgenic mice with 2-fold-elevated plasma HDL levels had more endotoxin bound to HDL, lower plasma cytokine levels, and improved survival rates compared with low-HDL mice. Intravenous infusion of HDL also protected mice, but only when given as reconstituted HDL prepared from phospholipid and either HDL apoprotein or an 18-amino acid peptide synthesized to mimic the structure of apolipoprotein A-I of HDL. Intact plasma HDL was mildly toxic, and HDL apoprotein was ineffective. The effectiveness of the reconstituted peptide renders very unlikely any significant contribution to protection by trace proteins in apo-HDL. These data suggest a simple leaflet insertion model for binding and neutralization of lipopolysaccharide by phospholipid on the surface of HDL. Plasma HDL may normally act to protect against endotoxin; this protection may be augmented by administration of reconstituted HDL or reconstituted peptides.
The c-rel protooncogene product represses NF-kappa B p65-mediated transcriptional activation of the long terminal repeat of type 1 human immunodeficiency virus.
Authors: Doerre S , Sista P , Sun S C , Ballard D W , Greene W C
Published: May 2006

The long terminal repeat (LTR) of the type 1 human immunodeficiency virus (HIV-1) and the 5' regulatory region of the gene encoding the interleukin 2 receptor alpha subunit (IL-2R alpha) share functional kappa B enhancer elements involved in the regulation of these inducible transcription units during T-cell activation. These kappa B enhancer elements are recognized by a structurally related family of interactive proteins that includes p50, p65, and the product of the c-rel protooncogene (c-Rel). Recent biochemical studies have shown that p65 and p50 form the prototypical NF-kappa B complex, which is rapidly translocated from the cytoplasm to the nucleus during T-cell activation. This intracellular signaling complex potently stimulates kappa B-directed transcription from either the HIV-1 LTR or the IL-2R alpha promoter via the strong transactivation domain present in p65. We now demonstrate that nuclear expression of human c-Rel, which is induced by either phorbol ester or tumor necrosis factor alpha with delayed kinetics relative to p65, markedly represses p65-mediated activation of these transcription units. These inhibitory effects of c-Rel correlate with its DNA-binding activity but not with its ability to heterodimerize with p50, suggesting that c-Rel inhibition involves competition with p50/p65 for occupancy of the kappa B enhancer element. Together, these findings suggest that one function of c-Rel is as a physiologic repressor of the HIV-1 LTR and IL-2R alpha promoters, serving to efficiently counter the strong transcriptional activating effects of p65.
Use of photoactivatable peptide substrates of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) to characterize a myristoyl-CoA-Nmt1p-peptide ternary complex and to provide evidence for an ordered reaction mechanism.
Authors: Rudnick D A , Rocque W J , McWherter C A , Toth M V , Jackson-Machelski E , Gordon J I
Published: May 2006

Nmt1p (EC 2.3.1.97) catalyzes the transfer of myristate (C14:0) from coenzyme A to the N-terminal glycine residue of a variety of eukaryotic cellular and viral proteins. Our recent studies of the 455-amino acid Saccharomyces cerevisiae acyltransferase (Nmt1p) suggested that its mechanism of catalysis is ordered Bi Bi with myristoyl-CoA binding occurring prior to binding of peptide and release of CoA occurring prior to release of the myristoyl-peptide. The interaction between enzyme and peptide has now been examined in greater detail by using photoactivatable octapeptide substrates containing 125I-labeled azidosalicyclic acid attached via an amide bond to the gamma-amino group of a diaminobutyrate residue located at position 2 or the epsilon-amino group of a lysine residue located at position 8. The photopeptides can be specifically crosslinked to chymotryptic fragments of Nmt1p in the presence but not in the absence of a nonhydrolyzable myristoyl-CoA analog, S-(2-oxo)pentadecyl-CoA. Labeling of the chymotryptic fragments is markedly reduced when GLYASKLS, a high-affinity substrate derived from residues 2-9 of S. cerevisiae ADP-ribosylation factor 2, or ALYASKLS, a competitive inhibitor (for peptide), is added with the iodinated photopeptide. These findings suggest that peptide affinity for the acyl-CoA-Nmt1p binary complex is much greater than it is for apoNmt1p, consistent with the ordered Bi Bi mechanism ascribed to Nmt1p. Finally, automated sequential Edman degradation of these chymotryptic fragments suggests that the peptide binding domain of Nmt1p may be composed of elements from two protease-resistant domains, Arg42-Try219 and Thr220-Leu455.
Oncostatin M is a mitogen for rabbit vascular smooth muscle cells.
Authors: Grove R I , Eberhardt C , Abid S , Mazzucco C , Liu J , Kiener P , Todaro G , Shoyab M
Published: May 2006

