bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of the prosystemin gene in transgenic tomato plants generates a systemic signal that constitutively induces proteinase inhibitor synthesis.
Authors: McGurl B , Orozco-Cardenas M , Pearce G , Ryan C A
Published: May 2006

Tomato plants (Lycopersicon esculentum, var. Better Boy) were stably transformed with a gene consisting of the open reading frame of a prosystemin cDNA under the regulation of the cauliflower mosaic virus 35S promoter. The leaves of the transgenic plants constitutively produced proteinase inhibitor I and II proteins, which accumulated over time to levels exceeding 1 mg/g of dry leaf weight. This phenotype contrasts with that of untransformed plants, which produce proteinase inhibitor proteins in leaves only in response to wounding or chemical inducers. The transgenic plants were also stunted, although they appeared normal in all other respects. Grafting the upper half (scion) of an untransformed tomato plant onto the lower half (root stock) of a tomato plant expressing the prosystemin transgene resulted in the constitutive expression of proteinase inhibitor proteins in the leaves of both the transformed root stock and the untransformed scion, demonstrating that expression of the prosystemin transgene generates a mobile wound signal. These results show that systemic signal propagation in the transgenic plants does not require wounding, and they support the proposed role of systemin as the mobile wound signal.
Enhanced tumor cell growth suppression by an N-terminal truncated retinoblastoma protein.
Authors: Xu H J , Xu K , Zhou Y , Li J , Benedict W F , Hu S X
Published: May 2006

The retinoblastoma (RB) gene encodes a nuclear phosphoprotein of 928 amino acids (pRB). Thus far, much effort in RB research has been focused on both the viral oncoprotein-binding domains and the C-terminal domain, whereas little is known about the N-terminal moiety of the protein. We report here that an N-terminal truncated RB protein of approximately 94 kDa (pRB94) exerts more potent cell growth suppression as compared to the full-length pRB protein in a diversity of tumor cell lines examined, including those having a normal endogenous RB gene. Tumor cells transfected with the pRB94-expressing plasmids displayed multiple morphological changes frequently associated with cellular senescence and/or apoptosis. They failed to enter S phase and rapidly died. The pRB94 expressed in recipient tumor cells had a longer half-life than the full-length pRB protein and tended to remain in an active un- or hypophosphorylated form. Since it has also been found that N-terminal truncated RB proteins often accumulated in growth-arrested and/or differentiated tumor cells, we suggest that N-terminal truncation of pRB may be one of the cellular mechanisms modulating the RB protein function in cell-cycle control.
Recombinant human hemoglobins designed for gene therapy of sickle cell disease.
Authors: McCune S L , Reilly M P , Chomo M J , Asakura T , Townes T M
Published: May 2006

Two human hemoglobins designed to inhibit the polymerization of sickle hemoglobin (Hb S; alpha 2 beta S2) have been produced. Mutations that disrupt the ability of Hb S to form polymers were introduced into the normal human beta-globin gene by site-specific mutagenesis. These mutations affect the axial and lateral contacts in the sickle fiber. The recombinant hemoglobin designated anti-sickling hemoglobin 1 (Hb AS1) contains the mutations beta 22 glutamic acid to alanine and beta 80 asparagine to lysine. Hb AS2 has the same beta 22 glutamic acid to alanine mutation combined with beta 87 threonine to glutamine. Human alpha- and beta AS-globin genes were separately fused downstream of beta-globin locus control region sequences and these constructs were coinjected into fertilized mouse eggs. Transgenic mouse lines that synthesize high levels of each anti-sickling hemoglobin were established and anti-sickling hemoglobins were purified from hemolysates and characterized. Both AS hemoglobins bind oxygen cooperatively and the oxygen affinities of these molecules are in the normal range. Delay time experiments demonstrate that Hb AS2 is a potent inhibitor of Hb S polymerization; therefore, locus control region beta AS2-globin gene constructs may be suitable for future gene therapy of sickle cell disease.
Characterization of three yeast copper-zinc superoxide dismutase mutants analogous to those coded for in familial amyotrophic lateral sclerosis.
Authors: Nishida C R , Gralla E B , Valentine J S
Published: May 2006

