bluuebunny/crossref_metadata_2025 huggingface dataset:
Prenyl modification of guanine nucleotide regulatory protein gamma 2 subunits is not required for interaction with the transducin alpha subunit or rhodopsin.
Authors: Wildman D E , Tamir H , Leberer E , Northup J K , Dennis M
Published: May 2006

Guanine nucleotide-binding regulatory protein (G protein) beta gamma dimers that were active in reconstitution assays were produced in insect cells using the baculovirus/Sf9 insect cell expression system. Sf9 cells were infected either singly or in combination with recombinant baculoviruses containing a human G-protein beta 1 gene or a bovine G-protein gamma 2 gene. It was possible to express the beta 1 and gamma 2 gene products independently of each other in this system, as determined by using immunological and metabolic labeling techniques. Further, the ability of recombinant beta and/or gamma chains to function in defined biochemical assays of beta gamma activity was assessed for membrane extracts and supernatant fractions from infected Sf9 cells. Extracts of cells expressing beta or gamma chain alone were inactive in these assays, whereas those from cells coinfected with beta 1 and gamma 2 did display activity. These assays were used to identify recombinant beta gamma dimer migration during chromatographic purification, and the recombinant dimers were purified to near homogeneity. Both the membrane-associated and soluble beta gamma dimers facilitated rhodopsin-catalyzed guanosine 5'-[gamma-thio]triphosphate binding to Gt alpha, the GTP-binding subunit of the retinal G protein transducin (K0.5 of 13 +/- 2 and 36 +/- 5 nM, respectively). Both recombinant beta gamma dimers also facilitated the pertussis toxin-catalyzed ADP-ribosylation of Gt alpha with equal potency (K0.5 of 9 +/- 1 and 10 +/- 3 nM for membrane and soluble dimers, respectively). [3H]Mevalonolactone labeling showed that the gamma 2 subunits of membrane-associated beta gamma dimers incorporated radiolabel, whereas in the soluble form they did not. Thus, prenyl modification of gamma 2 directs the membrane association of the beta 1 gamma 2 dimer and increases its apparent affinity for receptor, but it is not required for the functional interaction(s) of the dimer.
Biogeography of Tongan birds before and after human impact.
Authors: Steadman D W
Published: May 2006

Bones deposited in caves show that, before the arrival of humans, at least 27 species of land birds lived on the Tongan island of 'Eua, where 13 indigenous species live today. Six of these 13 species were recorded from pre-human strata; three others probably occurred on 'Eua in pre-human times but were not in the fossil sample; and four others probably colonized 'Eua since the arrival of humans approximately 3000 years ago. Of the 23 species of extinct or extirpated land birds recorded from 'Eua, the nearest geographic occurrences of conspecifics or most closely related congeners are from the Solomon Islands (1 species), New Caledonia (2 species), Fiji and/or Samoa (9 species), elsewhere in Tonga (8 species), or unknown (3 species). The avifauna of West Polynesia (Fiji-Tonga-Samoa) is more closely related to that of Melanesia than that of East Polynesia. There was little pre-human turnover in Tongan land birds. The arrival of humans has influenced the Tongan avifauna more than any climatic, tectonic, or biological event of the past approximately 100,000 years.
Protein prenylcysteine analog inhibits agonist-receptor-mediated signal transduction in human platelets.
Authors: Huzoor-Akbar  , Wang W , Kornhauser R , Volker C , Stock J B
Published: May 2006

Signal transduction components, including the Ras superfamily of low molecular weight GTP-binding proteins and the gamma subunits of heterotrimeric G proteins, are reversibly carboxyl methylated at C-terminal prenylcysteine residues. We have previously shown that the prenylcysteine analog N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC) inhibits carboxyl methylation of these proteins in human platelets. Here we show that concentrations of AFC that inhibit Ras carboxyl methylation (10-50 microM) also block responses to agonists such as ADP, collagen, arachidonic acid, U46619 (a stable analog of prostaglandin H2), thrombin, and guanosine 5'-[gamma-thio]triphosphate. AFC does not inhibit aggregation induced by effectors such as ionomycin, phorbol 12,13-dibutyrate, and bacterial phospholipase C that bypass G proteins to activate platelets at the level of cytosolic Ca2+ concentration and protein kinase C. These findings indicate that AFC inhibits agonist-receptor-mediated signal transduction in human platelets.
Dissociation of human mid-dorsolateral from posterior dorsolateral frontal cortex in memory processing.
Authors: Petrides M , Alivisatos B , Evans A C , Meyer E
Published: May 2006

Work with non-human primates had previously demonstrated that the mid-dorsolateral frontal cortex, which comprises cytoarchitectonic areas 46 and 9, plays a critical role in the performance of non-spatial self-ordered working memory tasks, whereas the immediately adjacent posterior dorsolateral frontal cortex (area 8) is critical for the learning and performance of visual conditional associative tasks. The present study used positron emission tomography with magnetic resonance imaging to demonstrate the existence, within the human brain, of these two functionally distinct subdivisions of the lateral frontal cortex. These findings provide direct evidence that, just as the monkey brain, the human lateral frontal cortex is functionally heterogeneous and that comparable anatomical areas underlie similar functions in the two species.
Supramolecular effects and molecular discrimination by macrocyclic hosts embedded in synthetic bilayer membranes.
Authors: Murakami Y , Hayashida O
Published: May 2006