The growth regulatory protein oncostatin M was initially discovered in macrophage-conditioned medium. We investigated the effects of oncostatin M on cultured rabbit aorta smooth muscle cells (SMCs) and found that the peptide stimulated an increase in the incorporation of [3H]thymidine into DNA. The magnitude of the stimulation was dependent on oncostatin M concentration and SMC confluency. In subconfluent cultures, 1-2 nM stimulated 4- to 5-fold increases in DNA synthesis after 20 hr. Other structurally related cytokines (granulocyte colony-stimulating factor, leukemia inhibitory factor, interleukin 6, ciliary neurotrophic factor) did not affect SMC DNA synthesis. After 5 or 8 days, oncostatin M caused a doubling in SMC number and also induced a transformed phenotype. The combination of oncostatin M and platelet-derived growth factor for 8 days resulted in a 4-fold increase in cell number, approximately the same increase in cell number as induced by the addition of 10% fetal calf serum. Further investigation suggested that the mitogenic effect of oncostatin M was in part due to tyrosine kinase activation. Within 1-2 min, the factor increased phosphotyrosine levels of several SMC proteins. In addition, detectable increases in diacylglycerol levels occurred within 2-5 min, reached 50% above control by 30 min, and remained elevated through 45 min of incubation with oncostatin M. SMC inositol phosphate levels were also elevated within 2 min and then returned to near control values by 20 min. Within 30 min, oncostatin M induced expression of the immediate-early gene EGR-1. These data indicate that oncostatin M may be an important, naturally occurring mitogen for vascular SMCs.
Identification of a 3'-->5' exonuclease activity associated with human RNA polymerase II.
Authors: Wang D , Hawley D K
Published: May 2006

Human RNA polymerase II is shown to be associated with a 3'-->5' exonuclease activity that removes nucleoside 5'-monophosphates from the 3' end of the transcripts in isolated ternary complexes. This activity is stimulated by SII, a protein that acts as a transcription elongation factor in vitro. In addition, we show that another transcription factor, TFIIF, stimulates a competing pyrophosphorolysis reaction. These findings raise interesting questions about the roles of these activities in vivo, including the possibility that this RNA polymerase may proofread the nascent transcript.
Ribosomal RNA antitermination in vitro: requirement for Nus factors and one or more unidentified cellular components.
Authors: Squires C L , Greenblatt J , Li J , Condon C , Squires C L
Published: May 2006

Using an in vitro transcription assay, we have successfully demonstrated read through of a Rho-dependent terminator by the ribosomal RNA antitermination system. The assay used a DNA template containing a promoter-antiterminator-terminator arrangement, RNA polymerase, termination factor Rho, antitermination factors NusA, NusB, NusE, and NusG, and a cellular extract depleted of NusB. Terminator read-through was highly efficient only in the presence of the extract and Nus factors, suggesting that an as yet uncharacterized cellular component is required for ribosomal antitermination. The NusB-depleted extract had no activity in the absence of NusB, confirming an absolute requirement for this protein in ribosomal RNA antitermination. The DNA template requirements were the same as those previously established in vivo; transcription of a wild-type boxA sequence is both necessary and sufficient to promote RNA polymerase modification into a terminator-resistant form.
Anti-immune complex antibodies enhance affinity and specificity of primary antibodies.
Authors: Ullman E F , Milburn G , Jelesko J , Radika K , Pirio M , Kempe T , Skold C
Published: May 2006

Antibodies have previously been described that enhance the binding of a second antibody to its antigen. The origin of this effect has been variously ascribed to binding to a neodeterminant on the Fc region, to a combined determinant representing portions of the second antibody and the immunogen, and to a ligand-induced conformation of the Fab fragment. This paper describes an antibody that recognizes an immune complex of an antibody to tetrahydrocannabinol (THC). The antibody binds the anti-THC antibody at an epitope recognized by an anti-idiotype antibody that is capable of blocking THC binding. The ability of various THC derivatives to enhance or inhibit binding taken together with equilibria and kinetic data support a model in which the anti-immune complex antibody interacts through adventitious binding to pendant groups on the THC derivatives. This type of interaction offers the opportunity to increase the sensitivity and specificity of immunoassays beyond the limits imposed by normal antibody binding. The implications of these findings with regard to earlier observations of anti-immune complex antibodies are discussed.
Stimulation of Mn peroxidase activity: a possible role for oxalate in lignin biodegradation.
Authors: Kuan I C , Tien M
Published: May 2006