Sequences encoding three copper-zinc superoxide dismutase (CuZnSOD) mutant proteins analogous to those coded for in familial amyotrophic lateral sclerosis (fALS) were constructed in the Saccharomyces cerevisiae CuZnSOD gene and expressed in yeast lacking CuZnSOD (sod1-). Gly85-->Arg CuZnSOD failed to rescue the oxygen-sensitive phenotype of sod1- yeast, but Gly93-->Ala CuZnSOD and Lys100-->Gly CuZnSOD were apparently fully functional in vivo. The Gly85-->Arg mutant protein was purified and its metal-binding properties and SOD activity were found to be significantly altered relative to wild type. The Gly93-->Ala CuZnSOD was likewise purified but, in contrast, demonstrated metal-binding comparable to wild type and activity 80% that of wild type. These results suggest that SOD activity of human fALS mutant CuZnSODs may vary considerably in vivo, with at least some of them retaining a considerable amount of activity. Alternative theories to increased free-radical damage should be considered in attempting to explain fALS.
A rodent model for Wilms tumors: embryonal kidney neoplasms induced by N-nitroso-N'-methylurea.
Authors: Sharma P M , Bowman M , Yu B F , Sukumar S
Published: May 2006

Embryonal kidney cell tumors develop in rats given the alkylating agent N-nitroso-N'-methylurea as neonates. These tumors resemble the childhood Wilms tumors in their histopathology. Deletions and mutations in the Wilms tumor suppressor gene, WT1, are present in up to 6% of childhood nephroblastomas. To investigate the role of WT1 in rat kidney tumorigenesis, we studied the genetic alterations in WT1 and its target genes. Point mutations were found in WT1 cDNA in 7 of 18 kidney tumors. Mesenchymal tumors contained G-->A transition mutations in codons 128, 364, and 372, typical of the methylating action of N-nitroso-N'-methylurea on DNA. Each of the four nephroblastomas contained the same T-->A mutation at codon 111 of WT1, reflective of transversion mutagenesis by N-nitroso-N'-methylurea in vivo. Like Wilms tumors, mRNA levels of WT1, IGF2, Pax-2, and MK genes were higher than newborn kidney in the majority of the tumors. The histopathology of the rat kidney tumors and the genetic alterations are reminiscent of those observed in Wilms tumors, establishing this as a relevant model system for the human disease.
Transmission of Creutzfeldt-Jakob disease from humans to transgenic mice expressing chimeric human-mouse prion protein.
Authors: Telling G C , Scott M , Hsiao K K , Foster D , Yang S L , Torchia M , Sidle K C , Collinge J , DeArmond S J , Prusiner S B
Published: May 2006

Transgenic (Tg) mice were constructed that express a chimeric prion protein (PrP) in which a segment of mouse (Mo) PrP was replaced with the corresponding human (Hu) PrP sequence. The chimeric PrP, designated MHu2MPrP, differs from MoPrP by 9 amino acids between residues 96 and 167. All of the Tg(MHu2M) mice developed neurologic disease approximately 200 days after inoculation with brain homogenates from three patients dying of Creutzfeldt-Jakob disease (CJD). Inoculation of Tg(MHu2M) mice with CJD prions produced MHu2MPrPSc (where PrPSc is the scrapie isoform of PrP); inoculation with Mo prions produced Mo-PrPSc. The patterns of MHu2MPrPSc and MoPrPSc accumulation in the brains of Tg(MHu2M) mice were different. About 10% of Tg(HuPrP) mice expressing HuPrP and non-Tg mice developed neurologic disease > 500 days after inoculation with CJD prions. The different susceptibilities of Tg(HuPrP) and Tg(MHu2M) mice to Hu prions indicate that additional species-specific factors are involved in prion replication. Diagnosis, prevention, and treatment of Hu prion diseases should be facilitated by Tg(MHu2M) mice.
Intron sequence directs RNA editing of the glutamate receptor subunit GluR2 coding sequence.
Authors: Egebjerg J , Kukekov V , Heinemann S F
Published: May 2006

