bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of mannosylglucosaminylinositol phospholipids in normal but not paroxysmal nocturnal hemoglobinuria cells.
Authors: Hirose S , Ravi L , Prince G M , Rosenfeld M G , Silber R , Andresen S W , Hazra S V , Medof M E
Published: May 2006

To identify mannosyl (Man)-containing intermediates of the human glycoinositol phospholipid (GPI) anchor pathway and examine their expression in paroxysmal nocturnal hemoglobinuria (PNH), mannolipid products deriving from in vitro guanosine diphosphate [3H]Man labeling of HeLa cell microsomes were characterized. The defined GPI species were correlated with products deriving from in vivo [3H]Man labeling of normal and (GPI-anchor defective) affected leukocytes. In vitro analyses in HeLa cells showed dolichol-phosphoryl (Dol-P)-[3H]Man and a spectrum of [3H]Man lipids exhibiting TLC mobilities approximating those of Trypanosoma brucei (Tryp) GPI precursors. Iatrobead HPLC separations and partial characterizations of the major isolated [3H]Man species (designated H1-H8) showed that all but H1 (Dol-P-Man) were sensitive to HNO2 deamination and serum GPI-specific phospholipase D digestion but were resistant to phosphatidylinositol-specific phospholipase C digestion unless previously deacylated with mild alkali. [3H]Man label in H3, H4, and H6 but not in H5 or H7 was efficiently released into the aqueous phase by jack bean alpha-mannosidase digestion. BioGel P-4 and AX-5 sizing of the dephosphorylated core glycan fragments of H6 and H7 gave values that coincided precisely with the corresponding glycan fragments from the fully assembled Tryp anchor donor A' (P2). Affected leukocytes from four patients with PNH supported formation of GlcNAc- and GlcN-PI but all failed to express H6 and H7 as well as H8 and two showed complete absence of earlier Man-containing intermediates. These findings argue that human intracellular GPI mannolipids are built on acylated inositol phospholipids, that H6 and H7 contain differentially phosphoethanolamine-substituted Man3-GlcN-inositol cores, and that PNH cells are defective in conversion of GlcN-PI into these more mature mannolipid structures.
The deep, hot biosphere.
Authors: Gold T
Published: May 2006

There are strong indications that microbial life is widespread at depth in the crust of the Earth, just as such life has been identified in numerous ocean vents. This life is not dependent on solar energy and photosynthesis for its primary energy supply, and it is essentially independent of the surface circumstances. Its energy supply comes from chemical sources, due to fluids that migrate upward from deeper levels in the Earth. In mass and volume it may be comparable with all surface life. Such microbial life may account for the presence of biological molecules in all carbonaceous materials in the outer crust, and the inference that these materials must have derived from biological deposits accumulated at the surface is therefore not necessarily valid. Subsurface life may be widespread among the planetary bodies of our solar system, since many of them have equally suitable conditions below, while having totally inhospitable surfaces. One may even speculate that such life may be widely disseminated in the universe, since planetary type bodies with similar subsurface conditions may be common as solitary objects in space, as well as in other solar-type systems.
Differential regulation of T helper phenotype development by interleukins 4 and 10 in an alpha beta T-cell-receptor transgenic system.
Authors: Hsieh C S , Heimberger A B , Gold J S , O'Garra A , Murphy K M
Published: May 2006

To address the mechanisms controlling T helper (Th) phenotype development, we used DO10, a transgenic mouse line that expresses the alpha beta T-cell receptor from an ovalbumin-reactive T hybridoma, as a source of naive T cells that can be stimulated in vitro with ovalbumin peptide presented by defined antigen-presenting cells (APCs). We have examined the role of cytokines and APCs in the regulation of Th phenotype development. Interleukin 4 (IL-4) directs development toward the Th2 phenotype, stimulating IL-4 and silencing IL-2 and interferon gamma production in developing T cells. Splenic APCs direct development toward the Th1 phenotype when endogenous IL-10 is neutralized with anti-IL-10 antibody. The splenic APCs mediating these effects are probably macrophages or dendritic cells and not B cells, since IL-10 is incapable of affecting Th phenotype development when the B-cell hybridoma TA3 is used as the APC. These results suggest that early regulation of IL-4 and IL-10 in a developing immune response and the identity of the initiating APCs are critical in determining the Th phenotype of the developing T cells.
Transfer of multiple sclerosis into severe combined immunodeficiency mice by mononuclear cells from cerebrospinal fluid of the patients.
Authors: Saeki Y , Mima T , Sakoda S , Fujimura H , Arita N , Nomura T , Kishimoto T
Published: May 2006

To investigate the mode of the pathogenesis of multiple sclerosis (MS), we transferred cerebrospinal fluid (CSF) cells, predominantly mononuclear cells, from MS patients at both exacerbation and remission stages of the disease into severe combined immunodeficiency mice by intracisternal injection. As controls, (i) CSF cells from patients with cervical spondylosis and (ii) peripheral blood mononuclear cells from normal individuals were transferred. Four to 6 weeks after transfer, most mice transferred with CSF cells from MS patients at the exacerbation stage of the disease developed paralysis and ataxia. The histopathological examination on the sacrificed mice revealed multiple scattered, discrete lesions localized in the white matter of the brainstems and spinal cords. These lesions were characterized by various degrees of tissue necrosis, involving inflammatory-cell infiltration. Most infiltrating cells were macrophages, although a smaller number of granulocytes appeared in several foci. Reactive astrocytic gliosis was also seen around the necrotic foci. Furthermore, these lesions exhibited demyelination. These histopathological changes are similar to those seen in MS. In contrast, none of the severe combined immunodeficiency mice transferred with CSF cells from MS patients at the remission stage of the disease, or with CSF cells from the patients with cervical spondylosis, or with peripheral blood mononuclear cells from normal individuals showed any such histopathological changes. These observations provide convincing direct evidence of encephalitogenicity of mononuclear cells in CSF from MS patients at the exacerbation stage of the disease.
Characterization of a periplasmic thiol:disulfide interchange protein required for the functional maturation of secreted virulence factors of Vibrio cholerae.
Authors: Peek J A , Taylor R K
Published: May 2006