Oxalate is produced by numerous wood-degrading fungi. Our studies here show that the white-rot fungus Phanerochaete chrysosporium produces extracellular oxalate under conditions that induce synthesis of the ligninolytic system. Little or no oxalate was detected in cultures grown under high nutrient nitrogen or carbon. This extracellular oxalate was identified and quantitated by HPLC. Its identity was further substantiated by its decomposition by the enzyme oxalate oxidase. The oxalate content of the extracellular fluid (peaking at 60 microM) paralleled the extracellular activity of the lignin-degrading enzyme, Mn peroxidase. Significantly, we demonstrated that oxalate, at physiological concentrations, substantially stimulated Mn peroxidase-catalyzed phenol red oxidation, presumably by its ability to chelate Mn. Stopped flow studies also indicate that oxalate accelerates the turnover of Mn peroxidase. Furthermore, we discovered that oxalate can support Mn peroxidase-catalyzed oxidations in the absence of exogenous H2O2 and in the presence of dioxygen. These results allow us to propose an important role for oxalate, a ubiquitous compound produced by wood-destroying fungi, in lignin biodegradation.
RuvA and RuvB proteins of Escherichia coli exhibit DNA helicase activity in vitro.
Authors: Tsaneva I R , Müller B , West S C
Published: May 2006

The SOS-inducible ruvA and ruvB gene products of Escherichia coli are required for normal levels of genetic recombination and DNA repair. In vitro, RuvA protein interacts specifically with Holliday junctions and, together with RuvB (an ATPase), promotes their movement along DNA. This process, known as branch migration, is important for the formation of heteroduplex DNA. In this paper, we show that the RuvA and RuvB proteins promote the unwinding of partially duplex DNA. Using single-stranded circular DNA substrates with annealed fragments (52-558 nucleotides in length), we show that RuvA and RuvB promote strand displacement with a 5'-->3' polarity. The reaction is ATP-dependent and its efficiency is inversely related to the length of the duplex DNA. These results show that the ruvA and ruvB genes encode a DNA helicase that specifically recognizes Holliday junctions and promotes branch migration.
Correlation between Src family member regulation by the protein-tyrosine-phosphatase CD45 and transmembrane signaling through the T-cell receptor.
Authors: Cahir McFarland E D , Hurley T R , Pingel J T , Sefton B M , Shaw A , Thomas M L
Published: May 2006

Stimulation of tyrosine phosphorylation is an early and important event in antigen-induced T-cell activation. T-cell clones deficient in expression of CD45, a transmembrane protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), are impaired in their ability to respond to either antigen or T-cell receptor cross-linking. Analysis of the CD45-deficient CD8+ T-cell clone L3M-93 demonstrates that the Src family members p56lck and p59fyn show increased immunoreactivity with anti-phosphotyrosine antibody and exhibit decreased kinase activity. The site of increased tyrosine phosphorylation in Src family members was identified by comparison of cyanogen bromide peptide maps. Phosphorylation of the C-terminal phosphopeptide, containing the negative regulatory site of tyrosine phosphorylation, from the CD45-deficient cells was increased 8-fold for p56lck and 2-fold for p59fyn. These data suggest that CD45 dephosphorylates the negative regulatory site of multiple Src family members in the cytotoxic T-lymphocyte clone L3 and show a correlation between the ability to respond efficiently to antigen and the dephosphorylation of Src family members by CD45.
Perivascular and intravenous administration of basic fibroblast growth factor: vascular and solid organ deposition.
Authors: Edelman E R , Nugent M A , Karnovsky M J
Published: May 2006