The Ca2+ permeability and the rectifying properties of the glutamate receptors assembled from the subunits GluR1-GluR4 depend upon a critical Arg in the GluR2 subunit located in a domain that has been proposed to span the membrane. The GluR2 subunit gene encodes a Gln (CAG) at this position, whereas the mRNA is edited so that it encodes an Arg (CGG) at this position [Sommer, B., Kohler, M., Sprengel, R. & Seeburg, P. H. (1991) Cell 67, 11-20]. The editing process is specific since only the GluR2 subunit RNA is edited even though the GluR1, GluR3, and GluR4 RNAs have a similar sequence. We show that this selective RNA editing depends upon a critical intron sequence in the GluR2 gene. This critical intron sequence is sufficient to cause editing of the GluR3 subunit exon in a chimera minigene constructed so that the GluR3 exon is placed upstream to the GluR2 intron sequence. Transfections of a neuronal cell line, N2a, with minigene constructs encoding different fragments of the GluR2 gene demonstrate that the 5' part of the 3' intron is essential for editing. Part of the exon and this critical intron sequence contains an inverted repeat that can fold into a structure consisting of three helical elements. Similar conclusions were reached by Higuchi, M., Single, F. n., Köhler, M., Sommer, B., Sprengel, R. & Seeburg, P. H. [(1993) Cell 75, 1361-1370]. These experiments demonstrate that the low Ca2+ permeability of the ionotropic non-N-methyl-D-aspartate glutamate receptors depends upon RNA editing, which requires a sequence in an intron 3' to the exon.
Linker histones H1 and H5 prevent the mobility of positioned nucleosomes.
Authors: Pennings S , Meersseman G , Bradbury E M
Published: May 2006

We have previously identified a generally occurring short-range mobility of nucleosome cores on DNA in relatively low ionic strength conditions. Here we report that this mobility of histone octamers positioned on constructs of 5S rDNA is suppressed by the binding of histone H1 or H5 to the nucleosome. Histone H5 is the more potent inhibitor of nucleosome mobility, in accordance with its higher affinity for chromatin. We propose that this reversible restraint on chromatin dynamics may play a role in local regulation of processes that require access to the DNA.
Split ubiquitin as a sensor of protein interactions in vivo.
Authors: Johnsson N , Varshavsky A
Published: May 2006

We describe an assay for in vivo protein interactions. Protein fusions containing ubiquitin, a 76-residue, single-domain protein, are rapidly cleaved in vivo by ubiquitin-specific proteases, which recognize the folded conformation of ubiquitin. When a C-terminal fragment of ubiquitin (C(ub)) is expressed as a fusion to a reporter protein, the fusion is cleaved only if an N-terminal fragment of ubiquitin (Nub) is also expressed in the same cell. This reconstitution of native ubiquitin from its fragments, detectable by the in vivo cleavage assay, is not observed with a mutationally altered Nub. However, if C(ub) and the altered Nub are each linked to polypeptides that interact in vivo, the cleavage of the fusion containing C(ub) is restored, yielding a generally applicable assay for kinetic and equilibrium aspects of in vivo protein interactions. This method, termed USPS (ubiquitin-based split-protein sensor), makes it possible to monitor a protein-protein interaction as a function of time, at the natural sites of this interaction in a living cell.
Expression of the gene for a small GTP binding protein in transgenic tobacco elevates endogenous cytokinin levels, abnormally induces salicylic acid in response to wounding, and increases resistance to tobacco mosaic virus infection.
Authors: Sano H , Seo S , Orudgev E , Youssefian S , Ishizuka K
Published: May 2006

