bluuebunny/crossref_metadata_2025 huggingface dataset:
Hereditary differences in the expression of the human glutathione transferase active on trans-stilbene oxide are due to a gene deletion.
Authors: Seidegård J , Vorachek W R , Pero R W , Pearson W R
Published: May 2006

Glutathione transferase (GT; EC 2.5.1.18) mRNA levels were measured in human liver samples by using mouse and human cDNA clones that encode class-mu and class-alpha GT. Although all the RNA samples examined contained class-alpha GT mRNA, class-mu GT mRNA was found only in individuals whose peripheral leukocytes expressed GT activity on the substrate trans-stilbene oxide. The mouse class-mu cDNA clone was used to identify a human class-mu GT cDNA clone, lambda GTH411. The amino acid sequence of the GT encoded by lambda GTH411 is identical with the 23 residues determined for the human liver GT-mu isoenzyme and shares 76-81% identity with mouse and rat class-mu GT isoenzymes. The mouse and human class-mu GT cDNA inserts hybridize with multiple BamHI and EcoRI restriction fragments in the human genome. One of these hybridizing fragments is missing in the DNA of individuals who lack GT activity on trans-stilbene oxide. Hybridizations with nonoverlapping subfragments of lambda GTH411 suggest that there are at least three class-mu genes in the human genome. One of these genes appears to be deleted in individuals lacking GT activity on trans-stilbene oxide.
Beta-amyloid precursor protein of Alzheimer disease occurs as 110- to 135-kilodalton membrane-associated proteins in neural and nonneural tissues.
Authors: Selkoe D J , Podlisny M B , Joachim C L , Vickers E A , Lee G , Fritz L C , Oltersdorf T
Published: May 2006

Progressive cerebral deposition of extracellular filaments composed of the beta-amyloid protein (beta AP) is a constant feature of Alzheimer disease (AD). Since the gene on chromosome 21 encoding the beta AP precursor (beta APP) is not known to be altered in AD, transcriptional or posttranslational changes may underlie accelerated beta AP deposition. Using two antibodies to the predicted carboxyl terminus of beta APP, we have identified the native beta APP in brain and nonneural human tissues as a 110- to 135-kDa protein complex that is insoluble in buffer and found in various membrane-rich subcellular fractions. These proteins are relatively uniformly distributed in adult brain, abundant in fetal brain, and detected in nonneural tissues that contain beta APP mRNA. Similarly sized proteins occur in rat, cow, and monkey brain and in cultured human HL-60 and HeLa cells; the precise patterns in the 110- to 135-kDa range are heterogeneous among various tissues and cell lines. Confirmation that the immunodetected tissue proteins are forms of beta APP was obtained when mammalian cells transfected with a full-length beta APP cDNA showed selectively augmented expression of 110- to 135-kDa proteins and specific immunocytochemical staining. Unexpectedly, the antibodies to the carboxyl terminus of beta APP labeled amyloid-containing senile plaques in AD brain. We conclude that the highly conserved beta APP molecule occurs in mammalian tissues as a heterogeneous group of membrane-associated proteins of approximately 120 kDa. Detection of the nonamyloidogenic carboxyl terminus within plaques suggests that proteolytic processing of the beta APP into insoluble filaments occurs locally in cortical regions that develop beta-amyloid deposits with age.
Orthorhombic crystals and three-dimensional structure of the potent toxin II from the scorpion Androctonus australis Hector.
Authors: Fontecilla-Camps J C , Habersetzer-Rochat C , Rochat H
Published: May 2006

Orthorhombic crystals (space group P212121, a = 45.94 A, b = 40.68 A, c = 29.93 A) of the potent scorpion alpha-toxin II from Androctonus australis Hector were grown using sterile techniques. The structure was solved by a combination of heavy-atom and model phasing. Subsequently, it was refined at 1.8 A resolution by a fast-Fourier restrained least-squares procedure. The crystallographic R factor is 0.152 for data with 7.0 A greater than d greater than 1.8 A and F greater than 2.5 sigma (F) and 0.177 when all data are considered. Eighty-nine solvent molecules have been incorporated into the model. The dense core formed by the alpha-helical and antiparallel beta-sheet moieties and three of the four disulfide bridges is similar in variant 3, a toxin purified from the North American scorpion Centruroides sculpturatus, and in toxin II. However, the two molecules differ markedly in the orientation of loops protruding from the core. Toxin II seems to contain several highly ordered solvent molecules. Eight of them occupy a cavity consisting of the C-terminal region and a loop found only in scorpion alpha-toxins. The highly reactive and pharmacologically important Lys-58 is found at one of the extremes of this cavity, where it establishes a series of hydrogen bonds with protein and solvent atoms. The reactivities of the five lysine residues of toxin II are highly correlated with the formation of hydrogen bonds, hydrophobic interactions, and salt links.
Redox reactivity of bacterial and mammalian ferritin: is reductant entry into the ferritin interior a necessary step for iron release?
Authors: Watt G D , Jacobs D , Frankel R B
Published: May 2006