A number of ToxR-regulated genes that encode products required for the biogenesis or function of the toxin-coregulated colonization pilus (TCP) of Vibrio cholerae have been identified previously by TnphoA fusions. In this study we have examined the role of the product of one of these genes, tcpG, to which a fusion results in a piliated cell lacking all of the in vivo and in vitro functions associated with TCP. Our results show that TcpG is not an ancillary pilus adhesin component as suggested by the mutant phenotype but instead is a 24-kDa periplasmic protein that shares active-site homology with several different bacterial thioredoxins and protein disulfide isomerase, as well as overall homology with the disulfide bond-forming DsbA periplasmic oxidoreductase protein of E. coli. Corresponding activity can be demonstrated in vitro for TcpG-enriched fractions from a wild-type strain but is absent in a similarly fractionated tcpG-phoA mutant. The phenotype conferred by a tcpG mutation was found to be pleiotropic in nature, also affecting the extracellular secretion of cholera toxin A subunit and a major protease. This suggests a general role for TcpG in allowing a group of virulence-associated (and perhaps other) proteins that contain disulfide bonds to assume a secretion or functionally competent state.
Purification and identification of bovine liver gamma-carboxylase.
Authors: Berkner K L , Harbeck M , Lingenfelter S , Bailey C , Sanders-Hinck C M , Suttie J W
Published: May 2006

The microsomal gamma-carboxylase catalyzes modification of a limited set of glutamyl residues to gamma-carboxyglutamyl residues in a vitamin K-dependent reaction that also utilizes O2 and CO2. We report the purification to apparent homogeneity of the bovine liver microsomal carboxylase. Affinity chromatography exploiting the association of the carboxylase with prothrombin precursor and carboxylase binding to the propeptide sequence were combined with ion-exchange chromatography and fractionation using immobilized lectins. A 3.5 x 10(5)-fold purification was obtained, which is the highest purification, by a factor of 35, yet reported for this enzyme. A single 98-kDa protein is obtained from this isolation. Carboxylase activity is associated with this protein by two different criteria. Antibodies prepared against the carboxylase detected the 98-kDa protein when used in Western analysis. In addition, the single 98-kDa protein was shown to comigrate with activity when electrophoresed in a nondenaturing gel system. The availability of purified preparations of carboxylase will facilitate an increased understanding of the complex biochemical reaction carried out by this protein.
Stable expression of mammalian type A gamma-aminobutyric acid receptors in mouse cells: demonstration of functional assembly of benzodiazepine-responsive sites.
Authors: Hadingham K L , Harkness P C , McKernan R M , Quirk K , Le Bourdellès B , Horne A L , Kemp J A , Barnard E A , Ragan C I , Whiting P J
Published: May 2006

The differential sensitivity of type A gamma-aminobutyric acid (GABAA) receptors to benzodiazepine ligands seen in the mammalian nervous system is thought to be generated by the existence of a number of different receptor subtypes, assembled from a range of closely related subunits (alpha 1-6, beta 1-3, gamma 1-3, and delta) encoded by discrete genes. The characteristics of a given subtype can be determined by the coexpression of cloned cDNAs encoding the subunits of interest. Two transient expression systems have so far been employed in the study of the ligand-binding characteristics and chloride channel properties of such GABAA receptors--Xenopus oocytes and transfected mammalian cells. Here we report on the use of a steroid-inducible promoter expression system for the production of a permanently transfected clonal cell line expressing the alpha 1 beta 1 gamma 2L GABAA receptor subtype. Using both immunoprecipitation by subunit-specific antisera and gel-exclusion chromatography, we have shown that the alpha 1, beta 1, and gamma 2L subunits coassemble to form receptor macromolecules that are of the same size as native GABAA receptors. Additionally, the recombinant receptors have the same benzodiazepine pharmacology as native alpha 1-containing GABAA receptors and function as GABA-gated chloride channels. Such cell lines expressing individual GABAA receptor subtypes will prove important tools in the study of the structure, function, and pharmacology of GABAA receptors and in the development of subtype-specific drugs.
Localization of a DNA repair gene (XRCC5) involved in double-strand-break rejoining to human chromosome 2.
Authors: Jeggo P A , Hafezparast M , Thompson A F , Broughton B C , Kaur G P , Zdzienicka M Z , Athwal R S
Published: May 2006