Hybrid assemblies were prepared in combinations of a synthetic lipid, involving an L-alanine residue interposed between an anionic head group and a hydrophobic double-chain segment, with cage-type cyclophanes having a three-dimensionally extended hydrophobic cavity. Incorporation behavior of the cage-type hosts into the multi- and single-walled vesicules was characterized by highly sensitive differential scanning calorimetry and dynamic light-scattering measurements as well as by gel-filtration chromatography and electronic absorption spectroscopy. Both hosts were found to be completely incorporated into a relatively polar domain close to the vesicular surface. Anionic guest molecules, such as naphthol yellow S, dimethylsulfonazo III, bromopyrogallol red, and orange G, were effectively incorporated into the hydrophobic cavities provided by the cage-type cyclophanes embedded in the single-walled bilayer membrane, as confirmed by circular dichroism spectroscopy; the formation constants for 1:1 host-guest complexes were in the order of 10(4)-10(5) mol.dm-3. This means that the binding affinities of the cage-type hosts are largely retained even when the hosts are incorporated into the vesicle. Moreover, the pyridinium moieties bound to the chiral L-valine residues in the bridging components of the hosts underwent conformational changes upon inclusion of the guest, as confirmed by circular dichroism spectroscopy and molecular mechanics and dynamics calculations.
Homozygous somatic Wt1 point mutations in sporadic unilateral Wilms tumor.
Authors: Coppes M J , Liefers G J , Paul P , Yeger H , Williams B R
Published: May 2006

Wilms tumor may be caused by loss of function of genes at different loci. A Wilms tumor suppressor gene, WT1, at chromosome 11 band p13, has recently been cloned and characterized. WT1 has been implicated in the development of Wilms tumor by virtue of mutations in patients with genitourinary anomalies and susceptibility to Wilms tumor. Homozygous intragenic mutations have been reported in Wilms tumors, but usually not in sporadic unilateral Wilms tumors, which constitute the majority of Wilms tumor cases. Using the single-strand conformational polymorphism assay, we have identified three sporadic unilateral Wilms tumors with homozygous point mutations: one with a de novo germ-line nonsense point mutation within WT1 exon 8, and two carrying a somatic mutation within WT1 exon 10. In all three cases loss of the wild-type allele was demonstrated by tumor loss of heterozygosity. This report provides an example of two somatic mutations in the same tumor expected to inactivate WT1 function.
Interaction of the peroxisome-proliferator-activated receptor and retinoid X receptor.
Authors: Gearing K L , Göttlicher M , Teboul M , Widmark E , Gustafsson J A
Published: May 2006

The rat peroxisome-proliferator-activated receptor (PPAR) was expressed in insect cells and was shown to bind to a cognate PPAR response element (PPRE) from the acyl-CoA oxidase gene. Upon purification, PPAR was no longer able to bind DNA, although binding could be restored by addition of insect cell extracts. We investigated whether the retinoid X receptor (RXR) could supplement for this accessory activity. The rat RXR alpha cDNA was cloned and it was found that addition of in vitro-translated RXR alpha to purified PPAR facilitated binding of PPAR to a PPRE. Furthermore, an additional activity, which appeared to be distinct from rRXR alpha, was found in COS cell nuclear extracts that enabled binding of PPAR to a PPRE. Transient expression of RXR alpha in CHO cells was found to be essential for the response of a chloramphenicol acetyltransferase reporter construct containing PPREs to activators of PPAR. These results raise the possibility of convergence of the PPAR and retinoid-dependent signaling pathways on promoters containing PPRE-like responsive elements.
Effects of genetic replacements of charged and H-bonding residues in the retinal pocket on Ca2+ binding to deionized bacteriorhodopsin.
Authors: Zhang Y N , el-Sayed M A , Bonet M L , Lanyi J K , Chang M , Ni B , Needleman R
Published: May 2006

Metal cations are known to be required for proton pumping by bacteriorhodopsin (bR). Previous studies found that bR has two high-affinity and four to six low-affinity Ca(2+)-binding sites. In our efforts to find the location of these Ca2+ sites, the effects of replacing charged (Asp-85, Asp-212, and Arg-82) and H-bonding (Tyr-185) residues in the retinal pocket on the color control and binding affinity of Ca2+ ions in Ca(2+)-regenerated bR were examined. The important results are as follows: (i) The removal of Ca2+ from recombinant bR in which charged residues were replaced by neutral ones shifted the retinal absorption to the blue, opposite to that observed in wild-type bR or in recombinant bR in which the H-bonding residue, Tyr-185, was replaced by a non-H-bonding amino acid (Phe). (ii) Similar to the observation in wild-type bR, the binding of Ca2+ to the second site gave the observed color change in the recombinant bR samples in which charged residues were replaced by neutral ones. (iii) The residue replacements had no effect on the affinity constants of the four to six weakly bound Ca2+. (iv) The two high-affinity sites exhibited reduced affinity with substitutions; while the extent of the reduction depended on the specific substitution, each site was reduced by the same factor for each of the charged residue substitutions but by different factors for the mutant where Tyr-185 was replaced with Phe(Y185F). The above results suggest that the two Ca2+ ions in the two high-affinity sites are within interaction distance with one another and with the charged residues in the retinal pocket. The results further suggest that, while the interaction between Tyr-185 and the high-affinity Ca2+ ions is relatively short range and specific (with more coupling to the Ca2+ ion in the second affinity site), between the charged residues and Ca2+ ions it seems to be of the electrostatic (e.g., ion-ion) long range, nonspecific type. Although neither Asp-85, Asp-212, nor Arg-82 is individually directly involved in the binding of Ca2+ in these two sites, they might all participate in it. Together with the protonated Schiff base, the charged residues along with Tyr-185 and one or two Ca2+ ions (and probably a few water molecules) seem to form an electrostatically coupled system that is part of a cavity that controls the color and function of bR.
Postproliferative transcription of the rat osteocalcin gene is reflected by vitamin D-responsive developmental modifications in protein-DNA interactions at basal and enhancer promoter elements.
Authors: Owen T A , Bortell R , Shalhoub V , Heinrichs A , Stein J L , Stein G S , Lian J B
Published: May 2006