The in vivo mitogenicity of basic fibroblast growth factor (bFGF) for arterial smooth muscle cells relies on the removal of endothelium, raising the question of whether the endothelium serves as a mechanical barrier preventing contact of circulating bFGF with underlying smooth muscle cells or as a biochemical barrier that produces a local inhibitor of bFGF activity. To better define the role of the intact endothelium in modulating the vascular and tissue deposition of bFGF, we compared the fate of intravenous injections of 125I-labeled bFGF with perivascular controlled growth factor release. Peak serum bFGF levels were detected within 1 min of injection, and the growth factor was cleared thereafter with a serum half-life of almost 3 min. Polymeric controlled release devices delivered bFGF to the extravascular space without transendothelial transport. Deposition within the blood vessel wall was rapidly distributed circumferentially and was substantially greater than that observed following intravenous injection. The amount of bFGF deposited in arteries adjacent to the release devices was 40 times that deposited in similar arteries in animals who received a single intravenous bolus of bFGF. Endothelial denudation had a minimal effect on deposition following perivascular release, and it increased deposition following intravenous delivery 2-fold. The presence of intimal hyperplasia increased deposition of perivascularly released bFGF 2.4-fold but decreased the deposition of intravenously injected bFGF by 67%. In contrast, bFGF was 5- to 30-fold more abundant in solid organs after intravenous injection than it was following perivascular release. Deposition was greatest in the kidney, liver, and spleen and was substantially lower in the heart and lung. Thus, bFGF is rapidly cleared following intravenous injection and is deposited within both solid organs and the walls of blood vessels. Unlike the mitogenic potential of bFGF within blood vessels, vascular deposition is virtually independent of the presence of endothelium. Perivascular delivery is far more efficient than intravenous delivery at depositing bFGF within the arterial wall, and an increased neointima may provide added substrate for potential bFGF deposition but has limited contact with intravascular growth factor as a result of dilutional and flow-mediated effects.
Calphotin: a Drosophila photoreceptor cell calcium-binding protein.
Authors: Martin J H , Benzer S , Rudnicka M , Miller C A
Published: May 2006

Monoclonal antibody 23E9 identifies a calcium-binding protein, calphotin, in photoreceptor cells of the Drosophila melanogaster compound eyes and ocelli. The antigen is restricted to a defined cytoplasmic region; it is not present in the rhabdomeres, nuclei, mitochondria, or rough endoplasmic reticulum. A corresponding cDNA recognizes a 3-kb mRNA with retinal specificity similar to the antigen and maps to band 86E/F-87A/B on chromosome 3. An open reading frame of 2595 bp encodes an estimated 85-kDa protein of unusual amino acid composition, with > 50% proline, alanine, and valine and very few basic residues. The C-terminal segment contains a leucine zipper motif uninterrupted by prolines. We found no significant similarities with the GenBank or National Biomedical Resource Foundation data bases. The location of the protein within a distinct cytoplasmic region suggests that it might function as a calcium-sequestering "sponge" to regulate the amount of free cytoplasmic calcium.
Regulation of peptide-calmodulin complexes by protein kinase C in vivo.
Authors: Hinrichsen R D , Blackshear P J
Published: May 2006

We used the freshwater protozoan Paramecium tetraurelia to investigate the potential regulation by protein kinase C of calmodulin interactions with binding peptides in intact cells. In these organisms, an action potential results in membrane depolarization and a period of backward swimming; repolarization and a return to forward swimming requires the presence of normal calmodulin. We postulated that injection of high-affinity calmodulin binding peptides might interfere with repolarization and thus prolong the period of membrane depolarization. Synthetic peptides spanning the protein kinase C phosphorylation site/calmodulin-binding domains of the myristoylated alanine-rich C-kinase substrate (MARCKS) and the MARCKS-related protein (also known as F52 or MacMARCKS) were injected into cells; these caused a 2- to 3-fold increase in the duration of backward swimming. Similar changes were seen with two other calmodulin-binding peptides. This behavioral response could be prevented by coinjecting calmodulin. Activation of Paramecium protein kinase C with an active phorbol ester completely reversed (within 3 min) the behavioral effects of the normal MARCKS and MARCKS-related protein peptides. Injection of a nonphosphorylatable peptide, in which alanines were substituted for serines, resulted in the usual behavioral response; however, this was not reversed by phorbol ester treatment. The corresponding aspartate-substituted peptide, which has a 10-fold lower affinity for calmodulin, did not prolong backward swimming. These data suggest that these peptides can form complexes with calmodulin at the calcium concentrations that prevail in intact Paramecium cells and that such complexes can be disrupted by protein kinase C phosphorylation of the peptides.
Gamma-tocopherol detoxification of nitrogen dioxide: superiority to alpha-tocopherol.
Authors: Cooney R V , Franke A A , Harwood P J , Hatch-Pigott V , Custer L J , Mordan L J
Published: May 2006