Tobacco plants transformed with rgp1, a gene encoding a Ras-related small GTP binding protein, were previously shown to exhibit a distinct reduction in apical dominance with increased tillering. These abnormal pheno-types were later found to be associated with elevated levels of endogenous cytokinins (zeatin and zeatin riboside). Analysis of the expression of several genes known to be affected by cytokinins identified a clear increase in the mRNA levels of genes encoding acidic pathogenesis-related proteins in both transgenic plants and their progenies. This increase was directly attributable to elevated levels of the acidic pathogenesis-related protein inducers, salicylic acid (SA) and salicylic acid beta-glucoside, due to an abnormal and sensitive response of the transgenic plants to wounding. In contrast, mRNA levels of the gene for proteinase inhibitor II, which is normally induced by wounding, were generally suppressed in the same wounded plants, probably due to SA overproduction. The changes in SA and pathogenesis-related protein levels in the transgenic plants resulted in a distinct increase in their resistance to tobacco mosaic virus infection. In normal plants, the wound and pathogen-induced signal transduction pathways are considered to function independently. However, the wound induction of SA in the transgenic plants suggests that overexpression of this small GTP binding protein somehow interferes with the normal signal pathways, possibly by affecting cytokinin biosynthesis, and results in cross-signaling between these two transduction systems.
DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution.
Authors: Stemmer W P
Published: May 2006

Computer simulations of the evolution of linear sequences have demonstrated the importance of recombination of blocks of sequence rather than point mutagenesis alone. Repeated cycles of point mutagenesis, recombination, and selection should allow in vitro molecular evolution of complex sequences, such as proteins. A method for the reassembly of genes from their random DNA fragments, resulting in in vitro recombination is reported. A 1-kb gene, after DNase I digestion and purification of 10- to 50-bp random fragments, was reassembled to its original size and function. Similarly, a 2.7-kb plasmid could be efficiently reassembled. Complete recombination was obtained between two markers separated by 75 bp; each marker was located on a separate gene. Oligonucleotides with 3' and 5' ends that are homologous to the gene can be added to the fragment mixture and incorporated into the reassembled gene. Thus, mixtures of synthetic oligonucleotides and PCR fragments can be mixed into a gene at defined positions based on homology. As an example, a library of chimeras of the human and murine genes for interleukin 1 beta has been prepared. Shuffling can also be used for the in vitro equivalent of some standard genetic manipulations, such as a backcross with parental DNA. The advantages of recombination over existing mutagenesis methods are likely to increase with the numbers of cycles of molecular evolution.
Spermine potentiation of recombinant N-methyl-D-aspartate receptors is affected by subunit composition.
Authors: Zhang L , Zheng X , Paupard M C , Wang A P , Santchi L , Friedman L K , Zukin R S , Bennett M V
Published: May 2006

The present study shows that both the NR1 and NR2 subunits critically affect spermine potentiation of heteromeric recombinant N-methyl-D-aspartate receptors. NR1(011), the most prominent NR1 splice variant in rat forebrain, and NR1(100), prominent in midbrain, were expressed in Xenopus oocytes singly and in combination with NR2A, NR2B, and NR2C subunits. As for NR1(011) homomers, NR1(011)/NR2B receptors exhibited spermine potentiation by two mechanisms: by increasing glycine affinity and by increasing current through receptors with bound N-methyl-D-aspartate and glycine. NR1(011)/NR2A receptors exhibited only the increase in glycine affinity, and NR1(011)/NR2C receptors exhibited neither. As for NR1(100) homomers, NR1(100)/NR2B and NR1(100)/NR2A receptors exhibited spermine potentiation only by increasing the glycine affinity. Spermine produced no potentiation of NR1(100)/NR2C receptors. Thus, the NR2B subunit "permits" both forms of spermine potentiation, the NR2A subunit permits spermine potentiation only by increasing the glycine affinity, and th NR2C subunit permits neither form of potentiation. Spermine actions on NR1/NR2 showed little voltage dependence. These observations are of interest because the NR1 and NR2 subunits are differentially distributed and developmentally regulated. At early postnatal ages, NR2B subunit mRNA was more highly expressed than NR2A and NR2C mRNAs in hippocampus, neocortex, and caudate-putamen. These findings account for many of the observed differences among neurons in polyamine actions and suggest that these actions will vary in a cell-specific and age-related manner.
The role of phosphoenolpyruvate in the simultaneous uptake of fructose and 2-deoxyglucose by Escherichia coli.
Authors: Kornberg H , Lambourne L T
Published: May 2006