In the osteocalcin (OC) gene promoter, both independent positive and negative regulatory elements, as well as others with contiguous [TATA/glucocorticoid-responsive elements (GRE)] or overlapping [TATA/GRE, vitamin D-responsive enhancer elements (VDRE)/AP-1, and OC box/AP-1] domains, are sites for modifications in protein-DNA interactions. In the present studies, we have examined nuclear protein extracts from fetal rat calvarial cells that undergo a developmental sequence of bone cell differentiation. Our results demonstrate modifications in protein-DNA interactions that relate to the developmental stages of the osteoblast and support developmental regulation of OC gene transcription. Basal expression of the OC gene is associated with sequence-specific protein-DNA interactions at the OC box, VDRE, and TATA/GRE box. Distinct differences are observed in proliferating osteoblasts, where the OC gene is not transcribed compared to postproliferative, differentiated osteoblasts that transcribe the OC gene. Furthermore, the protein-DNA complexes that reflect hormonal control are also developmentally regulated, mediating both the transcriptionally active and repressed states of the OC gene. For example, in proliferating osteoblasts, a vitamin D receptor-antibody-sensitive complex is formed that is different from the DNA binding complex induced by vitamin D postproliferatively when the OC gene is transcribed. Mutational analysis of the steroid hormone binding domain and the overlapping AP-1 site at the VDRE supports mutually exclusive occupancy by Fos-Jun heterodimers and vitamin D receptor. Such protein-DNA interactions at the VDRE are consistent with repression of competency for vitamin D-mediated transcriptional enhancement in proliferating osteoblasts expressing high levels of Fos and Jun.
The short form of the CheA protein restores kinase activity and chemotactic ability to kinase-deficient mutants.
Authors: Wolfe A J , Stewart R C
Published: May 2006

Escherichia coli expresses two forms of the chemotaxis-associated CheA protein, CheAL and CheAS, as the result of translational initiation at two distinct, in-frame initiation sites in the gene cheA. The long form, CheAL, plays a crucial role in the chemotactic signal transduction mechanism by phosphorylating two other chemotaxis proteins: CheY and CheB. CheAL must first autophosphorylate at amino acid His-48 before transferring its phosphono group to these other signal transduction proteins. The short form, CheAS, lacks the N-terminal 97 amino acids of CheAL and, therefore, does not possess the site of autophosphorylation. Here we demonstrate that although it lacks the ability to autophosphorylate, CheAS can mediate phosphorylation of kinase-deficient variants of CheAL each of which retains a functional autophosphorylation site. This transphosphorylation enables these kinase-deficient CheAL variants to phosphorylate CheY. Because it mediates this activity, CheAS can restore to kinase-deficient E. coli cells the ability to tumble and, thus, to perform chemotaxis in swarm plate assays.
ARIA, a protein that stimulates acetylcholine receptor synthesis, also induces tyrosine phosphorylation of a 185-kDa muscle transmembrane protein.
Authors: Corfas G , Falls D L , Fischbach G D
Published: May 2006

Motoneurons promote the accumulation of acetylcholine receptors (AChRs) at developing neuromuscular synapses. The AChR-inducing activity protein ARIA, which is purified from chicken brain and increases the synthesis of AChRs in chicken myotubes, may play a crucial role in this process. Here we show that ARIA induces the rapid tyrosine phosphorylation of a M(r) 185,000 protein (p185) in muscle cells. Phosphorylation of p185 correlates with AChR induction at each stage of ARIA purification. Moreover, medium conditioned by spinal cord motoneurons stimulates AChR synthesis and p185 phosphorylation. Studies with membrane-impermeant reagents and 125I-labeled ARIA indicate that p185 is a transmembrane ARIA-receptor tyrosine kinase. Our data suggests that muscle AChR synthesis can be regulated through tyrosine phosphorylation.
Mechanism of action of des-His1-[Glu9]glucagon amide, a peptide antagonist of the glucagon receptor system.
Authors: Post S R , Rubinstein P G , Tager H S
Published: May 2006