In the vitamin E group, alpha-tocopherol is generally considered to be the most potent antioxidant with the highest vitamin bioactivity, yet gamma-tocopherol is produced in greater amounts by many plants and is the principal tocopherol in the United States diet. This report describes a fundamental difference in the chemical reactivities of alpha-tocopherol and gamma-tocopherol with nitrogen dioxide (NO2), which leads to the formation of a nitrosating agent from alpha-tocopherol, but not from gamma-tocopherol. Nitric oxide (NO) is a major product of the reaction of gamma-tocopherol with NO2, while alpha-tocopherol reacts with NO2 to form an intermediate tocopheroxide analogue. The biological significance of gamma-tocopherol is suggested by limited epidemiological data as well as the observation that it is a more potent inhibitor than alpha-tocopherol of neoplastic transformation during the postinitiation phase in 3-methylcholanthrene-treated C3H/10T1/2 murine fibroblasts. This latter property suggests the superiority of gamma-tocopherol in a mammalian biological assay and a role for endogenous NO production in promotion of neoplastic transformation.
Neural expression and chromosomal mapping of Neu differentiation factor to 8p12-p21.
Authors: Orr-Urtreger A , Trakhtenbrot L , Ben-Levy R , Wen D , Rechavi G , Lonai P , Yarden Y
Published: May 2006

Neu differentiation factor (NDF/heregulin) is a 44-kDa glycoprotein that interacts with the Neu/ErbB-2 receptor tyrosine kinase to increase its phosphorylation on tyrosine residues. In vitro NDF promotes differentiation of certain mammary tumor cell lines to milk-producing cells. As a first step toward understanding the physiological role of NDF, we performed in situ hybridization analyses to determine mRNA distribution in the mouse embryo and to map the gene to human karyotypes. In 14.5-day-postcoitum mouse embryos, NDF expression is confined predominantly to the central and peripheral nervous system, including the neuroepithelium that lines the lateral ventricles of the brain, the ventral horn of the spinal cord, and the intestinal as well as dorsal root ganglia. Other tissues that contain NDF transcripts are the adrenal gland, liver, and distinct cell layers of the dermis and germinal ridge. In situ hybridization of a 3H-labeled probe to human metaphase spreads localized the NDF gene to the short arm of chromosome 8 at bands p12-p21.
Genetic analysis of diabetes and insulitis in an interspecific cross of the nonobese diabetic mouse with Mus spretus.
Authors: de Gouyon B , Melanitou E , Richard M F , Requarth M , Hahn I H , Guenet J L , Demenais F , Julier C , Lathrop G M , Boitard C
Published: May 2006

The nonobese diabetic (NOD) mouse is a widely used model for genetic studies of insulin-dependent diabetes mellitus due to the similarities between the murine and human diseases. To aid in the localization and identification of diabetes-related susceptibility genes, we have constructed an interspecific backcross between NOD and Mus spretus (SEG/Pas) mice. Although no diabetic animals were observed in the first backcross generation of (SEG/Pas x NOD) x NOD (BC1), the incidence of insulitis (lymphocyte infiltration of the islets of Langerhans) exceeded 20% after injections of cyclophosphamide, a treatment that provokes an acute form of diabetes in NOD mice. Insulitis, a prediabetic condition, is a useful phenotype in studies of diabetes susceptibility. In the second backcross (BC2) generation, 8% of the animals became diabetic and 76% were found to have insulitis. Genetic mapping studies in the BC2 families confirmed the importance of the major histocompatibility complex region on the severity of insulitis and suggested that additional susceptibility loci were linked to markers on mouse chromosomes 3, 6, and 15. Mus spretus crosses have been an important tool in recent advances in murine genetics, and our results extend their usefulness to the study of a multifactorial disease.
Cloning and expression of the gene from Candida albicans that encodes a high-affinity corticosteroid-binding protein.
Authors: Malloy P J , Zhao X , Madani N D , Feldman D
Published: May 2006

We have previously demonstrated the presence of a corticosteroid-binding protein (CBP) in Candida albicans and speculated on its homology to the glucocorticoid receptor. To explore this relationship further, we cloned the CBP gene. Our strategy employed sequencing enzymatically derived peptide fragments from purified CBP and using this information to synthesize degenerate oligonucleotide primers for use in the PCR. A 117-bp fragment amplified from C. albicans DNA was then used to screen a genomic library. Hybridizing clones were isolated, and DNA sequencing revealed an open reading frame of 1467 bp which encoded a protein with a molecular weight of 55,545. Southern analysis demonstrated that the gene was present at a unique locus within chromosome R of the Candida genome, while Northern analysis showed that the gene was expressed in C. albicans as a 1.8-kb transcript. CBP was over-expressed in Saccharomyces cerevisiae, and it exhibited an apparent dissociation constant (Kd) of 7 nM for [3H]corticosterone and displayed a steroid hormone binding profile comparable to that of the native protein. Searches of the data banks revealed little overall similarity to other cloned genes. However, the amino acid sequence contains a dinucleotide-binding fingerprint. In conclusion, we have cloned the gene encoding the CBP from C. albicans and have shown that the expressed protein has the properties of the native CBP. A comparison of the cloned gene to members of the steroid-thyroid-retinoic acid receptor gene superfamily showed that CBP is unrelated to these hormone receptors.
Recently recovered Kenyapithecus mandible and its implications for great ape and human origins.
Authors: McCrossin M L , Benefit B R
Published: May 2006