Nonmetabolizable glucose analogs inhibit the growth of Escherichia coli on a wide variety of carbon sources. This phenomenon was investigated with particular reference to the effect of 2-deoxyglucose (2DG) on growth on fructose as sole carbon source. When the inhibitor is supplied in sufficiently low concentrations, the initial arrest of growth is overcome; this relief of inhibition is aided by means that increase the availability of phosphoenolpyruvate (PEP) to the growing cells, such as the use of L-aspartate instead of ammonium chloride as sole nitrogen source for growth, and the introduction of the pps+ allele into a pps- strain. Studies with [14C]2DG showed that the analog or its 6-phosphate as such did not inhibit growth but that 2DG exerted its effect by competing for intracellular PEP and lowering its concentration below that needed to sustain growth. Direct measurements of the PEP-dependent phosphorylation of 2DG and of fructose by permeabilized E. coli showed that the apparent Km for PEP was nearly 7 times higher for 2DG that it was for fructose, although the apparent Vmax for 2DG was nearly 3 times that for fructose; this explains the ability of cells to overcome the inhibition by low, but not by high, concentrations of 2DG.
Functional nonequivalence of sperm in Drosophila pseudoobscura.
Authors: Snook R R , Markow T A , Karr T L
Published: May 2006

We report on a form of sperm polymorphism, termed polymegaly, that occurs in species of the Drosophila obscura group. Individual males of species in this group characteristically produce more than one discrete length of nucleated, motile sperm. Hypotheses suggested to explain the evolutionary significance of sperm polymorphism have been either nonadaptive or adaptive, with the latter focusing on sperm competition or nutrient provisioning. These hypotheses assume all sperm types fertilize eggs; however, no data have been gathered to test this assumption. We found that two size classes of sperm are produced and transferred to females in approximately equal numbers by the male; only long sperm persist in significant numbers in female sperm storage organs. Furthermore, we used a DNA-specific dye (bisbenzimide) and sperm-specific antibodies to ask if both sperm types fertilize eggs in Drosophila pseudoobscura. Confocal microscopy and immunofluorescent analyses of fertilized eggs using anti-sperm polyclonal antisera demonstrated that only long sperm participate in fertilization. These data falsify those hypotheses in which all sperm types are assumed to be functionally equivalent (fertilize eggs). Any remaining or new hypotheses for the evolutionary significance of polymegaly must incorporate these findings. Several new areas of research are suggested.
Human immunodeficiency virus type 1 strains in the lungs of infected individuals evolve independently from those in peripheral blood and are highly conserved in the C-terminal region of the envelope V3 loop.
Authors: Itescu S , Simonelli P F , Winchester R J , Ginsberg H S
Published: May 2006

To determine whether human immunodeficiency virus type 1 (HIV-1) strains in the lungs of infected individuals are derived from proviral forms contemporaneously present in the peripheral blood or whether they evolve independently as an autonomous pool of viral quasispecies, HIV-1 envelope V3 domain structures at these sites were analyzed and compared. The V3 loop proviral nucleotide and inferred amino acid sequences from lung bronchoalveolar lavage, where HIV-1 is primarily found in macrophages, were more homogeneous within individuals than those from unseparated peripheral blood mononuclear cells, where virus is predominantly in T cells. Comparison between individuals revealed that strains from bronchoalveolar lavage, but not from peripheral blood mononuclear cells, contained V3 domain nucleotide sequences with a great degree of homogeneity in the C-terminal region and a highly conserved, negatively charged amino acid motif. This V3 loop C-terminal structure could be important in the ability of HIV-1 to infect alveolar macrophages. Phylogenetic analyses of V3 domain nucleotide sequences in cells of monocyte/macrophage lineage at both sites revealed the strains in lung macrophages to have evolved further from a presumed ancestral species than those in blood monocytes and to differ considerably in the inferred V3 loop amino acid structures. These results show that, as disease progression occurs, viral strains in monocyte/macrophage lineage cells within the lung and blood microenvironments are not in a state of unrestricted bidirectional traffic but, instead, evolve independently.
C1-Cx revisited: intramolecular synergism in a cellulase.
Authors: Din N , Damude H G , Gilkes N R , Miller R C , Warren R A , Kilburn D G
Published: May 2006