Complementation of the repair defect in hamster xrs mutants has been achieved by transfer of human chromosome 2 using the method of microcell-mediated chromosome transfer. The xrs mutants belong to ionizing radiation complementation group 5, are highly sensitive to ionizing radiation, and have an impaired ability to rejoin radiation-induced DNA double-strand breaks. Both phenotypes were corrected by chromosome 2, although the correction of radiation sensitivity was only partial. Complementation was achieved in two members of this complementation group, xrs6 and XR-V15B, derived independently from the CHO and V79 cell lines, respectively. The presence of human chromosome 2 in complemented clones was examined cytogenetically and by PCR analysis with primers directed at a human-specific long interspersed repetitive sequence or chromosome 2-specific genes. Complementation was observed in 25/27 hybrids, one of which contained only the q arm of chromosome 2. The two noncomplementing hybrids were missing segments of chromosome 2. The use of a back-selection system enabled the isolation of clones that had lost the human chromosome and these regained radiation sensitivity. Transfer of several other human chromosomes did not result in complementation of the repair defect in XR-V15B. These data show that the gene defective in xrs cells, XRCC5, which is involved in double-strand break rejoining, is located on human chromosome 2q.
Photoinduced electron transfer from tris(2,2'-bipyridyl)ruthenium to cytochrome c oxidase.
Authors: Nilsson T
Published: May 2006

Flash photolysis has been used to effect electron transfer from tris(2,2'-bipyridyl)ruthenium(II) to cytochrome c oxidase in the presence of a sacrificial electron donor, aniline. The observation that photoreduction occurs only at low ionic strength and high pH indicates that an electrostatic complex between the ruthenium compound and the enzyme is the reactive species. The reaction was followed at 830, 605, and 445 nm. The initial absorbance changes observed suggest that the copper ion CuA is the preferred electron acceptor and that electron transfer from the excited ruthenium complex takes place in less than 1 microsecond. Some rapid cytochrome a reduction is also observed. Absorbance changes after the initial transients suggest that the reduced CuA then equilibrates with cytochrome a with a rate constant of 2 x 10(4) s-1. Comparison of the absorbance changes at 605 and 445 nm and the kinetic difference spectrum in the Soret region indicate that no reduction of cytochrome a3 takes place. With the oxidized enzyme, no further reactions were detected, whereas, in the peroxide and ferryl intermediates, cytochrome a reoxidizes on a millisecond time scale. The reaction appears biphasic in both intermediates, with rate constants in the range 2 x 10(2) to 4 x 10(3) s-1. This is considerably slower than the maximal rates observed for electron transfer between cytochrome a and the bimetallic site found in earlier work and suggests rate limitation by other processes. The rates obtained for the slower phases are close to the rate for catalysis of cytochrome c oxidation.
Intravesicular acidification correlates with binding of ADP-ribosylation factor to microsomal membranes.
Authors: Zeuzem S , Feick P , Zimmermann P , Haase W , Kahn R A , Schulz I
Published: May 2006

The ADP-ribosylation factor (ARF), a highly conserved low molecular weight GTP-binding protein, has been implicated to function in intracellular protein transport to and within the Golgi complex. In pancreatic acinar cells the ARF is confined to the cytoplasmic faces of trans-Golgi stack membranes, a compartment known to maintain a low intravesicular pH, which is established by a chloride-dependent MgATP-driven proton pump. The present study shows that MgATP (2mM), but neither adenosine 5'-[gamma-thio]triphosphate in the presence of Mg2+ nor ATP in the absence of Mg2+, increases transfer of ARF from the surrounding medium into the vesicle membranes. The specific vacuolar-type proton pump inhibitor bafilomycin B1 (10 nM), the protonophore carbonylcyanide m-chlorophenylhydrazone (10 microM), and replacement of chloride in the incubation buffer by acetate or nitrate resulted in an almost complete inhibition of the MgATP-dependent association of ARF to the vesicle membranes. The results demonstrate that redistribution of ARF to the vesicle membrane correlates with the intravesicular pH established by a vacuolar-type H(+)-ATPase. The intravesicular pH appears to be one mechanism by which certain low molecular weight GTP-binding proteins become relocated from the cytosol to their specific membrane vesicles.
Coregulation of purine and histidine biosynthesis by the transcriptional activators BAS1 and BAS2.
Authors: Daignan-Fornier B , Fink G R
Published: May 2006

We have found cross-pathway regulation between purine and histidine biosynthesis in yeast. The transcription factors BAS1 and BAS2/PHO2, which are also regulators of the histidine pathway, participate in the regulation of the purine biosynthetic pathway. Analysis of four genes of the purine pathway (ADE1, ADE2, ADE5,7, and ADE8) shows that their expression is repressed by adenine. The maximal basal and induced expression of these purine genes requires the presence of both BAS1 and BAS2. The factor BAS1 has been shown to bind at a site containing the TGACTC hexanucleotide motif in the ADE2 and ADE5,7 promoters. This motif is required for both basal and induced activation of the ADE2 gene by BAS1 and BAS2.
Neurosteroids: pregnenolone in human sciatic nerves.
Authors: Morfin R , Young J , Corpéchot C , Egestad B , Sjövall J , Baulieu E E
Published: May 2006

The characterization and quantification of pregnenolone in human sciatic nerves were undertaken, following previous demonstration of the synthesis of this steroid in rat brain oligodendrocytes, to explore the hypothesis that Schwann cells may demonstrate the same biosynthetic activity. Pregnenolone was definitively identified by mass spectrometry and quantified by specific radioimmunoassay. Its concentration (mean +/- SD, 63.9 +/- 45.9 ng/g of wet tissue, n = 12) was greater than or equal to 100 times the plasma level and concentration found in tendons and muscle. No correlation was found with sex or age. Free dehydroepiandrosterone as well as sulfate and fatty acid esters of pregnenolone and dehydroepiandrosterone were also measured. Results are discussed in terms of the concept that these "neurosteroids" may be synthesized in the peripheral nervous system.
Unequal human immunodeficiency virus type 1 reverse transcriptase error rates with RNA and DNA templates.
Authors: Boyer J C , Bebenek K , Kunkel T A
Published: May 2006