We report here a Kenyapithecus africanus juvenile mandible recovered from middle Miocene (ca. 14-16 million years) deposits of Maboko Island (Lake Victoria), Kenya. Symphyseal and dental attributes of the mandible distinguish K. africanus, a species widely regarded as the earliest known member of the great ape and human clade, from other Miocene large-bodied hominoids. The Maboko Island mandible exhibits a markedly proclined symphyseal axis, massive inferior transverse torus, mesiodistally narrow, high-crowned, and strongly procumbent lateral incisor, and molars with cingula restricted to the median buccal cleft. Although the presence of some of these conditions in Kenyapithecus was suggested earlier, the fragmentary and ill-preserved nature of previously known specimens led certain authorities to doubt their validity. Our assessment of mandibular and dental morphology indicates that K. africanus diverged after Proconsul and Griphopithecus but prior to the last common ancestor of Sivapithecus, extant great apes, and humans. The robustly constructed mandibular symphysis and anterior dentition suggest that incisal biting played as important a role as thick molar enamel in the dietary adaptations of K. africanus.
Casimir light: the source.
Authors: Schwinger J
Published: May 2006

The release of Casimir energy in filling a dielectric hole is identified as the source of coherent sonoluminescence. Qualitative agreement with recently acquired data is found for the magnitude and shape of the spectrum.
Dynamic and distributed properties of many-neuron ensembles in the ventral posterior medial thalamus of awake rats.
Authors: Nicolelis M A , Lin R C , Woodward D J , Chapin J K
Published: May 2006

The traditional view that the map of the face in the ventral posterior medial thalamus (VPM) is static and highly discrete was derived largely from qualitative studies that reported only small, robust, and nonoverlapping receptive fields (RFs). Here, by using more quantitative techniques, we have provided evidence for an alternative hypothesis: the RFs in the VPM are large and overlapping and tend to shift as a function of post-stimulus time. These results were obtained through simultaneous recordings of up to 23 single neurons across the whisker representation in the VPM of rats. Under both awake and anesthetized conditions, these neurons responded robustly at short (4-6 ms) and/or long (15-25 ms) latencies to discrete vibromechanical stimulation of single facial whiskers. Computer graphics were used to construct three-dimensional plots depicting the magnitudes of neuronal responses to stimulation of each of several whiskers as a function of post-stimulus time. These "spatiotemporal RFs" demonstrated that (i) the RFs of VPM neurons are quite large, covering up to 20 whiskers and (ii) the spatial locations of these RFs may shift dramatically over the first 35 ms of post-stimulus time, especially from the caudal-most to the rostral-most whiskers on the face. These results suggest that the VPM contains a dynamic and distributed representation of the face, in which stimulus information is coded in both spatial and temporal domains.
Targeted modification of the apolipoprotein B gene results in hypobetalipoproteinemia and developmental abnormalities in mice.
Authors: Homanics G E , Smith T J , Zhang S H , Lee D , Young S G , Maeda N
Published: May 2006

Familial hypobetalipoproteinemia is an autosomal codominant disorder resulting in a dramatic reduction in plasma concentrations of apolipoprotein (apo) B, cholesterol, and beta-migrating lipoproteins. A benefit of hypobetalipoproteinemia is that mildly affected individuals may be protected from coronary vascular disease. We have used gene targeting to generate mice with a modified Apob allele. Mice containing this allele display all of the hallmarks of human hypobetalipoproteinemia: they produce a truncated apoB protein, apoB70, and have markedly decreased plasma concentrations of apoB, beta-lipoproteins, and total cholesterol. In addition, the mice manifest several characteristics that are occasionally observed in human hypobetalipoproteinemia, including reduced plasma triglyceride concentrations, fasting chylomicronemia, and reduced high density lipoprotein cholesterol. An unexpected finding is that the modified Apob allele is strongly associated with exencephalus and hydrocephalus. These mice should help increase our understanding of hypobetalipoproteinemia, atherogenesis, and the etiology of exencephalus and hydrocephalus.
Class II-positive hematopoietic cells cannot mediate positive selection of CD4+ T lymphocytes in class II-deficient mice.
Authors: Markowitz J S , Auchincloss H , Grusby M J , Glimcher L H
Published: May 2006