We have investigated the mechanisms through which des-His1-[Glu9]glucagon amide functions as a peptide antagonist of the glucagon receptor/adenylyl cyclase system. Studies with radiolabeled peptides identified that (i) the antagonist bound to intact hepatocytes according to a single first-order process, whereas the rate of association of glucagon with the same preparation could be described only by the sum of two first-order processes; (ii) the interaction of the antagonist with saponin-permeabilized hepatocytes was not affected by the addition of GTP to the incubation medium or by the elimination of Mg2+, whereas the interaction of glucagon with the same cell preparation was modified significantly by the presence of the nucleotide or by the absence of the divalent metal ion; (iii) the dissociation of antagonist from intact hepatocytes incubated in buffer was complete, whereas that of agonist was not; and (iv) the antagonist bound to intact hepatocytes at steady state according to a single binding isotherm (as did both agonist and antagonist in permeabilized hepatocytes), whereas glucagon bound to the intact cell system with two clearly defined apparent dissociation constants. A model is presented for the mechanism of action of the glucagon antagonist in which the analog binds to glucagon receptors in a Mg(2+)- and GTP-independent fashion and in which resulting ligand-receptor complexes fail to undergo sequential adjustments necessary for the stimulation of adenylyl cyclase.
The interleukin 2 CD28-responsive complex contains at least three members of the NF kappa B family: c-Rel, p50, and p65.
Authors: Ghosh P , Tan T H , Rice N R , Sica A , Young H A
Published: May 2006

Optimal activation of T cells requires at least two signals. One signal can be delivered by the antigen-specific T-cell receptor, and the second signal is provided by the costimulatory molecule(s) delivered by the antigen-presenting cell. CD28 is a T-cell surface molecule and stimulation through this protein plays an important role in delivering the second activation signal. In this report, we show that in human peripheral blood T cells, CD28-mediated signal transduction involves the rel family proteins--c-Rel, p50, and p65. Treatment of peripheral blood T cells with phorbol 12-myristate 13-acetate (PMA) and anti-CD28 monoclonal antibody (mAb) results in augmentation of nuclear c-Rel, p50, and p65, and this augmentation can occur in the presence of the immunosuppressant cyclosporin A. It is also shown in this report that, in response to PMA/anti-CD28 mAb or anti-CD3/anti-CD28 mAb, c-Rel, p50, and p65 are associated with CD28-responsive element present in the promoter of the human interleukin 2 gene. The functional significance of c-Rel involvement in the CD28-responsive complex is demonstrated by transient transfection analysis, where cotransfection of c-Rel augments the level of expression of a chloramphenicol acetyltransferase reporter gene linked to the CD28-responsive element.
Comparison of variable region 3 sequences of human immunodeficiency virus type 1 from infected children with the RNA and DNA sequences of the virus populations of their mothers.
Authors: Scarlatti G , Leitner T , Halapi E , Wahlberg J , Marchisio P , Clerici-Schoeller M A , Wigzell H , Fenyö E M , Albert J , Uhlén M
Published: May 2006

We have compared the variable region 3 sequences from 10 human immunodeficiency virus type 1 (HIV-1)-infected infants to virus sequences from the corresponding mothers. The sequences were derived from DNA of uncultured peripheral blood mononuclear cells (PBMC), DNA of cultured PBMC, and RNA from serum collected at or shortly after delivery. The infected infants, in contrast to the mothers, harbored homogeneous virus populations. Comparison of sequences from the children and clones derived from DNA of the corresponding mothers showed that the transmitted virus represented either a minor or a major virus population of the mother. In contrast to an earlier study, we found no evidence of selection of minor virus variants during transmission. Furthermore, the transmitted virus variant did not show any characteristic molecular features. In some cases the transmitted virus was more related to the virus RNA population of the mother and in other cases it was more related to the virus DNA population. This suggests that either cell-free or cell-associated virus may be transmitted. These data will help AIDS researchers to understand the mechanism of transmission and to plan strategies for prevention of transmission.
Gradients of transgene expression directed by the human myoglobin promoter in the developing mouse heart.
Authors: Parsons W J , Richardson J A , Graves K H , Williams R S , Moreadith R W
Published: May 2006

Prior studies using transient transfection assays in cultured avian and murine skeletal myotubes indicate that the proximal 2-kb segment of the 5' flanking region of the human myoglobin gene contains transcriptional control elements sufficient to direct muscle-specific and developmentally regulated expression of reporter genes. To examine the function of the human myoglobin gene promoter during development of skeletal and cardiac myocytes in the intact animal, a 2.0-kb myoglobin gene upstream fragment was fused to an Escherichia coli lacZ reporter gene and injected into fertilized mouse oocytes. beta-Galactosidase (beta-gal) activity was detected selectively in cardiac and skeletal myocytes of fetal and adult transgenic mice. A distinctive spatial pattern of myoglobin promoter activity was observed in fetal hearts: beta-gal staining was more pronounced within the left ventricular subendocardium than within the subepicardium and was essentially undetectable in the ventricular trabeculae or atria. Expression of endogenous myoglobin mRNA and protein, assessed by in situ hybridization and immunohistochemistry, demonstrated a similar spatial pattern. In contrast, hearts from adult transgenic mice demonstrated essentially homogeneous expression of beta-gal and of endogenous myoglobin mRNA and protein throughout the myocardium, including the trabeculae and atria. These data indicate that the 2.0-kb upstream region of the human myoglobin gene includes cis-acting regulatory elements sufficient to direct transgene expression during murine cardiac development that is myocyte-specific and responsive to positional cues in a similar manner to the endogenous myoglobin gene.
Nitric oxide mediates cytokine-induced inhibition of insulin secretion by human islets of Langerhans.
Authors: Corbett J A , Sweetland M A , Wang J L , Lancaster J R , McDaniel M L
Published: May 2006