Both mammalian and bacterial ferritin undergo rapid reaction with small-molecule reductants, in the absence of Fe2+ chelators, to form ferritins with reduced (Fe2+) mineral cores. Large, low-potential reductants (flavoproteins and ferredoxins) similarly react anaerobically with both ferritin types to quantitatively produce Fe2+ in the ferritin cores. The oxidation of Fe2+ ferritin by large protein oxidants [cytochrome c and Cu(II) proteins] also occurs readily, yielding reduced heme and Cu(I) proteins and ferritins with Fe3+ in their cores. These latter oxidants also convert enthetically added Fe2+, bound in mammalian or bacterial apo- or holoferritin, to the corresponding Fe3+ state in the core of each ferritin type. Because the protein reductants and oxidants are much larger than the channels leading into the mineral core attached to the ferritin interior, we conclude that redox reactions involving the Fe2+/Fe3+ components of the ferritin core can occur without direct interaction of the redox reagent at the mineral core surface. Our results also suggest that the oxo, hydroxy species of the core, composed essentially of Fe(O)OH, arise exclusively from solvent deprotonation. The long-distance ferritin-protein electron transfer observed in this study may occur by electron tunneling.
A recombinase from Drosophila melanogaster embryos.
Authors: Eisen A , Camerini-Otero R D
Published: May 2006

We have partially purified a DNA strand-exchange activity (recombinase) from nuclear extracts of Drosophila melanogaster embryos. The protein fraction forms a joint molecule between a circular single-strand DNA and a homologous linear duplex DNA that is resolved from the substrates by agarose gel electrophoresis. A strand-exchange activity can be obtained from nuclear extracts from embryos as old as 24 hr. The activity is similar to that partially purified from human cells [Hsieh, P., Meyn, S.M. & Camerini-Otero, R.D. (1986) Cell 44, 885-894]. It is homology-dependent, requires Mg2+, appears to be directional in that it prefers to displace the 3' end of the noncomplementary strand, and does not require exogenous ATP. Forty nanograms of protein in the partially purified DNA strand-exchange fraction from D. melanogaster embryos can completely convert 50 ng of substrate single-strand DNA into joint molecules in 10 min. In the electron microscope, joint molecules are seen to consist of a circular single-strand DNA molecule attached to only one end of a linear duplex DNA molecule; a displaced strand is also seen. The region of heteroduplex formation can be as long as 600 base pairs. The demonstration of a strand-exchange activity from wild-type D. melanogaster embryos invites analysis of recombination-defective mutants to explore the role of DNA strand exchange in homologous recombination.
Accurate simulation of protein dynamics in solution.
Authors: Levitt M , Sharon R
Published: May 2006

Simulation of the molecular dynamics of a small protein, bovine pancreatic trypsin inhibitor, was found to be more realistic when water molecules were included than when in vacuo: the time-averaged structure was much more like that observed in high-resolution x-ray studies, the amplitudes of atomic vibration in solution were smaller, and fewer incorrect hydrogen bonds were formed. Our approach, which provides a sound basis for reliable simulation of diverse properties of biological macromolecules in solution, uses atom-centered forces and classical mechanics.
Covalent binding of the endogenous estrogen 16 alpha-hydroxyestrone to estradiol receptor in human breast cancer cells: characterization and intranuclear localization.
Authors: Swaneck G E , Fishman J
Published: May 2006

The interactions of 16 alpha-hydroxyestrone (16 alpha-OHE1), a metabolite of estradiol (E2), with estrogen receptors (ERs) were compared in this study to the classic E2-receptor mechanism in human breast cancer cells MCF-7 in culture. When MCF-7 cells were incubated with radioinert 16 alpha-OHE1 or its 3H-labeled form for 4 weeks, the estrogen bound extensively and irreversibly in a time-dependent fashion to nuclear protein species that correspond to the ER. Here we show that the interactions of 16 alpha-OHE1 with the ER are different from those of E2 with the receptor. Dissociation of tritiated E2-ER or 16 alpha-OHE1-ER complexes, salt extraction, DNase and proteinase K digestion, and ethanol treatment demonstrated that the binding of 16 alpha-OHE1 to the ER corresponds to two different forms: a classical noncovalent interaction similar to that of E2, and a covalent adduct formation between the metabolite and the ER. These complexes localized preferentially in nuclear matrix components as revealed by cell fractionation and probing with a monoclonal anti-ER antibody. [3H]16 alpha-OHE1-ER complexes analyzed by polyacrylamide gel electrophoresis demonstrated a radiolabeled band at approximately 66 kDa that was absent when the exposure of cells was done in the presence of E2 in competition and that was also absent in [3H]E2 incubations. The present results when considered together with our previous findings of elevated activities of estrogen 16 alpha-hydroxylase, the enzyme responsible for the formation of 16 alpha-OHE1, in breast cancer patients and in women at enhanced risk for the disease, suggest that covalent modification of the ER may be one mechanism of malignant transformation in estrogen target tissues.
Translation of the sequence AGG-AGG yields 50% ribosomal frameshift.
Authors: Spanjaard R A , van Duin J
Published: May 2006