Generation of immunocompetent alpha/beta T-cell receptor-positive T cells from CD4+CD8+ thymocytes depends upon their interaction with thymic major histocompatibility complex (MHC) molecules. This process of positive selection provides mature T cells that can recognize antigens in the context of self-MHC proteins. Previous studies investigating haplotype restriction in thymic and bone-marrow chimeras concluded that radioresistant thymic cortical epithelium directs the positive selection of thymocytes. There is controversy, however, as to whether intra- or extrathymic radiosensitive bone marrow-derived macrophage and dendritic cells also can mediate positive selection. To determine whether CD4+ T cells can be positively selected by hematopoietic cells, we generated chimeric animals expressing MHC class II molecules on either bone marrow-derived or thymic stromal cells by using a recently produced strain of MHC class II-deficient mice. CD4+ T cells developed only when class II MHC molecules were expressed on radioresistant thymic cells. In contrast to what recently has been observed for the selection of CD8+ T lymphocytes, MHC class II-positive bone marrow-derived cells were unable to mediate the selection of CD4+ T cells when the thymic epithelium lacked MHC class II expression. These data suggest that CD4+ and CD8+ T cells may be generated by overlapping, but not identical, mechanisms.
Attempts to restore scrapie prion infectivity after exposure to protein denaturants.
Authors: Prusiner S B , Groth D , Serban A , Stahl N , Gabizon R
Published: May 2006

A wealth of experimental evidence argues that infectious prions are composed largely, if not entirely, of the scrapie isoform of the prion protein. We attempted to restore scrapie infectivity after exposure to protein denaturants including urea, chaotropic salts, and SDS. None of the procedures restored infectivity. Dialysis to remove slowly chaotropic ions and urea failed to restore scrapie infectivity. Attempts to create monomers of the scrapie isoform of the prion protein under nondenaturing conditions using a wide variety of detergents have been unsuccessful, to date, except for one report claiming that scrapie infectivity could be recovered from 12% polyacrylamide gels after SDS/PAGE [Brown, P., Liberski, P. P., Wolff, A. & Gajdusek, D. C. (1990) Proc. Natl. Acad. Sci. USA 87, 7240-7244]. We found that < 0.001% of the infectious prion titer could be recovered from the region of a polyacrylamide gel where the denatured proteinase K-resistant core of the scrapie isoform of the prion protein and other 30-kDa proteins migrate. We conclude that under the denaturing conditions used for SDS/PAGE, the scrapie isoform of the prion protein is denatured and little or no renaturation occurs upon injection of fractions eluted from gels into animals for bioassays.
Calmodulin and calmodulin-binding proteins in hair bundles.
Authors: Walker R G , Hudspeth A J , Gillespie P G
Published: May 2006

Calcium ion plays an important role in the hair cell's mechanoelectrical transduction process; in particular, Ca2+ controls adaptation to protracted mechanical stimuli. Because calmodulin is a ubiquitous intracellular receptor for Ca2+ and has been shown to accumulate at the tips of stereocilia, we determined its concentration and identified the proteins with which it interacts in the hair bundle. By performing quantitative immunoblot analysis on isolated bundles, we ascertained that the average concentration of calmodulin within each stereocilium is approximately 70 microM. Extraction experiments disclosed that, in the presence of 20 microM Ca2+, 50% of the calmodulin is bound to detergent-soluble receptors. To distinguish these receptors, we developed an assay that utilizes calmodulin crosslinked to alkaline phosphatase. This technique is approximately 100-fold more sensitive than calmodulin-binding assays that employ 125I- or biotin-labeled calmodulin. When used with chemiluminescence detection in a blot-overlay assay, the calmodulin-alkaline phosphatase conjugate identified hair-bundle proteins of molecular masses 25, 35, 145, 175, 240, and 350 kDa. We examined the subcellular distribution of these receptors; all but the 240-kDa molecule are soluble in a nonionic detergent. The relatively high concentration of calmodulin and the presence of several calmodulin-binding proteins provide evidence for a role of calmodulin in hair bundles.
Opposite regulation of gene transcription and cell proliferation by c-Myc and Max.
Authors: Gu W , Cechova K , Tassi V , Dalla-Favera R
Published: May 2006