Endoglucanase A (CenA) from the bacterium Cellulomonas fimi is composed of a catalytic domain and a nonhydrolytic cellulose-binding domain that can function independently. The individual domains interact synergistically in the disruption and hydrolysis of cellulose fibers. This intramolecular synergism is distinct from the well-known intermolecular synergism between individual cellulases. The catalytic domain corresponds to the hydrolytic Cx system and the cellulose-binding domain corresponds to the nonhydrolytic C1 system postulated by Reese et al. [Reese, E. T., Sui, R. G. H. & Levinson, H. S. (1950) J. Bacteriol. 59, 485-497] to be required for the hydrolysis of cellulose.
Stable delivery of physiologic levels of recombinant erythropoietin to the systemic circulation by intramuscular injection of replication-defective adenovirus.
Authors: Tripathy S K , Goldwasser E , Lu M M , Barr E , Leiden J M
Published: May 2006

A number of inherited and acquired serum protein deficiencies including hemophilias A and B, diabetes mellitus, and the erythropoietin-responsive anemias are currently treated with repeated subcutaneous or intravenous infusions of purified or recombinant proteins. The development of an in vivo gene-transfer approach to deliver physiologic levels of recombinant proteins to the systemic circulation would represent a significant advance in the treatment of these disorders. Here we describe the construction of a replication-defective adenovirus (AdEF1hEpo) containing the human erythropoietin (hEpo) cDNA under the transcriptional control of the cellular elongation factor 1 alpha (EF1 alpha) promoter and the 4F2 heavy chain (4F2HC) enhancer. Neonatal CD-1 and adult SCID mice injected once intramuscularly (i.m.) with 10(7) to 10(9) plaque-forming units (pfu) of this virus displayed significant dose-dependent elevations of serum hEpo levels and increased hematocrits, which were stable over the 4-month time course of these experiments. Adenovirus injected i.m. remained localized at the site of injection and there was no evidence of either systemic infection or a localized inflammatory response. These results suggest that i.m. injection of recombinant replication-defective adenovirus vectors may serve as a paradigm for the treatment of human serum protein deficiencies.
Panglobal distribution of a single clonal lineage of the Irish potato famine fungus.
Authors: Goodwin S B , Cohen B A , Fry W E
Published: May 2006

More than 300 isolates of the Irish potato famine fungus, Phytophthora infestans, collected in 20 countries on five continents, were analyzed for genetic variation at the mating type and two allozyme loci. A subset of more than 200 isolates was also analyzed for DNA "fingerprint" variation. A surprising result was that a single clonal lineage dominated most populations worldwide. All of the variation within this lineage appeared to have arisen by mitotic recombination or by mutation. In addition to the most common clonal lineage, a number of different, but apparently closely related, lineages occurred in the United States and Canada. The low levels of gene diversity in the derived populations compared to the presumed ancestral population in central Mexico indicate that P. infestans went through extreme genetic bottlenecks during its dispersal. The genetic data are consistent with the hypothesis that the initial migration of P. infestans in the 1840s was from Mexico to the United States and that only a single genetic individual was transported to Europe and subsequently to the rest of the world. If this hypothesis is correct, then the Irish potato famine was caused by a single clonal genotype of P. infestans.
Localization of neuropeptide Y Y1 receptors in the rat nervous system with special reference to somatic receptors on small dorsal root ganglion neurons.
Authors: Zhang X , Bao L , Xu Z Q , Kopp J , Arvidsson U , Elde R , Hökfelt T
Published: May 2006