We have inserted the sequence 5'-AAG-GAGGU-3', which is complementary to the 3' terminus of Escherichia coli 16S rRNA, in a reading frame and analyzed its effect on the accuracy and overall rate of translation in vivo. Translation over the sequence yields a 50% ribosomal frameshift if the reading phase is A-AGG-AGG-U. The other two possible frames do not give shifts. The introduction of a UAA stop codon before (UAA-AGG-AGG-U) but not after (A-AGG-AGG-UAA) the AGG codons abolishes the frameshift. The change in the reading phase occurs exclusively to the +1 direction. Efficient frameshifting is also induced by the sequence A-AGA-AGA-U. The arginine codons AGG and AGA are read by minor tRNA. Suppression of frameshifting takes place when a gene for minor tRNA(Arg) is introduced on a multicopy plasmid. We suggest that frameshifting during translation of the A-AGG-AGG-U sequence is due to the erroneous decoding of the tandem AGG codons and arises by depletion of tRNA(Arg). The complementarity of tandem AGG codons to the 3' terminus of 16S rRNA is a coincidence and apparently not related to the shift. Replacing the AGG-AGG sequence by the optimal arginine codons CGU-CGU does not increase the overall rate of translation.
Genomic organization of the human T-cell antigen-receptor alpha/delta locus.
Authors: Satyanarayana K , Hata S , Devlin P , Roncarolo M G , De Vries J E , Spits H , Strominger J L , Krangel M S
Published: May 2006

Two clusters of overlapping cosmid clones comprising about 100 kilobases (kb) at the human T-cell antigen-receptor alpha/delta locus were isolated from a genomic library. The structure of the germ-line V delta 1 variable gene segment was determined. V delta 1 is located 8.5 kb downstream of the V alpha 13.1 gene segment, and both V segments are arranged in the same transcriptional orientation. The V alpha 17.1 segment is located between V delta 1 and the D delta, J delta, C delta region (containing the diversity, joining, and constant gene segments). Thus, V delta and V alpha segments are interspersed along the chromosome. The germ-line organization of the D delta 2, J delta 1, and J delta 2 segments was determined. Linkage of C delta to the J alpha region was established by identification of J alpha segments within 20 kb downstream of C delta. The organization of the locus was also analyzed by field-inversion gel electrophoresis. The unrearranged V delta 1 and D delta, J delta, C delta regions are quite distant from each other, apparently separated by a minimum of 175-180 kb.
Stable genetic transformation of intact
Nicotiana
cells by the particle bombardment process
Authors: Klein Theodore M. , Harper Elisabeth C. , Svab Zora , Sanford John C. , Fromm Michael E. , Maliga Pal
Published: May 2006

We show that the genetic transformation of
Nicotiana tabacum
can be achieved by bombarding intact cells and tissues with DNA-coated particles. Leaves or suspension culture cells were treated with tungsten microprojectiles carrying plasmid DNA containing a neomycin phosphotransferase gene. Callus harboring the foreign gene was recovered from the bombarded tissue by selection on medium containing kanamycin. Kanamycin-resistant plants have subsequently been regenerated from the callus derived from leaves. Transient expression of an introduced β-glucuronidase gene was used to assess the efficiency of DNA delivery by microprojectiles. The frequency of cells that were stably transformed with the neomycin phosphotransferase gene was a few percent of the cells that transiently expressed the β-glucuronidase gene. These results show that gene transfer by high-velocity microprojectiles is a rapid and direct means for transforming intact plant cells and tissues that eliminates the need for production of protoplasts or infection by
Agrobacterium
.
Group-specific, major histocompatibility complex class I-restricted cytotoxic responses to human immunodeficiency virus 1 (HIV-1) envelope proteins by cloned peripheral blood T cells from an HIV-1-infected individual.
Authors: Koenig S , Earl P , Powell D , Pantaleo G , Merli S , Moss B , Fauci A S
Published: May 2006

Freshly separated unfractionated peripheral blood mononuclear cells (PBMC) and cloned cell lines from a healthy human immunodeficiency virus 1 (HIV-1)-seropositive individual were examined for cytotoxic responses to HIV proteins expressed by recombinant vaccinia viruses. It was found that freshly isolated PBMC recognize variant envelope proteins of HIV-1 but not a more distantly related envelope protein derived from the simian immunodeficiency virus (SIVmac). Although the effector cells were predominantly CD8+, both MHC-matched and -unmatched target cells were lysed. Cytotoxic T lymphocyte (CTL) clones were found to lyse cells expressing HIV-1 envelope or reverse transcriptase. In contrast to the cytotoxic response detected with PBMC, the cloned CTLs were major histocompatibility complex (MHC) class I restricted. Our finding that a cloned CTL line lysed cells expressing highly divergent HIV envelopes strongly suggested that a conserved epitope was recognized. Identification of these shared epitopes may assist in designing a vaccine for HIV-1 that could stimulate MHC-restricted cytotoxic responses.
Comb wax mediates the acquisition of nest-mate recognition cues in honey bees
Authors: Breed Michael D. , Williams Kristina R. , Fewell Jennifer H.
Published: May 2006

Honey bees,
Apis mellifera
, acquire nest-mate recognition cues from wax, the predominant material used in nest construction. Exposure of a newly emerged worker bee to wax-comb substrate significantly reduced the acceptability of that worker to sister bees. Cues acquired from the comb provided colony-specific information about the identity of worker bees; moreover, the effect of comb exposure has been previously shown to override individually produced cues. Food odors (anise oil), when dissolved in paraffin wax, affected worker-recognition characteristics but food odors did not affect these characteristics when fed to bees in sugar candy. Paraffin wax alone did not affect the recognition cues of bees, showing that the wax can be a neutral medium for the transmission of cues. The wax comb in the colony and the hydrocarbon outer layer of the bee cuticle may be a continuous medium for any hydrocarbon-soluble substances used by honey bees in nest-mate recognition; if so, a mechanism by which environmental cues are acquired by honey bees is provided.
Heteroatom-substituted fatty acid analogs as substrates for N-myristoyltransferase: an approach for studying both the enzymology and function of protein acylation.
Authors: Heuckeroth R O , Glaser L , Gordon J I
Published: May 2006

Myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that transfers the myristoyl (14:0) moiety from myristoyl CoA thioester to nascent proteins, is remarkably specific for both peptide and fatty acyl CoA substrates. To investigate the interaction of NMT with fatty acyl CoA substrates, we have synthesized 10 oxygen- or sulfur-substituted fatty acid analogs. These analogs differ dramatically in hydrophobicity from naturally occurring fatty acids of similar length. As acylpeptides, sulfur-substituted myristic acid analogs migrate on reverse-phase HPLC like 11:0 or 12:0 fatty acids, while oxygen-substituted analogs migrate like 9:0 to 11:0 fatty acids. CoA thioesters of several of these analogs serve as good NMT substrates in vitro, implying that NMT selects fatty acyl substrates primarily on the basis of chain length rather than hydrophobicity. Myristelaidoyl (14:1, delta 9,10-trans) CoA is also a significantly better substrate than myristoleoyl (14:1, delta 9,10-cis) CoA. The fatty acyl group bound to NMT profoundly influences the rate of acylpeptide formation and the affinity of NMT for peptide substrates. However, the peptide substrate bound to NMT does not produce significant alterations in the enzyme's affinity for myristoyl CoA. In vitro characterization of these heteroatom substituted analogs suggests that they will be efficiently incorporated into proteins in vivo and may markedly alter acylprotein targeting and function.
Structure of the reaction center from Rhodobacter sphaeroides R-26 and 2.4.1: symmetry relations and sequence comparisons between different species.
Authors: Komiya H , Yeates T O , Rees D C , Allen J P , Feher G
Published: May 2006

Photosynthetic reaction centers from purple bacteria exhibit an approximate twofold symmetry axis, which relates both the cofactors and the L and M subunits. For the reaction center from Rhodobacter sphaeroides, deviations from this twofold symmetry axis have been quantitated by superposing, by a 180 degrees rotation, the cofactors of the B branch onto the A branch and the M subunit onto the L subunit. An alignment of the sequences of the L and M subunits from four purple bacteria, one green bacterium, and the D1 and D2 subunits of a photosystem II-containing green alga is presented. The residues that are conserved in all six species are shown in relation to the structure of Rb. sphaeroides and their possible role in the function of the reaction center is discussed. A method is presented for characterizing the exposure of alpha-helices to the membrane based on the periodicity of conserved residues. This method may prove useful for modeling the three-dimensional structures of membrane proteins.
Erythropoietin changes the globin program of an interleukin 3-dependent multipotential cell line.
Authors: Enver T , Nakamoto B , Karlinsey J , Josephson B , Greenberger J , Papayannopoulou T
Published: May 2006

B6SUtA is a factor-dependent murine cell line of adult origin displaying the functional properties of a multipotent hematopoietic stem cell. We analyzed the globin programs of B6SUtA cells undergoing erythroid differentiation in both suspension and clonal cultures. In the absence of added erythropoietin, a small number of hemoglobinized cells were present, and these expressed predominantly embryonic globin. Addition of erythropoietin increased the number and maturation of hemoglobinized cells and led to a preferential augmentation of adult globin. Analysis of individual B6SUtA erythroid bursts showed that embryonic and adult globin can be expressed in cells derived from a single progenitor. Furthermore, by studying globin expression in cultured cells from mouse embryos, we found that the globin programs of B6SUtA cells are similar to those of erythroid progenitors at the period of transition from yolk sac to fetal liver erythropoiesis. Since B6SUtA cells are derived from adult bone marrow and they have the capacity to express embryonic globin, we speculate that the globin locus is not irreversibly modified during development and that adult cells at early stages of erythroid differentiation can transiently express ontogenetically primitive globin programs.
Evidence that a point mutation in dihydrofolate reductase-thymidylate synthase confers resistance to pyrimethamine in falciparum malaria.
Authors: Peterson D S , Walliker D , Wellems T E
Published: May 2006

Analysis of a genetic cross of Plasmodium falciparum and of independent parasite isolates from Southeast Asia, Africa, and South America indicates that resistance to pyrimethamine, an antifolate used in the treatment of malaria, results from point mutations in the gene encoding dihydrofolate reductase-thymidylate synthase (EC 1.5.1.3 and EC 2.1.1.45, respectively). Parasites having a mutation from Thr-108/Ser-108 to Asn-108 in DHFR-TS are resistant to the drug. The Asn-108 mutation occurs in a region analogous to the C alpha-helix bordering the active site cavity of bacterial, avian, and mammalian enzymes. Additional point mutations (Asn-51 to Ile-51 and Cys-59 to Arg-59) are associated with increased pyrimethamine resistance and also occur at sites expected to border the active site cavity. Analogies with known inhibitor/enzyme structures from other organisms suggest that the point mutations occur where pyrimethamine contacts the enzyme and may act by inhibiting binding of the drug.
Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization.
Authors: Aronsson M , Fuxe K , Dong Y , Agnati L F , Okret S , Gustafsson J A
Published: May 2006