Cytokines have been implicated as immunological effector molecules that mediate beta cell destruction associated with insulin-dependent diabetes mellitus. In this report we demonstrate that the cytokine combination of human recombinant interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma) induces the formation of nitric oxide by human islets. This combination of cytokines stimulates both the formation of the nitric oxide derivative, nitrite, and the accumulation of cGMP by human islets. The nitric oxide synthase inhibitor NG-monomethyl-L-arginine prevents formation of both cGMP and nitrite. IL-1 beta and IFN-gamma are sufficient to induce nitric oxide formation by human islets, whereas TNF-alpha potentiates nitrite production. This combination of cytokines (IL-1 beta, TNF-alpha, and IFN-gamma) also influences insulin secretion by human islets. Pretreatment of human islets with low concentrations of this cytokine combination (IL-1 beta at 15 units/ml, 0.7 nM TNF-alpha, and IFN-gamma at 150 units/ml) appears to slightly stimulate insulin secretion. Higher concentrations (IL-1 beta at 75 units/ml, 3.5 nM TNF-alpha, and IFN-gamma at 750 units/ml) inhibit insulin secretion from human islets, and the inhibitory effect is prevented by NG-monomethyl-L-arginine. This higher concentration of cytokines also induces the formation of an electron paramagnetic resonance-detectable g = 2.04 axial feature by human islets that is characteristic of the formation of an iron-dithio-dinitrosyl complex. The formation of this complex is prevented by NG-monomethyl-L-arginine, thus confirming that this cytokine combination induces the formation of nitric oxide by human islets. These results indicate that nitric oxide mediates the inhibitory effects of cytokines on glucose-stimulated insulin secretion by human islets and suggest that nitric oxide may participate in beta-cell dysfunction associated with insulin-dependent diabetes mellitus.
Isolation of proteins associated with kinetoplast DNA networks in vivo.
Authors: Xu C , Ray D S
Published: May 2006

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomes, is a highly condensed disc-shaped network of catenated DNA circles consisting of maxicircles, the equivalent of conventional mitochondrial DNA, and several thousand smaller circular DNAs termed minicircles. Upon cell lysis, kDNA expands, giving rise to a two-dimensional network of catenated circles with an overall diameter close to that of the whole cell. To identify proteins associated with the condensed form of kDNA in the cell, proteins were reversibly crosslinked to kDNA in whole cells of Crithidia fasciculata by formaldehyde treatment. Crosslinked networks were purified and found to retain a condensed structure which becomes fully expanded upon proteinase K treatment or reversal of the crosslinks by heating at 65 degrees C. Five low molecular weight proteins released from the kDNA by heat treatment were purified by polyacrylamide gel electrophoresis and their amino-terminal sequences were determined. PCR amplification and sequence analysis of cDNA sequences between these amino-terminal sequences and the miniexon (spliced leader) sequence present at the 5' end of all C. fasciculata mRNAs predicts the presence of 9-amino acid presequences with features characteristic of mitochondrial presequences on three of the proteins. Two of these proteins are lysine-rich basic proteins. These findings suggest that basic proteins may play a role in the condensation of kDNA in the kinetoplast and that these proteins are imported into the kinetoplast by a mechanism involving a cleavable presequence.
Enhanced immunity to human immunodeficiency virus (HIV) envelope elicited by a combined vaccine regimen consisting of priming with a vaccinia recombinant expressing HIV envelope and boosting with gp160 protein.
Authors: Cooney E L , McElrath M J , Corey L , Hu S L , Collier A C , Arditti D , Hoffman M , Coombs R W , Smith G E , Greenberg P D
Published: May 2006

Transmission studies have suggested that an optimal human immunodeficiency virus type 1 (HIV-1) vaccine should induce both neutralizing antibodies and cytolytic T cells to eliminate free virus and infected cells. A phase I trial in healthy HIV-1-seronegative persons was conducted with a combination HIV-1 vaccine regimen (strain IIIB) consisting of priming with a recombinant vaccinia (vac/env) virus expressing HIV-1 envelope and boosting with a gp160 glycoprotein derived from a recombinant baculovirus (rgp160). T-cell and antibody responses detected after immunization with either vac/env alone or rgp160 alone were generally of low magnitude and transient, and no subject developed neutralizing antibodies. In contrast, recipients of the combination regimen demonstrated in vitro T-cell proliferative responses to homologous HIV-1 antigens that were 3- to 10-fold higher than responses with either vaccine alone, and these responses were sustained for > 18 months in 75% of recipients. Moreover, both CD8+ and CD4+ cytolytic T cells were detected. Antibody responses (titer, 1:800 to 1:102,400) to homologous HIV envelope developed in all recipients of the combination regimen, and neutralizing antibodies were detected in 7 of 13. Thus, immunization with a live virus vaccine followed by boosting with a soluble protein offers promise for inducing the broad immunity needed in an HIV vaccine.
Mass spectrometric and Edman sequencing of lipocortin I isolated by two-dimensional SDS/PAGE of human melanoma lysates.
Authors: Hall S C , Smith D M , Masiarz F R , Soo V W , Tran H M , Epstein L B , Burlingame A L
Published: May 2006