Sequence variation in the type 1 human immunodeficiency virus (HIV-1) results, in part, from inaccurate replication by reverse transcriptase. Although this enzyme is error-prone during synthesis in vitro with DNA templates, the fidelity of RNA-dependent DNA synthesis relevant to minus-strand replication in the virus life cycle has not been examined extensively. In the present study, we have developed a system to determine the fidelity of transcription and reverse transcription and have used it to compare the fidelity of DNA synthesis by the HIV-1 reverse transcriptase with RNA and DNA templates of the same sequence. Overall, fidelity was several-fold higher with RNA than with DNA. Sequence analysis of mutants generated with the two substrates revealed that differences in error rates were substantial for specific errors. Fidelity with RNA was greater than 10-fold higher for substitution and minus-one nucleotide errors at five different homopolymeric positions. Because such errors likely result from template-primer slippage, this result suggests that misaligned intermediates are formed and/or used less frequently with an RNA template-DNA primer than with a DNA template-DNA primer. The results also suggest that HIV-1 reverse transcriptase synthesis with an RNA template-DNA primer was error-prone during incorporation of the first two nucleotides, perhaps due to aberrant enzyme-substrate interactions as synthesis initiates. The unequal error rates with RNA and DNA templates suggest that mistakes during minus- and plus-strand DNA synthesis may not contribute equally to the mutation rate of HIV-1. The data also provide estimates of substitution and frameshift error rates during transcription by T7 RNA polymerase.
New transients in the electron-transfer dynamics of photolyzed mixed-valence CO-cytochrome c oxidase.
Authors: Einarsdóttir O , Dawes T D , Georgiadis K E
Published: May 2006

Electron transfer following photolysis of CO from mixed-valence (cytochrome a3+ Cu2+A cytochrome a2+3-CO Cu+B) cytochrome oxidase (ferrocytochrome-c; oxygen oxidoreductase, EC 1.9.3.1) was studied on time scales of nanoseconds to milliseconds by multichannel time-resolved optical absorption spectroscopy. In this method, the optical absorption was measured at many wavelengths simultaneously by using an optical spectrometric multichannel analyzer system. The high-quality time-resolved difference spectra showed a large increase on a microsecond time scale in the visible region centered at approximately 520 nm and in the UV region centered at approximately 390 nm. These absorbance changes were not observed after photodissociation of CO from the fully reduced enzyme and therefore are attributed to intramolecular electron transfer. Simultaneously, there was a blue shift and a small increase in the alpha band, which is attributed to the reduction of cytochrome alpha. Approximately one-third of the absorbance change at 520 nm can be attributed to reduction of cytochrome a. The absorbance changes associated with the 520- and the 390-nm bands are on the same time scale (t1/2 approximately 2 microseconds) as the dissociation of CO from Cu+B reported previously by time-resolved infrared spectroscopy. The position and shape of these bands are reasonable for charge-transfer transitions involving copper(II). We suggest that the absorbance increase at 520 nm, which cannot be attributed to a reduction of cytochrome a, may represent a charge transfer involving Cu2+B accompanying the oxidation of Cu+B to Cu2+B. The absorbance increase at 390 nm is also partially attributed to this transition. These results suggest that Cu2+B may be observed spectrophotometrically in the electron-transfer dynamics of cytochrome oxidase.
Gravitational symmetry breaking in microtubular dissipative structures.
Authors: Tabony J , Job D
Published: May 2006

Reduction-diffusion theories can account for both morphogenesis and the sensitivity of biological systems to weak fields. They predict that gravity can cause the symmetry breaking that is necessary for pattern formation. Microtubules play an important role in organizing the cell, and recent studies hae shown that they can form in vitro dissipative structures. We have found that these structures show patterns of microtubular orientation that are gravity dependent and that the gravitational field causes symmetry breaking. This behavior, which cannot be explained by convection, is in accordance with the theory of dissipative structures. These results suggest that microtubular dissipative structures may play an important role both in morphogenesis and in accounting for the sensitivity of biological systems to weak fields. They aso provide another explanation for biological gravitropism.
RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase.
Authors: Tuerk C , MacDougal S , Gold L
Published: May 2006

High-affinity ligands of the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) were isolated by the SELEX procedure (systematic evolution of ligands by exponential enrichment) from RNA populations randomized at 32 positions. Analysis of these ligands revealed a pseudoknot consensus with primary sequence bias at some positions. We demonstrated that at least one of the ligands inhibits cDNA synthesis by HIV reverse transcriptase but fails to inhibit other reverse transcriptases. These experiments highlight the power of SELEX to yield highly specific ligands that reduce the activity of target proteins. Such ligands may provide therapeutic reagents for viral and other diseases.
Inhibition of thymocyte apoptosis and negative antigenic selection in bcl-2 transgenic mice.
Authors: Siegel R M , Katsumata M , Miyashita T , Louie D C , Greene M I , Reed J C
Published: May 2006