The localization and distribution of mRNA encoding the glucocorticoid receptor (GR) was investigated in tissue sections of the adult male rat brain by in situ hybridization and RNA blot analysis. GR mRNA levels were measured by quantitative autoradiography with 35S- and 32P-labeled RNA probes, respectively. Strong labeling was observed within the pyramidal nerve cells of the CA1 and CA2 areas of the hippocampal formation, in the granular cells of the dentate gyrus, in the parvocellular nerve cells of the paraventricular hypothalamic nucleus, and in the cells of the arcuate nucleus, especially the parvocellular part. Moderate labeling of a large number of nerve cells was observed within layers II, III, and VI of the neocortex and in many thalamic nuclei, especially the anterior and ventral nuclear groups as well as several midline nuclei. Within the cerebellar cortex, strong labeling was observed all over the granular layer. In the lower brainstem, strong labeling was found within the entire locus coeruleus and within the mesencephalic raphe nuclei rich in noradrenaline and 5-hydroxytryptamine cell bodies, respectively. A close correlation was found between the distribution of GR mRNA and the distribution of previously described GR immunoreactivity. These studies open the possibility of obtaining additional information on in vivo regulation of GR synthesis and how the brain may alter its sensitivity to circulating glucocorticoids.
Hydrogen production and deuterium-proton exchange reactions catalyzed by
Desulfovibrio
nickel(II)-substituted rubredoxins
Authors: Saint-Martin Pascal , Lespinat Paul A. , Fauque Guy , Berlier Yves , LeGall Jean , Moura Isabel , Teixeira Miguel , Xavier Antonio V. , Moura Jose J. G.
Published: May 2006

The nickel tetrahedral sulfur-coordinated core formed upon metal replacement of the native iron in
Desulfovibrio
sp. rubredoxins is shown to mimic the reactivity pattern of nickel-containing hydrogenases with respect to hydrogen production, deuterium-proton exchange, and inhibition by carbon monoxide.
Some mismatch repair activities in Escherichia coli.
Authors: Radicella J P , Clark E A , Fox M S
Published: May 2006

Heterozygous bacteriophage lambda DNA molecules, whose replication requires mismatch correction of a mutant nucleotide in the transcribed strand, provide an assay for localized mismatch repair in Escherichia coli. We describe two systems: one removes the A in C.A or G.A mismatches and the other removes one or the other C in a C.C mismatch. Mutations disabling the first system result in a mutator phenotype that may be identical to mutY.
In vitro transcription of eukaryotic genes is affected differently by the degree of DNA supercoiling.
Authors: Hirose S , Suzuki Y
Published: May 2006

In a posterior silk gland extract, covalently closed circular (ccc) DNA is in a superhelical state that supports more transcription of fibroin gene than does linear DNA. A HeLa cell extract showed neither the supercoiling activity nor the preference for the transcription of ccc DNA over linear DNA. These activities could be added to the HeLa cell extract. Phosphocellulose fractionation of the posterior silk gland extract yielded a flow-through fraction and a 0.6 M KCl eluate fraction that were required for the supercoiling and for the efficient transcription of the ccc template in the acceptor HeLa cell extract. The 0.6 M KCl fraction had a DNA topoisomerase II activity, and the flow-through fraction contained a supercoiling factor that, with the aid of topoisomerase II, introduced negative supercoils into ccc DNA. When both fractions were added to the posterior silk gland extract, more supercoiling occurred than with the extract alone. Several genes were optimally transcribed under various extents of supercoiling. The fibroin gene and adenovirus 2 major late promoter were fully transcribed as partially supercoiled templates. The sericin gene required more supercoiling for full transcription, whereas no preference for supercoiling was seen with the transcription of hsp70. These results suggest that DNA topology plays a role in the regulation of gene expression.
Structure and expression of spinach leaf cDNA encoding ribulosebisphosphate carboxylase/oxygenase activase.
Authors: Werneke J M , Zielinski R E , Ogren W L
Published: May 2006

Ribulosebisphosphate carboxylase/oxygenase activase is a recently discovered enzyme that catalyzes the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase ["rubisco"; ribulose-bisphosphate carboxylase; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] in vivo. Clones of rubisco activase cDNA were isolated immunologically from spinach (Spinacea oleracea L.) and Arabidopsis thaliana libraries. Sequence analysis of the spinach and Arabidopsis cDNAs identified consensus nucleotide binding sites, consistent with an ATP requirement for rubisco activase activity. A derived amino acid sequence common to chloroplast transit peptides was also identified. After synthesis of rubisco activase in vitro, the transit peptide was cleaved and the protein was transported into isolated chloroplasts. Analysis of spinach and Arabidopsis nuclear DNA by hybridization indicated a single rubisco activase gene in each species. Leaves of spinach and Arabidopsis wild type contained a single 1.9-kilobase rubisco activase mRNA. In an Arabidopsis mutant lacking rubisco activase protein, mRNA species of 1.7 and 2.1 kilobases were observed under conditions of high-stringency hybridization with a wild-type cDNA probe. This observation indicates that the lesion in the mutant arises from an error in mRNA processing.
Efficient algorithms for molecular sequence analysis.
Authors: Karlin S , Morris M , Ghandour G , Leung M Y
Published: May 2006

Efficient (linear time) algorithms are described for identifying global molecular sequence features allowing for errors including repeats, matches between sequences, dyad symmetry pairings, and other sequence patterns. A multiple sequence alignment algorithm is also described. Specific applications are given to hepatitis B viruses and the J5-C (J, joining; C, constant) region of the immunoglobulin kappa gene.
Transposable element Tc1 of Caenorhabditis elegans recognizes specific target sequences for integration.
Authors: Mori I , Benian G M , Moerman D G , Waterston R H
Published: May 2006