We have integrated preparative two-dimensional polyacrylamide gel electrophoresis with high-performance tandem mass spectrometry and Edman degradation. By using this approach, we have isolated and identified, by partial sequencing, a human melanoma protein (34 kDa, pI 6.4) as lipocortin I. To our knowledge, this protein was not previously known to be associated with melanoma cells. The identity of the protein was confirmed by two-dimensional immunoblot analysis. High-energy collision-induced dissociation analysis revealed the sequence and acetylation of the N-terminal tryptic peptide and an acrylamide-modified cysteine in another tryptic peptide. Thus, knowledge concerning both the primary structure and covalent modifications of proteins isolated from two-dimensional gels can be obtained directly by this approach, which is applicable to a broad range of biological problems.
Scatter factor induces blood vessel formation in vivo.
Authors: Grant D S , Kleinman H K , Goldberg I D , Bhargava M M , Nickoloff B J , Kinsella J L , Polverini P , Rosen E M
Published: May 2006

Scatter factor (also known as hepatocyte growth factor) is a glycoprotein secreted by stromal cells that stimulates cell motility and proliferation. In vitro, scatter factor stimulates vascular endothelial cell migration, proliferation, and organization into capillary-like tubes. Using two different in vivo assays, we showed that physiologic quantities of purified native mouse scatter factor and recombinant human hepatocyte growth factor induce angiogenesis (the formation of new blood vessels). The angiogenic activity was blocked by specific anti-scatter factor antibodies. Scatter factor induced cultured microvascular endothelial cells to accumulate and secrete significantly increased quantities of urokinase, an enzyme associated with development of an invasive endothelial phenotype during angiogenesis. We further showed that immunoreactive scatter factor is present surrounding sites of blood vessel formation in psoriatic skin. These findings suggest that scatter factor may act as a paracrine mediator in pathologic angiogenesis associated with human inflammatory disease.
Immunocytochemical analysis of the coiled body in the cell cycle and during cell proliferation.
Authors: Andrade L E , Tan E M , Chan E K
Published: May 2006

Coiled bodies (CBs) are small, round structures found in the nucleoplasm of most eukaryotic cells. Human autoantibodies to a 80-kDa protein, p80-coilin, are immunohistologic markers for CBs. A polyclonal rabbit antiserum (R288) raised against recombinant p80-coilin was shown to have similar immunochemical properties as human autoantibodies and was used to analyze the expression of p80-coilin-associated CBs in cell cultures synchronized by double thymidine block, nocodazole arrest, serum starvation, or hormonal deprivation. By employing thymidine block and nocodazole arrest of HeLa cells, CBs were observed in immunofluorescent studies to be largest in size in the S and G2 phases of the cell cycle. These large CBs might have coalesced into one or two such structures per cell from smaller and more numerous CBs of three to eight per cell during the mid G1 phase of the cell cycle. No CB-like structures were observed in mitosis and early G1. However, immunoblotting analyses showed that the total amount of p80-coilin remained approximately the same throughout the cell cycle. When HeLa cells were separated into soluble and particulate fractions, p80-coilin was detected predominantly in the soluble fraction in mitosis and early G1, whereas it was present predominantly in the particulate fraction in late G1, S, and G2 when structurally distinct CBs were observed. In the analysis of CBs in two experimental models of cell proliferation (reversal of 3T3 serum starvation and FRTL-5 thyrotropin deprivation), proliferating cells contained larger, brighter, and more numerous CBs as well as a > 2-fold increase in the total amount of p80-coilin compared to that in quiescent cells. The expression of p80-coilin in quiescent cells induced to proliferate and the cyclic formation and breakdown of CBs might be consistent with the notion that CBs may be specialized centers related to the maturation of mRNA, but this evidence is indirect and needs further definitive study.
Acceptor end binding domain interactions ensure correct aminoacylation of transfer RNA.
Authors: Weygand-Durasević I , Schwob E , Söll D
Published: May 2006

The recognition of the acceptor stem of tRNA(Gln) is an important element ensuring the accuracy of aminoacylation by Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18). On the basis of known mutations and the crystal structure of the tRNA(Gln).GlnRS complex, we mutagenized at saturation two motifs in the acceptor end binding domain of GlnRS. Mutants with lowered tRNA specificity were then selected in vivo by suppression of a glutamine-specific amber mutation (lacZ1000) with an amber suppressor tRNA derived from tRNA(1Ser). The mischarging GlnRS mutants obtained in this way retain the ability to charge tRNA(Gln), but in addition, they misacylate a number of noncognate amber suppressor tRNAs. The critical residues responsible for specificity are Arg-130 and Glu-131, located in a part of GlnRS that binds the acceptor stem of tRNA(Gln). On the basis of the spectrum of tRNAs capable of being misacylated by such mutants we propose that, in addition to taking part in productive interactions, the acceptor end binding domain contributes to recognition specificity by rejecting noncognate tRNAs through negative interactions. Analysis of the catalytic properties of one of the mischarging enzymes, GlnRS100 (Arg-130-->Pro, Glu-131-->Asp), indicates that, while the kinetic parameters of the mutant enzyme are not dramatically changed, it binds noncognate tRNA(Glu) more stably than the wild-type enzyme does (Kd is 1/8 that of the wild type). Thus, the stability of the noncognate complex may be the basis for mischarging in vivo.
Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia.
Authors: Lois C , Alvarez-Buylla A
Published: May 2006

Subventricular zone (SVZ) cells proliferate spontaneously in vivo in the telencephalon of adult mammals. Several studies suggest that SVZ cells do not differentiate after mitosis into neurons or glia but die. In the present work, we show that SVZ cells labeled in the brains of adult mice with [3H]thymidine differentiate directly into neurons and glia in explant cultures. In vitro labeling with [3H]thymidine shows that 98% of the neurons that differentiate from the SVZ explants are derived from precursor cells that underwent their last division in vivo. This report identifies the SVZ cells as neuronal precursors in an adult mammalian brain.
Coherent 40-Hz oscillation characterizes dream state in humans.
Authors: Llinás R , Ribary U
Published: May 2006