The bcl-2 gene, which is overexpressed in human follicular B-cell lymphomas, has been found to extend cellular lifespan through inhibition of apoptosis, or programmed cell death. However, the physiological role of the Bcl-2 protein in lymphocyte development is unclear. We have established a transgenic mouse line that expresses high levels of the Bcl-2 protein in both cortical and medullary thymocytes, disrupting the normal pattern of expression of this gene. We found that in these mice, immature thymocytes became resistant to apoptosis mediated by corticosteroids and calcium ionophores. Untreated thymocytes also exhibited a survival advantage in suspension cultures compared with controls. In addition, overexpression of bcl-2 enabled a proportion of thymocytes and peripheral T cells to escape the process of clonal deletion, which normally eliminates self-reactive T cells during thymocyte maturation. These findings implicate the Bcl-2 protein in regulating the lifespan of maturing thymocytes and in the antigenic-selection process.
Partial loss of function mutations in DnaK, the Escherichia coli homologue of the 70-kDa heat shock proteins, affect highly conserved amino acids implicated in ATP binding and hydrolysis.
Authors: Wild J , Kamath-Loeb A , Ziegelhoffer E , Lonetto M , Kawasaki Y , Gross C A
Published: May 2006

A set of 37 mutations in DnaK, the Escherichia coli homologue of the 70-kDa heat shock proteins, was isolated using a selection for high constitutive expression of heat shock proteins. Of these, 11 mutants were able to carry out some but not all functions of DnaK. These partial function mutants were divided into two classes. Class I mutants are recessive and permit replication of bacteriophage lambda and growth of cells up to 40 degrees C. Class II mutants are dominant, do not permit growth of lambda, and are temperature-sensitive for growth above 34 degrees C. Mutations in both classes alter amino acids that are highly conserved in the 70-kDa heat shock protein family. The dominant negative mutations provide strong genetic evidence that at least one form of DnaK is multimeric. Moreover, every dominant negative mutation occurs at an amino acid that has been hypothesized to be intimately involved in the process of ATP binding and hydrolysis. Our findings provide strong support for the hypothesis that such mutations are excellent tools for identifying amino acids that play critical roles in protein function.
Biochemical complementation of chalcone synthase mutants defines a role for flavonols in functional pollen.
Authors: Mo Y , Nagel C , Taylor L P
Published: May 2006

Chalcone synthase catalyzes the initial step of that branch of the phenylpropanoid pathway that leads to flavonoids. A lack of chalcone synthase activity has a pleiotropic effect in maize and petunia mutants: pollen fertility as well as flavonoid synthesis is disrupted. Both maize and petunia mutants are self-sterile due to a failure to produce a functional pollen tube. The finding that the mutant pollen is partially functional on wild-type stigmas led to the isolation and identification of kaempferol as a pollen germination-inducing constituent in wild-type petunia stigma extracts. We show that adding micromolar quantities of kaempferol to the germination medium or to the stigma at pollination is sufficient to restore normal pollen germination and tube growth in vitro and full seed set in vivo. Further we show that the rescue ability resides in particular structural features of a single class of compounds, the flavonol aglycones. This finding identifies another constituent of plant reproduction and suggests that addition or removal of the flavonol signal during pollen germination and tube growth provides a feasible way to control plant fertility.
Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion.
Authors: Moll U M , Riou G , Levine A J
Published: May 2006

Twenty-seven cases of inflammatory breast cancer were screened for the presence of the p53 protein by immunocytochemical methods using a monoclonal antibody directed against the p53 protein. Three groups were detected: 8 cases (30%) had high levels of p53 in the nucleus of the cancer cells; 9 cases (33%) had a complete lack of detectable staining; 10 cases (37%) showed a pattern of cytoplasmic staining with nuclear sparing. Nucleotide sequence analysis of p53 cDNAs derived from the samples with cytoplasmic staining revealed only wild-type p53 alleles in 6 out of 7 cases. An eighth case was determined to be wild type by a single-strand conformation polymorphism. In contrast, the samples containing nuclear p53 contained a variety of missense mutations and a nonsense mutation. The p53 cDNAs from 3 of the tumors that lacked detectable p53 staining were analyzed, and all 3 had wild-type nucleotide sequences. Interestingly, a case of normal lactating breast tissue also showed intense cytoplasmic staining for p53 with nuclear sparing. These data suggest that some breast cancers that contain the wild-type form of p53 protein may inactivate its tumor-suppressing activity by sequestering this protein in the cytoplasm, away from its site of action in the cell nucleus. The detection of cytoplasmic p53 in normal lactating breast tissue could suggest that this is the mechanism employed in specific physiological situations to permit transient cell proliferation. This observation could explain how some breast cancer tissues inactivate p53 function without mutation.
Specificity of antisense oligonucleotides in vivo.
Authors: Woolf T M , Melton D A , Jennings C G
Published: May 2006

Antisense oligonucleotides are widely used as inhibitors of gene expression in cultured cells and have been proposed as potential therapeutic agents, but it is not known to what extent they are specific for their intended target RNAs. Statistical considerations indicate that if oligonucleotides can form hybrids with mRNA molecules in vivo by means of short or imperfect regions of complementarity, then the specificity of oligonucleotides as antisense reagents will be greatly compromised. We have used Xenopus oocytes as a model system in which to investigate the potential specificity of antisense oligonucleotides in vivo. We injected perfect and partially matched antisense oligonucleotides into oocytes and measured the resulting degradation of the target RNA in each case. On the basis of the extent to which antisense oligonucleotides can cause cleavage of RNAs at imperfectly matched target sites, we conclude that in this system it is probably not possible to obtain specific cleavage of an intended target RNA without also causing at least the partial destruction of many nontargeted RNAs.
The T-DNA-linked VirD2 protein contains two distinct functional nuclear localization signals.
Authors: Tinland B , Koukolíková-Nicola Z , Hall M N , Hohn B
Published: May 2006