The frequency of movement of Tc1, a 1.6-kilobase transposable element in the nematode Caenorhabditis elegans, is under genetic control, and Tc1 insertion sites are widely but nonrandomly distributed. The usually high frequency of insertions at multiple sites in the gene unc-22 suggested that this gene might be particularly rich in preferred target sites. To discover the features of Tc1 target sites, we have sequenced the sites of seven independent Tc1 transpositions into unc-22 and three other sites. Our comparison of these and two other sites from the literature indicates that in all cases Tc1 integrates at the dinucleotide T-A when it is flanked both 5' and 3' by particular preferred nucleotides. Our analysis revealed the following consensus target for Tc1 integration: G-A-K-A-T-A-T-G-T, in which K = G or T. This target site sequence specificity has implications both for the mechanism of Tc1 transposition and the use of Tc1 in cloning genes by transposon-tagging.
Receptors for prolactin, somatostatin, and luteinizing hormone-releasing hormone in experimental prostate cancer after treatment with analogs of luteinizing hormone-releasing hormone and somatostatin.
Authors: Kadar T , Redding T W , Ben-David M , Schally A V
Published: May 2006

Membrane receptors for luteinizing hormone-releasing hormone (LH-RH), somatostatin, and prolactin (PRL) were investigated in the Dunning R-3327H rat prostate adenocarcinoma specimens after in vivo treatment with microcapsules of the agonist [D-Trp6]LH-RH and the somatostatin analog RC-160. The LH-RH receptors showed a low-binding affinity (Kd = 54 nM) and high capacity (Bmax = 12.0 pmol/mg). Treatment with the [D-Trp6]LH-RH decreased the binding affinity (Kd = 0.52 microM). Specific somatostatin receptors, with Kd = 1.3 nM and Bmax = 543 fmol/mg, were also found. Treatment with [D-Trp6]LH-RH lowered Bmax to 44 fmol/mg, and administration of RC-160 reduced Kd to 30 nM. After the combined treatment with the two analogs, Kd and Bmax were decreased. Specific PRL receptors (Kd = 0.72 nM; Bmax = 161 fmol/mg) were also detected. Treatment with either analog reduced Bmax by 50%, but a much greater reduction of PRL binding capacity was revealed after in vitro dissociation of the bound endogenous PRL by MgCl2. The dramatic fall in the total number of PRL receptors after combination treatment with both analogs could be partially responsible for the decrease in the weight and volume of prostate tumors. The findings support the concept that analogs of LH-RH and somatostatin can inhibit tumors directly through their own respective receptors. One of several mechanisms of the antineoplastic activity of these analogs could be the elimination of tumor growth-promoting effect of PRL by the reduction of the total number of PRL receptors.
Glutathione S-transferase Ya subunit gene: identification of regulatory elements required for basal level and inducible expression.
Authors: Telakowski-Hopkins C A , King R G , Pickett C B
Published: May 2006

The function of the 5'-flanking region of a rat glutathione S-transferase Ya subunit structural gene has been examined in homologous and heterologous cells. By using the 5'-flanking region of the Ya subunit gene fused to the structural gene encoding chloramphenicol acetyltransferase, we have identified two cis-acting regulatory elements in the upstream region of the Ya gene. One element is required for maximum basal level expression in homologous cells, whereas maximum basal level expression in homologous cells, whereas the second element is required for inducible expression of the Ya gene by planar aromatic compounds such as beta-naphthoflavone. The cis-acting element required for inducible expression of the Ya gene by beta-naphthoflavone is functional only in cells with normal dioxin receptors.
Structure of ferredoxin I from Azotobacter vinelandii.
Authors: Stout G H , Turley S , Sieker L C , Jensen L H
Published: May 2006

The structure of Azotobacter vinelandii ferredoxin I (Av FdI, 106 amino acids) has been redetermined, based on x-ray diffraction data from tetragonal crystals of the native protein and two heavy atom derivatives. The current model differs greatly from the one previously reported and is in agreement with arguments based on various spectroscopic and other methods. The unit cell parameters are a = b = 55.62 A and c = 95.51 A, whereas the space group was found to be P4(1)2(1)2 instead of P4(3)2(1)2. The sequence of the first half of Av FdI is closely homologous with ferredoxin from Peptococcus aerogenes (Pa Fd, 54 amino acids) and the fold of the corresponding chain is almost identical. The ligands of the 3Fe complex are Cys-8, -16, and -49, corresponding to three of the four ligands in complex I of Pa Fd; the ligands of the 4Fe complex are Cys-20, -39, -42, and -45, corresponding to the four ligands in complex II of Pa Fd.
Fibronectin as a carrier for the transglutaminase from human erythrocytes.
Authors: Lorand L , Dailey J E , Turner P M
Published: May 2006

Nondenaturing electrophoresis was used to demonstrate that, immediately upon exposure to plasma, the transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) from erythrocytes undergoes a significant shift in mobility. The plasma effect shows saturable characteristics and depends entirely on the presence of fibronectin in plasma, indicative of complex formation between this protein and transglutaminase. The results suggest a specific carrier function for fibronectin that might be of physiological importance in determining the fate of a tissue transglutaminase accidentally discharged into plasma.
Drosophila kinesin: characterization of microtubule motility and ATPase.
Authors: Saxton W M , Porter M E , Cohn S A , Scholey J M , Raff E C , McIntosh J R
Published: May 2006