c-Myc and Max are nuclear phosphoproteins capable of forming DNA-binding, homo- and heteropolymeric complexes in vitro and in vivo. Using a transient cotransfection assay involving c-Myc and Max expression vectors and a reporter gene plasmid containing the Myc/Max binding site, we find that Max represses transcription, whereas a significant stimulation is obtained when Max is coexpressed with c-Myc. Analysis of specific mutants indicates that transcriptional activation requires both the c-Myc and the Max dimerization and DNA-binding domains, as well as the c-Myc transactivation function; transcriptional repression by Max requires both DNA binding and dimerization. Analogously, in stably transfected human B-lymphoblastoid cell lines, overexpressed c-Myc and Max synergize to cause malignant transformation, whereas overexpression of Max alone leads to growth inhibition. These results indicate that the c-Myc and Max are transcriptional regulators with the ability to oppositely regulate target-gene expression and cell proliferation, most likely as the result of the opposite effects of heterodimeric c-Myc-Max (positive) versus homodimeric Max (negative) complexes.
In vitro assembly of multiprotein complexes containing alpha, beta, and gamma tubulin, heat shock protein HSP70, and elongation factor 1 alpha.
Authors: Marchesi V T , Ngo N
Published: May 2006

We have isolated two sets of multiprotein complexes from supernatants from high-speed centrifugation of nocodazole-arrested CHO cells. One set, assembled in vitro after a 37 degrees C incubation in the presence of ATP or GTP, is composed of equivalent amounts of alpha- and beta-tubulin and a 50-kDa protein, provisionally identified as elongation factor 1 alpha. These complexes, which are heterogeneous in size when analyzed by sucrose gradient ultracentrifugation, also contain the cognate form of heat shock protein HSP70 and gamma-tubulin, a tubulin isoform of low abundance, along with other proteins known to be involved in the regulation of mitosis. Similar but distinct complexes assemble in vitro if the same extracts are incubated at 37 degrees C without added nucleotides; multiprotein complexes generated under these conditions lack HSP70 but contain instead a 43-kDa protein identified as an actin isoform. Both sets of assembled complexes exhibit a globular substructure when analyzed by electron microscopy, and their size distribution suggests that they assemble by the step-wise addition of smaller precursors. The properties of these multiprotein complexes and their presence in cells arrested in a stage between prophase and metaphase suggest that they may be precursors to mitotic centrosomes and are possibly involved in mitotic spindle nucleation.
T7 RNA polymerase mutants with altered promoter specificities.
Authors: Raskin C A , Diaz G A , McAllister W T
Published: May 2006

The amino acid at position 748 in T7 RNA polymerase (RNAP) functions to discriminate base pairs at positions -10 and -11 in the promoter. We have constructed a series of T7 RNAP mutants having all possible amino acid substitutions at this position. Surprisingly, most (13/19) substitutions result in active RNAPs, and many of these exhibit altered promoter specificities. Identification of mutant RNAPs with altered specificities expands the repertoire of highly specific phage RNAPs that are available for use in phage RNAP-based transcription systems and highlights the complexity of sequence-specific DNA recognition.
Beta-arrestin and arrestin are recognized by autoantibodies in sera from multiple sclerosis patients.
Authors: Ohguro H , Chiba S , Igarashi Y , Matsumoto H , Akino T , Palczewski K
Published: May 2006

Multiple sclerosis (MS), one of the most common chronic neurologic diseases, is characterized by the presence of multiple plaques of demyelination throughout the central nervous system. Although the etiology of the disease has not been established, it is believed to involve autoimmune mechanisms. We have examined sera from patients with MS for the presence of antibodies to antigens from brain and retina. Immunoblot analysis of soluble fraction of proteins from bovine brain revealed a prominent band at 45 kDa stained with sera of 8-14 patients with MS. In two patients with MS, serum antibody titers during relapse were higher compared with those when the patients were in remission. These antibodies were undetectable in cerebrospinal fluid of our MS patients and additionally were absent in sera of patients with other neurological diseases and normal control subjects. Furthermore, immunoblot analysis of the soluble fraction from bovine retinal rod outer segments revealed a prominent protein band at 48 kDa stained with MS sera. This antigen was purified to homogeneity from bovine retinal outer segments and identified as arrestin. Additionally, sera from MS patients reacted with purified beta-arrestin 1, a 45-kDa protein homologous to arrestin that is found in various tissues. Using limited proteolysis of arrestin and a competitive ELISA test with a synthetic peptide, we identified the recognition site(s) for antibodies in sera of MS patients at a dominant immunogenic site on arrestin located at the C-terminal region of the molecule. We suggest that the presence of circulating antibodies reactive with beta-arrestin or arrestin may be related to the course of MS progression.