Magnetic recording from five normal human adults demonstrates large 40-Hz coherent magnetic activity in the awake and in rapid-eye-movement (REM) sleep states that is very reduced during delta sleep (deep sleep characterized by delta waves in the electroencephalogram). This 40-Hz magnetic oscillation has been shown to be reset by sensory stimuli in the awake state. Such resetting is not observed during REM or delta sleep. The 40 Hz in REM sleep is characterized, as is that in the awake state, by a fronto-occipital phase shift over the head. This phase shift has a maximum duration of approximately 12-13 msec. Because 40-Hz oscillation is seen in wakefulness and in dreaming, we propose it to be a correlate of cognition, probably resultant from coherent 40-Hz resonance between thalamocortical-specific and nonspecific loops. Moreover, we proposed that the specific loops give the content of cognition, and a nonspecific loop gives the temporal binding required for the unity of cognitive experience.
Experience-dependent plasticity in adult rat barrel cortex.
Authors: Diamond M E , Armstrong-James M , Ebner F F
Published: May 2006

This study tested the hypothesis that the receptive fields (RFs) of neurons in the adult sensory cortex are shaped by the recent history of sensory experience. Sensory experience was altered by a brief period of "whisker pairing": whiskers D2 and either D1 or D3 were left intact, while all other whiskers on the right side of the face were trimmed close to the fur. The animals were anesthetized 64-66 h later and the responses of single neurons in contralateral cortical barrel D2 to stimulation of whisker D2 (the center RF) and the four neighboring whiskers (D1, D3, C2, and E2; the excitatory surround RF) were measured. Data from 79 cells in four rats with whiskers paired were compared to data from 52 cells in four rats with untrimmed whiskers (control cases). During the period of whisker pairing, the RFs of cells in barrel D2 changed in three ways: (i) the response to the center RF, whisker D2, increased by 39%, (ii) the response to the paired surround RF whisker increased by 85-100%, and (iii) the response to all clipped (unpaired) surround RF whiskers decreased by 9-42%. In the control condition, the response of barrel D2 cells to the two neighboring whiskers, D1 and D3, was equal. After whisker pairing, the response to the paired neighbor of D2 was more than twice as large as the response to the cut neighbor of D2. These findings indicate that a brief change in the pattern of sensory activity can alter the configuration of cortical RFs, even in adult animals.
Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes.
Authors: Cristiano R J , Smith L C , Woo S L
Published: May 2006

We have combined a receptor-mediated DNA delivery system with the endosomal lysis ability of adenovirus and shown that DNA can be delivered into primary hepatocytes, resulting in a high level of gene expression. When asialoorosomucoid conjugated with poly(L-lysine) was used to deliver the Escherichia coli beta-galactosidase gene into primary hepatocytes through binding with the hepatic asialoglycoprotein receptor, only a low level of beta-galactosidase was detectable, with less than 0.1% of the hepatocytes being transfected. This level of activity can be greatly enhanced by the cointernalization of the DNA.protein complex with a replication-defective adenovirus, resulting in 100% of the hepatocytes staining blue with 5-bromo-4-chloro-3-indolyl beta-D-galactoside. Quantitative analysis of beta-galactosidase expression also showed a 1000-fold enhancement of activity. To test the applicability of this DNA delivery system for the correction of phenylketonuria, a metabolic disorder that causes severe mental retardation in children, we have delivered the human phenylalanine hydroxylase (PAH) gene to hepatocytes derived from a PAH-deficient mouse strain and demonstrated complete reconstitution of enzymatic activity. This method shows great promise for efficient gene delivery to the liver for correction of hepatic disorders.
Suppression of apoptosis in a cytotoxic T-cell line by interleukin 2-mediated gene transcription and deregulated expression of the protooncogene bcl-2.
Authors: Deng G , Podack E R
Published: May 2006

Absence of interleukin 2 (IL-2) from IL-2-dependent cells, such as the cytotoxic T-cell line CTLL2, causes DNA fragmentation and programmed cell death (apoptosis). We found that, upon initiation, DNA degradation proceeds rapidly. IL-2-deprived CTLL2 cells can be rescued from apoptosis by the addition of IL-2 2 h prior to the onset of detectable DNA breakdown. Addition of inhibitors of transcription with IL-2 abolished the IL-2-mediated rescue of CTLL2 cells. Thus it appears that IL-2-mediated gene transcription is necessary for survival. Deregulated expression of a protooncogene, bcl-2, inhibits apoptosis of cells dependent on other hematopoietic growth factors. To determine whether bcl-2 was active in CTLL2 cells, we transfected CTLL2 cells with a plasmid containing bcl-2 cDNA expressed under the metallothionein promoter and observed prolonged survival of the transfected cells upon IL-2 deprivation. Cell growth, however, was arrested in the G0/G1 or G2/M phases of the cell cycle. The prolonged survival of bcl-2 transfectants allowed the analysis of endogenous bcl-2 mRNA levels by Northern blot analysis. The expression of endogenous bcl-2 was down-regulated within 8 h of IL-2 withdrawal and was not detected after 3 days. Addition of IL-2 induced endogenous bcl-2 expression within 8 h. Full recovery of bcl-2 expression was achieved by 24 h after IL-2 addition. We conclude that the survival of death-prone CTLL2 cells may be viewed as IL-2-dependent suppression of suicide, probably by the IL-2-induced expression of the cellular bcl-2 gene.
Polymerization of murine macrophage inflammatory protein 1 alpha inactivates its myelosuppressive effects in vitro: the active form is a monomer.
Authors: Mantel C , Kim Y J , Cooper S , Kwon B , Broxmeyer H E
Published: May 2006