Agrobacterium tumefaciens causes neoplastic growth in plants by transferring a piece of DNA, called T-DNA, into the nucleus of the plant cell. The virulence protein VirD2 of A. tumefaciens is tightly linked to the T-DNA and is thought to direct it to the plant genome. Here we show that the VirD2 protein contains two nuclear localization signals that are functional both in yeast and in plant cells. One signal is located in the N-terminal part of the protein and resembles a single-cluster-type nuclear localization signal. The second signal is near the C terminus and is a bipartite-type nuclear localization signal. The involvement of these sequences in the entry of the T-DNA into the nucleus is discussed.
Purification and characterization of the diphtheria toxin repressor.
Authors: Schmitt M P , Twiddy E M , Holmes R K
Published: May 2006

The diphtheria toxin repressor gene (dtxR) encodes a protein (DtxR) that regulates transcription of the diphtheria toxin gene (tox) by an iron-dependent mechanism. Cloned dtxR was expressed in Escherichia coli from the phage T7 gene 10 promoter, and DtxR was purified. Specific binding of DtxR to the tox+ operator was dependent on reduction of DtxR and the presence of ferrous ions. DtxR protected a sequence of approximately 30 nucleotide pairs, partially overlapping the tox promoter and containing a region of dyad symmetry, from digestion by DNase I. DtxR exhibited very little binding to the mutant tox-201 operator region and failed to bind to the promoter/operator region of the ferric uptake regulation (fur) gene of E. coli.
Immunophotodiagnosis of colon carcinomas in patients injected with fluoresceinated chimeric antibodies against carcinoembryonic antigen.
Authors: Folli S , Wagnières G , Pèlegrin A , Calmes J M , Braichotte D , Buchegger F , Chalandon Y , Hardman N , Heusser C , Givel J C
Published: May 2006

Based on previous experiments in nude mice, showing that fluoresceinated monoclonal antibodies against carcinoembryonic antigen localized specifically in human carcinoma xenografts and could be detected by laser-induced fluorescence, we performed a feasibility study to determine whether this immunophotodiagnosis method could be applied in the clinic. Six patients, with known primary colorectal carcinoma, received an i.v. injection of 4.5 or 9 mg of mouse-human chimeric anti-carcinoembryonic antigen monoclonal antibody coupled with 0.10-0.28 mg of fluorescein (molar ratio 1/10 to 1/14). The monoclonal antibody was also labeled with 0.2-0.4 mCi of 125I (1 Ci = 37 GBq). Photodetection of the tumor was done ex vivo on surgically resected tissues for the six patients and in vivo by fluorescence rectosigmoidoscopy for the sixth patient. Upon laser irradiation, clearly detectable heterogeneous green fluorescence from the dye-antibody conjugate was visually observed on all six tumors; almost no such fluorescence was detectable on normal mucosa. The yellowish tissue autofluorescence, which was emitted from both tumor and normal mucosa, could be subtracted by real-time image processing. Radioactivity measurements confirmed the specificity of tumor localization by the conjugate; tissue concentrations of up to 0.059% injected dose per g of tumor and 10 times less (0.006%) per g of normal mucosa were found. The overall results demonstrate the feasibility of tumor immunophotodiagnosis at the clinical level.
How reverse turns may mediate the formation of helical segments in proteins: an x-ray model.
Authors: Perczel A , Foxman B M , Fasman G D
Published: May 2006

The three-dimensional structure of a protein is the assembly of different secondary structural elements, such as alpha-helices, beta-pleated sheets, and beta-turns. Although the conformation of hundreds of proteins has been elaborated in the solid state, only a vague understanding of the mechanism of their conformational folding is known. One facet of this topic is the conformational interconversion of one or more beta-turns to a helical structure (and vice versa), which may also be related to the formation of helix-turn-helix motifs often observed in globular proteins. Based on a comprehensive structural analysis of proteins, Sundaralingam and Sekharudu [Sundaralingam, M. & Sekharudu, Y. C. (1989) Science 244, 1333-1337] previously suggested that "structure-water" molecules in proteins may mediate such a conformational change. An x-ray crystal structure determination of t-butoxycarbonyl (Boc)-Val-Ser-NHCH3 reveals (i) an ideal type I beta-turn backbone conformation and (ii) a hydrogen-bond network more typical of an alpha-helix than a beta-turn conformation. The molecular packing of this simple beta-turn model reported here provides a plausible and simple alternative of how a beta-turn-like conformation may serve as a conformational template for helical-structure formation (and vice versa) during the folding procedure.
Alloalbuminemia in Sweden: structural study and phenotypic distribution of nine albumin variants.
Authors: Carlson J , Sakamoto Y , Laurell C B , Madison J , Watkins S , Putnam F W
Published: May 2006