Preparations of kinesin, a microtubule-based force-producing protein, have been isolated from Drosophila melanogaster embryos by incubation of microtubules with a nonhydrolyzable ATP analogue and gel filtration of proteins released from the microtubules by ATP. These preparations induced MgATP-dependent microtubule gliding in vitro with a Km for MgATP of 44 microM and a Vmax for gliding of 0.9 micron/sec. Samples of Drosophila proteins that were active in motility assays possessed an average ATPase activity in solution of 17 nmol/min per mg that increased to an average of 106 nmol/min per mg in the presence of microtubules. The major polypeptides that copurified with these activities showed relative molecular masses of 115 kDa and 58 kDa. An antiserum raised against the 115-kDa polypeptide also recognized the 110-kDa component of squid kinesin preparations and the 130-kDa component of sea urchin kinesin preparations.
Protection of mice from fatal measles encephalitis by vaccination with vaccinia virus recombinants encoding either the hemagglutinin or the fusion protein.
Authors: Drillien R , Spehner D , Kirn A , Giraudon P , Buckland R , Wild F , Lecocq J P
Published: May 2006

Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.
Construction and characterization of glutaredoxin-negative mutants of Escherichia coli.
Authors: Russel M , Holmgren A
Published: May 2006

Deoxyribonucleotides, the precursors of DNA, are formed de novo by ribonucleotide reductase, and in vitro thioredoxin or glutathione plus glutaredoxin have been isolated as hydrogen donors. The in vivo hydrogen donor for ribonucleotide reductase is not known. To study this, the Escherichia coli glutaredoxin gene (255 base pairs) was inactivated by inserting a 2-kilobase kanamycin-resistance fragment into the coding sequence of the cloned gene. The inactivated gene was inserted into the E. coli chromosome and mapped to about 18.5 min. A gene replacement technique was used to obtain a strain, A407, that lacked glutaredoxin by radioimmunoassay and by enzymatic assay with ribonucleotide reductase. Glutaredoxin was found not to be essential for viability of E. coli. Thioredoxin is also not essential for viability, as had been shown earlier, but a double mutant lacking glutaredoxin and thioredoxin could not be obtained by P1 transduction on a defined medium, indicating that either thioredoxin or glutaredoxin is essential. In rich medium, very slowly growing, unstable transductants were obtained that at high frequency gave rise to better growing cells. One such isolate, A410, was shown to still lack glutaredoxin and thioredoxin.
Role of algal eukaryotes in subtidal columnar stromatolite formation
Authors: Awramik Stanley M. , Riding Robert
Published: May 2006

Columnar stromatolites were abundant and widespread in the Proterozoic but are exceedingly rare in modern seas. Consequently, the stromatolites at Hamelin Pool in Shark Bay, Western Australia, have been widely interpreted as unique modern analogs of ancient stromatolites constructed by complex communities of cyanobacteria. However, the Shark Bay columnar stromatolites contain sediment that is unusually coarse for stromatolites both ancient and modern, and the subtidal columnar stromatolites have a significant component of algal eukaryotes dominated by motile diatoms with mucilaginous tubes. This suggests that Shark Bay columnar stromatolites are not strict analogs for most ancient cyanobacterial stromatolites, least of all for those from subtidal environments. We argue that algal eukaryotes may play a substantial role in the formation and maintenance of subtidal columnar stromatolites at Shark Bay and are capable of trapping coarse sediment. In contrast, cyanobacteria have difficulty in trapping coarse sediment and produce essentially fine-grained stromatolites. We propose that there are two major types or end members of Recent marine stromatolites: (
i
) eualgal-cyanobacterial stromatolites that are generally coarse-grained, and (
ii
) cyanobacterial stromatolites that are generally fine-grained.
Tris(tetramethylphenanthroline)ruthenium(II): a chiral probe that cleaves A-DNA conformations.
Authors: Mei H Y , Barton J K
Published: May 2006

lambda-Tris(3,4,7,8-tetramethyl-1,10-phenanthroline)ruthenium(II) [lambda-Ru(TMP)2+3] was found to be a distinctive molecular tool to examine the local variations in conformation along the strand. The metal complex binds cooperatively to A-form helices of various base sequences under conditions where little or no binding was found to analogous B-form DNAs. Photoactivated DNA cleavage may be coupled to this conformation-specific binding by taking advantage of the photophysical properties of ruthenium(II) complexes. lambda-Ru(TMP)2+3 cleaves preferentially 3H-labeled A-form polynucleotides upon irradiation with visible light. The photoinduced DNA strand scission is likely to be mediated by singlet oxygen, which leads to a preferential cleavage of guanine residues. Comparative mapping of cleavage sites on a linear pBR322 fragment for tris(phenanthroline)ruthenium(II), which binds to B-DNA and cleaves also by sensitization of singlet oxygen, and for Ru(TMP)2+3 shows the selective binding of lambda-Ru(TMP)2+3 to conformationally distinct sites along the fragment. These sites correspond to 5- to 13-base-pair homopyrimidine stretches.
Crystalline cytochrome c oxidase of bovine heart mitochondrial membrane: composition and x-ray diffraction studies.
Authors: Yoshikawa S , Tera T , Takahashi Y , Tsukihara T , Caughey W S
Published: May 2006