Macrophage inflammatory protein (MIP) 1 alpha has myelosuppressive and myeloprotective activity. That MIP-1 alpha polymerizes is known; this phenomenon was evaluated in terms of myelosuppression by assessing the effects of recombinant murine MIP-1 alpha on colony formation of murine and human myeloid progenitor cells in vitro. The following results are reported: (i) Polymerization is diluent- and concentration-dependent. (ii) Monomeric MIP-1 alpha is the active suppressive form for myeloid progenitor cells in vitro. (iii) Polymerized MIP-1 alpha is inactive and does not interfere with suppression by monomeric MIP-1 alpha. (iv) MIP-1 alpha has approximately 1000-fold higher specific activity than has been reported, but its effects are still specific for immature subsets of myeloid progenitors. (v) Suppression is initiated during the DNA-synthesis phase of the cell cycle. We conclude that polymerization of MIP-1 alpha might be a control mechanism that limits the myelosuppressive effects of monomeric MIP-1 alpha.
Structural biopolymer preservation in Miocene leaf fossils from the Clarkia site, northern Idaho.
Authors: Logan G A , Boon J J , Eglinton G
Published: May 2006

The 17- to 20-million-year-old locality at Clarkia, northern Idaho, is renowned for yielding amplifiable DNA from a magnolia leaf fossil. In-source pyrolysis-mass spectrometry and pyrolysis-gas chromatography/mass spectrometry now reveal that molecular preservation of biomacromolecules is highly selective; structural polysaccharides, cutin polyesters, and proteins were not preserved in detectable quantity in the leaf tissues, whereas both lignin and an aliphatic biopolymer were detected. This study points up the need for improved understanding of the precise modes and extent of preservation of biomacromolecules in fossil materials and sediments.
Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.
Authors: Bortell R , Owen T A , Shalhoub V , Heinrichs A , Aronow M A , Rochette-Egly C , Lutz Y , Stein J L , Lian J B , Stein G S
Published: May 2006

The bone-specific osteocalcin (OC) gene is transcribed only after completion of proliferation in normal diploid calvarial-derived osteoblasts during extracellular matrix mineralization. In contrast, the OC gene is expressed constitutively in both proliferating and nonproliferating ROS 17/2.8 osteosarcoma cells. To address molecular mechanisms associated with these tumor-related modifications in transcriptional control, we examined sequence-specific interactions of transactivation factors at key basal and hormone-responsive elements in the OC gene promoter. In ROS 17/2.8 cells compared to normal diploid osteoblasts, the absence of a stringent requirement for cessation of proliferation to support both induction of OC transcription and steroid hormone-mediated transcriptional modulation is reflected by modifications in transcription factor binding at (i) the two primary basal regulatory elements, the OC box (which contains a CCAAT motif as a central core) and the TATA/glucocorticoid-responsive element domain, and (ii) the vitamin D-responsive element. Particularly striking are two forms of the vitamin D receptor complex that are present in proliferating osteoblasts and osteosarcoma cells. Both forms of the complex are sensitive to vitamin D receptor antibody and retinoic X receptor antibody. After the down-regulation of proliferation, only the lower molecular weight complex is found in normal diploid osteoblasts. Both forms of the complex are present in nonproliferating ROS 17/2.8 cells with increased representation of the complex exhibiting reduced electrophoretic mobility that is phosphorylation-dependent.
Soluble tumor necrosis factor receptor: inhibition of human immunodeficiency virus activation.
Authors: Howard O M , Clouse K A , Smith C , Goodwin R G , Farrar W L
Published: May 2006

The inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) has been shown to stimulate human immunodeficiency virus type 1 (HIV-1) replication in both chronically and acutely infected T lymphocytes and monocytes. Transcriptional activation of the HIV long terminal repeat and subsequent increase in virus production are linked to TNF activation of the cellular transcription factor NF-kappa B. Here we report the use of two forms of soluble recombinant type 1 (p80) TNF receptor to inhibit TNF-induced HIV activation in vitro. One receptor form is a monomer containing the entire 236 residues of the extracellular (ligand-binding) portion of p80. A second receptor form is a chimeric homodimer containing these residues fused to a truncated human IgG1 immunoglobulin heavy chain and, thus, resembles a bivalent antibody without light chains. These recombinant receptor proteins were tested for their ability to inhibit TNF-alpha-induced expression of HIV-1 in chronically infected human cell lines. We also examined the ability of the soluble receptors to limit the activation of the HIV-long terminal repeat transcription. The soluble TNF receptor dimer was most effective at blocking TNF-alpha-induced HIV-1 expression in both monocytoid and lymphoid cells. The molar ratio of TNF-receptor dimer to TNF-alpha found to be most effective was, at least, 5:1. We conclude that at specific TNF/soluble TNF-receptor dimer ratios, TNF-alpha-induced HIV-1 transcription and expression can be limited in vitro.