Plasma samples exhibiting alloalbuminemia on electrophoresis at pH 8.6 were requested from clinical laboratories throughout Sweden. Nine variants, each representing a different single point mutation, were found in 100 apparently unrelated Swedes. The overall prevalence of alloalbuminemia was estimated at 1:1700. Mutations were identified by protein-structural analysis followed by allele-specific DNA hybridization to verify the most common types. Slightly retarded (+1) mobility was seen in 80 cases. Of these, 71 had the Arg(-2)----Cys proalbumin variant previously called Malmö I proalbumin. Thirteen examples of the second most frequent type, the substitution Lys313----Asn and a mobility change of -1 charge unit, were found, as well as six cases of Glu570----Lys (albumin B) and a single case of Arg-1----Gln (proalbumin Christchurch). Five previously unreported types of alloalbuminemia were identified: four instances of Glu376----Gln, which is the second known mutation at this site; two examples of Asp550----Ala, the second mutation reported at this site; and one example each of Asp63----Asn, Gln268----Arg, and Asn318----Lys. Other mutations were identified among eight subjects of foreign descent. The high frequency and relatively uniform geographic distribution of the Arg-2----Cys mutation suggest that it may have occurred in a founder individual many generation ago in Sweden.
Yeast artificial chromosomes for the molecular analysis of the familial polyposis APC gene region.
Authors: Hampton G M , Ward J R , Cottrell S , Howe K , Thomas H J , Ballhausen W G , Jones T , Sheer D , Solomon E , Frischauf A M
Published: May 2006

Two yeast artificial chromosomes (YACs) spanning a total distance of 1.1 megabase pairs of DNA around the MCC (for mutated in colorectal carcinoma) and APC (for adenomatous polyposis coli) genes at 5q21 have been isolated and characterized. Starting from the MCC gene, a strategy was undertaken to identify constitutional submicroscopic deletions in familial adenomatous polyposis patients that might considerably narrow down the position of the APC gene. To this end, YACs identified by the MCC gene were screened across a chromosome 5-specific cosmid library to provide a source of DNA probes for genomic scanning. The cosmids isolated from these experiments were used to screen a panel of somatic cell hybrids containing chromosome 5 segregated from patients suspected to carry putative interstitial deletions. This screening approach led to the confirmation of a small heterozygous deletion in a polyposis patient that overlaps one of the two isolated YACs. This YAC has been shown to contain the entire APC gene, in addition to a significant portion of DNA flanking the 5' end of the gene, and should therefore prove a valuable resource for functional studies by transfer to colorectal tumor-derived cell lines.
Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses.
Authors: Koonin E V , Gorbalenya A E , Purdy M A , Rozanov M N , Reyes G R , Bradley D W
Published: May 2006

Computer-assisted comparison of the nonstructural polyprotein of hepatitis E virus (HEV) with proteins of other positive-strand RNA viruses allowed the identification of the following putative functional domains: (i) RNA-dependent RNA polymerase, (ii) RNA helicase, (iii) methyltransferase, (iv) a domain of unknown function ("X" domain) flanking the papain-like protease domains in the polyproteins of animal positive-strand RNA viruses, and (v) papain-like cysteine protease domain distantly related to the putative papain-like protease of rubella virus (RubV). Comparative analysis of the polymerase and helicase sequences of positive-strand RNA viruses belonging to the so-called "alpha-like" supergroup revealed grouping between HEV, RubV, and beet necrotic yellow vein virus (BNYVV), a plant furovirus. Two additional domains have been identified: one showed significant conservation between HEV, RubV, and BNYVV, and the other showed conservation specifically between HEV and RubV. The large nonstructural proteins of HEV, RubV, and BNYVV retained similar domain organization, with the exceptions of relocation of the putative protease domain in HEV as compared to RubV and the absence of the protease and X domains in BNYVV. These observations show that HEV, RubV, and BNYVV encompass partially conserved arrays of distinctive putative functional domains, suggesting that these viruses constitute a distinct monophyletic group within the alpha-like supergroup of positive-strand RNA viruses.
Chemotactic sorting to cAMP in the multicellular stages of Dictyostelium development.
Authors: Traynor D , Kessin R H , Williams J G
Published: May 2006

Dictyostelium transformants that overproduce the extracellular form of cyclic nucleotide phosphodiesterase and so accumulate a reduced amount of cAMP are blocked in development after aggregation in the form of a tight mound, prior to formation of the apical tip. In such mounds, prespore cell differentiation is repressed, and the apical accumulation of prestalk cells is greatly retarded. When a source of cAMP is placed below the arrested mounds, prestalk cells that would normally migrate in an apical direction instead sort downwards to the substratum. Thus, by acting as the chemoattractant that draws prestalk cells to the apex, cAMP signaling directs the formation of a patterned structure.
Induction of tolerance to an IgG autoantibody.
Authors: Offen D , Spatz L , Escowitz H , Factor S , Diamond B
Published: May 2006

Nonautoimmune mice transgenic for the heavy chain of an IgG2b anti-double-stranded-DNA antibody express the transgene in lymphoid organs and display partial allelic exclusion of this gamma 2b transgene. The spleens of these mice are characterized by marked B-cell depletion. Although there are B cells in these mice that express the transgene and recognize double-stranded DNA, they are anergic in vivo. Recovery from the state of anergy occurs in vitro after lipopolysaccharide stimulation. Thus this transgenic model demonstrates the induction of self tolerance to an IgG autoantibody.
The endothelin-converting enzyme from human umbilical vein is a membrane-bound metalloprotease similar to that from bovine aortic endothelial cells.
Authors: Ahn K , Beningo K , Olds G , Hupe D
Published: May 2006