The integral mitochondrial membrane protein cytochrome c oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.1) was crystallized from solutions of the protein from bovine heart isolated as described earlier [Yoshikawa, S. & Caughey, W. S. (1982) J. Biol. Chem. 257, 412-420]. Crystallinity was demonstrated by x-ray diffraction. Microcrystals (tetragonal prisms, 0.02 mm in the largest dimension) were obtained in high yield with retention of activity and contained Fe, Cu, Zn, and Mg in approximate atom ratios of 1.0:1.25:0.5:0.5, respectively. Analysis of the amino acid residues and the tightly bound detergent support an apparent molecular mass of about 200 kDa, of which 150 kDa is protein (1316 +/- 66 amino acids) and 50 kDa is detergent (Brij 35). Seven major polypeptides are evident by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Adjustments in buffer concentration and other conditions have yielded much larger green crystals, hexagonal bipyramids; a crystal 0.3 X 0.5 X 0.7 mm gave x-ray diffractions as high as 8-A resolution and a space group of P6(2) or P6(4), and cell dimensions of a = b = 174.5 A, c = 282.2 A, alpha = beta = 90 degrees, and gamma = 120 degrees were obtained. A reasonable value of 3.1 A3/Da for Vm, the average space per dalton of protein in the crystal, was obtained for the asymmetric unit, which contains four irons and is a dimer of two minimal catalytic units. Cylindrical dimers (80 X 100 A) estimated from two-dimensional electron diffraction studies [Fuller, S. D., Capaldi, R. A. & Henderson, R. (1979) J. Mol. Biol. 134, 305-327] pack well in the crystal lattice with the symmetry of the space group of the crystal. The crystallization procedure developed is useful in purification of the enzyme and shows promise for the production of crystals of sufficiently high order to gain improved structural information from x-ray diffraction.
Identification of the glycosaminoglycan-attachment site of mouse invariant-chain proteoglycan core protein by site-directed mutagenesis.
Authors: Miller J , Hatch J A , Simonis S , Cullen S E
Published: May 2006

The invariant chain (Ii), a nonpolymorphic glycoprotein that associates with the immunoregulatory Ia proteins encoded by the major histocompatibility complex, has a proteoglycan form (Ii-CS) that bears a chondroitin sulfate glycosaminoglycan. In this proteoglycan form, Ii may remain associated with Ia at the cell surface. Inhibitors that prevent the addition of glycosaminoglycan to Ii have been found to depress antigen-presenting function. Ii does not have multiple candidate glycosaminoglycan-attachment sites, and we used site-directed mutagenesis to replace a candidate serine glycosaminoglycan-acceptor site with alanine at position 201 in the murine Ii protein. Transfection of the normal or altered gene into Ii-negative COS-7 cells showed that equivalent amounts of core Ii protein and its acidic, terminally glycosylated forms were synthesized, but the Ala-201 mutant Ii did not give rise to Ii-CS. The mutant protein had apparently normal transport through the Golgi compartment and associated stably with Ia molecules. Thus, this mutation directly identifies the site of glycosaminoglycan addition and shows that it can be eliminated without adversely affecting the overall biosynthesis of Ii.
Permanency of response to selection for quantitative characters in finite populations.
Authors: Cockerham C C , Tachida H
Published: May 2006

To study the permanency of response to selection for a quantitative character in finite populations and the nature of the genetic effects that contribute to this response, we have used the covariance between ancestors and descendents within populations. Effects and variances are defined for an initial equilibrium random mating monoecious population that gives rise to replicate finite populations. After a prescribed history of restricted population size, the replicate populations are expanded, and the covariance between ancestors and descendents is quantified in terms of descent measures and genetic components in the initial population as a means of determining the additive variance within populations. Several dominance components including joint dominance effects of loci contribute to the additive variance, some of which can be negative. There is always a positive contribution of additive by additive variance to the additive variance within populations, which can be large. With the new definitions of components of genetic variance within populations, selection response is formulated in the same manner as for the initial random mating population, but the components have been modified considerably by the restricted population size.
[D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P, a potent bombesin antagonist in murine Swiss 3T3 cells, inhibits the growth of human small cell lung cancer cells in vitro.
Authors: Woll P J , Rozengurt E
Published: May 2006

In the search for a more potent bombesin antagonist, we found [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P to be effective in mouse fibroblasts and to inhibit the growth of small cell lung cancer, a tumor that secretes bombesin-like peptides that may act as autocrine growth factors. In murine Swiss 3T3 cells, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P proved to be a bombesin antagonist as judged by the following criteria: (i) inhibition of DNA synthesis induced by gastrin-releasing peptide and other bombesin-like peptides; (ii) inhibition of 125I-labeled gastrin-releasing peptide binding to the bombesin/gastrin-releasing peptide receptor; (iii) reduction in cross-linking of the Mr 75,000-85,000 protein putatively a component of the bombesin/gastrin-releasing peptide receptor; (iv) blocking of early cellular events that precede mitogenesis--calcium mobilization and inhibition of epidermal growth factor binding. [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P was 5-fold more potent than the antagonist [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P. [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P also inhibits mitogenesis induced by vasopressin but not that induced by a variety of other mitogens. Both antagonists reversibly inhibited the growth of small cell lung cancer in vitro in a concentration-dependent manner. Peptide antagonists could, therefore, have far-reaching therapeutic implications.