A phosphoramidon-sensitive, membrane-bound metalloprotease that cleaves big endothelin 1 (big-ET-1) to ET-1 was obtained from human umbilical vein endothelial cells and also from bovine aortic endothelial cells by isolation of plasma-membrane vesicles free of lysosomes. The enzyme was characterized by RIA with an antibody specific for ET-1 and also by reverse-phase HPLC. For both sources, the pH rate profile of the membrane fraction had a very sharp maximum at pH 7.0; little or no activity was seen at more acidic pH values. In contrast, the cytosolic fraction had a major peak at acidic pH values, as well as a broad peak in the neutral region. The activity at pH 7.0 in the membrane fraction was shown by reverse-phase HPLC to produce ET-1 and C-terminal fragment as products. This activity was abolished by phosphoramidon, EDTA, and 1,10-phenanthroline but was not inhibited by pepstatin A, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, leupeptin, or E-64--consistent with the characteristics of a metalloprotease. These results suggest that this activity is from the physiologically relevant, phosphoramidon-inhibitable, endothelin-converting enzyme. The activity found at neutral pH values in the cytosolic fraction was only partially inhibited by EDTA and 1,10-phenanthroline but was not inhibited by phosphoramidon. The membrane-bound endothelin-converting enzyme from human umbilical vein endothelial cells and bovine aortic endothelial cells showed marked similarities, including IC50 values for phosphoramidon of 2.7 and 1.8 microM and Km values for big-ET-1 of 45.4 and 20.9 microM, respectively. The apparent molecular mass by gel filtration was approximately 300-350 kDa for the enzyme from either source. This report characterizes human endothelin-converting enzyme, which may be an important therapeutic target for cardiovascular disease.
Triple-helix formation by oligonucleotides containing the three bases thymine, cytosine, and guanine.
Authors: Giovannangéli C , Rougée M , Garestier T , Thuong N T , Hélène C
Published: May 2006

A homopurine-homopyrimidine sequence of human immunodeficiency virus (HIV) proviral DNA was chosen as a target for triple-helix-forming oligonucleotides. An oligonucleotide containing three bases (thymine, cytosine, and guanine) was shown to bind to its target sequence under physiological conditions. This oligonucleotide is bound in a parallel orientation with respect to the homopurine sequence. Thymines recognize A.T base pairs to form T.A.T base triplets and guanines recognize a run of G.C base pairs to form G.G.C base triplets. A single 5-methylcytosine was shown to stabilize the triple helix when incorporated in a stretch of thymines; it recognizes a single G.C base pair in a run of A.T base pairs. These results provide some of the rules required for choosing the more appropriate oligonucleotide sequence to form a triple helix at a homopurine-homopyrimidine sequence of duplex DNA. A psoralen derivative attached to the oligonucleotide containing thymine, 5-methylcytosine, and guanine was shown to photoinduce cross-linking of the two DNA strands at the target sequence in a plasmid containing part of the HIV proviral DNA sequence. Triplex formation and cross-linking were monitored by inhibition of Dra I restriction enzyme cleavage. The present results provide a rational basis for the development of triplex-forming oligonucleotides targeted to specific sequences of the HIV provirus integrated in its host genome.
Protein folding funnels: a kinetic approach to the sequence-structure relationship.
Authors: Leopold P E , Montal M , Onuchic J N
Published: May 2006

A lattice model of protein folding is developed to distinguish between amino acid sequences that do and do not fold into unique conformations. Although Monte Carlo simulations provide insights into the long-time processes involved in protein folding, these simulations cannot systematically chart the conformational energy surface that enables folding. By assuming that protein folding occurs after chain collapse, a kinetic map of important pathways on this surface is constructed through the use of an analytical theory of probability flow. Convergent kinetic pathways, or "folding funnels," guide folding to a unique, stable, native conformation. Solution of the probability flow equations is facilitated by limiting treatment to diffusion between geometrically similar collapsed conformers. Similarity is measured in terms of a reconfigurational distance. Two specific amino acid sequences are deemed foldable and nonfoldable because one gives rise to a single, large folding funnel leading to a native conformation and the other has multiple pathways leading to several stable conformers. Monte Carlo simulations demonstrate that folding funnel calculations accurately predict the fact of and the pathways involved in folding-specific sequences. The existence of folding funnels for specific sequences suggests that geometrically related families of stable, collapsed conformers fulfill kinetic and thermodynamic requirements of protein folding.
A defective replicase gene induces resistance to cucumber mosaic virus in transgenic tobacco plants.
Authors: Anderson J M , Palukaitis P , Zaitlin M
Published: May 2006

Nicotiana tabacum cv. Turkish Samsun NN plants were transformed with a modified and truncated replicase gene encoded by RNA-2 of cucumber mosaic virus strain Fny. The replicase gene had been modified by deleting a 94-base-pair region spanning nucleotides 1857-1950; the deletion also caused a shift in the open reading frame, resulting in a truncated translation product approximately 75% as large as the full-length protein. Upon transformation via Agrobacterium tumefaciens, transgenic plants were obtained that were resistant to virus disease when challenged with either cucumber mosaic virus virions or RNA at concentrations up to 500 micrograms/ml or 50 micrograms/ml, respectively, the highest concentrations tested. This resistance was absolute, as neither symptoms nor virus could be detected in uninoculated leaves, even after prolonged incubation (120 days after inoculation). These data suggest, therefore, that such a "replicase-mediated" resistance strategy may be applicable to other plant and animal viruses.