Immunohistochemical staining with an antiserum against the neuropeptide Y (NPY)-Y1 receptor (Y1-R) protein was shown in rat small dorsal root ganglion (DRG) neurons, which also were Y1-R mRNA-positive and calcitonin gene-related peptide (CGRP)-positive. The Y1-R-like immunoreactivity was almost exclusively located in the somatic plasmalemma and in the perinuclear region. Intracellular recording showed that the Y1 agonist [Leu31,Pro34]NPY evoked an outward current in small DRG neurons, suggesting a functional somatic Y1-R. No evidence for axonal transport of Y1-R protein was obtained after analysis of the dorsal horn for double staining with CGRP, after dorsal rhizotomy, or after compression of dorsal roots and the sciatic nerve. It is proposed that blood-borne NPY released from sympathetic nerves and adrenal medulla is the endogenous ligand for the Y1 receptors on the small DRG neurons. Y1-R-positive neurons were also seen in the dorsal horn of spinal cord, the hypothalamic arcuate nucleus, pyramidal cells in the cerebral cortex, Purkinje and basket cells in the cerebellar cortex, and in many other brain regions.
Solution scattering from 50S ribosomal subunit resolves inconsistency between electron microscopic models.
Authors: Svergun D I , Pedersen J S , Serdyuk I N , Koch M H
Published: May 2006

Models of the 50S ribosomal subunit from electron microscopy on isolated particles and on ordered sheets display significantly different features. A model of the shape of the native Escherichia coli 50S subunit in solution and of its RNA-rich core at 4-nm resolution has been produced by using methods for joint interpretation of x-ray and neutron small-angle scattering data obtained by contrast variation. The good agreement between the shape of the entire 50S subunit and the electron microscopic models of isolated particles and between the RNA-rich core and the model obtained from ordered sheets leads to the conclusion that the latter, which is based on the subjective contouring of density maps, is heavily biased toward the RNA.
Nuclear accumulation of interferon gamma.
Authors: Bader T , Weitzerbin J
Published: May 2006

Examination of the interferon gamma (IFN-gamma) amino acid sequence revealed two conserved basic amino acid clusters similar to the prototype nuclear localization signal. We followed the fate of cell surface receptor-bound IFN-gamma in murine leukemia L1210 cells. A time- and temperature-dependent accumulation of murine IFN-gamma in the cell nucleus could be demonstrated by autoradiography and indirect immunofluorescence after the rapid isolation of nuclei. Human IFN-gamma was also internalized and translocated to the nucleus of murine L1210 cells transfected with and expressing the human IFN-gamma receptor, but it appeared to be retained by the nucleus only transiently. IFN-gamma molecules chemically crosslinked to their cell surface receptor remain capable of being translocated to the nucleus even as part of a receptor-ligand complex. Thus, the bipartite nuclear localization signal sequence appears to be functional and suggests that nuclear targeting could participate in IFN-gamma signal transduction.
Directing transcription of an RNA polymerase III gene via GAL4 sites.
Authors: Marsolier M C , Chaussivert N , Lefebvre O , Conesa C , Werner M , Sentenac A
Published: May 2006

A yeast chimeric RNA polymerase III transcription system was constructed to explore the ordered, multistep process of gene activation in vivo. A promoter-deficient U6 RNA gene harboring GAL4-binding sites could be reactivated by fusing the GAL4 DNA-binding domain to components of the general transcription factor TFIIIC (tau) or TFIIIB. Expression of chimeric tau 138 or tau 131 (but not tau 95) subunits activated transcription from GAL4-binding sites located at various positions, including upstream of or within the gene. The function(s) of the B block binding domain of TFIIIC was provided by the fused GAL4-(1-147) domain. The GAL4-(1-147)-TFIIIB70 fusion protein acted at a distance like an activator of transcription. In contrast, none of the 10 different GAL4-(1-147)-polymerase subunit fusions was able to induce transcription, suggesting that RNA polymerase recruitment is not sufficient to initiate transcription.
Rapid humanization of the Fv of monoclonal antibody B3 by using framework exchange of the recombinant immunotoxin B3(Fv)-PE38.
Authors: Benhar I , Padlan E A , Jung S H , Lee B , Pastan I
Published: May 2006

B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv region of carcinoma-specific antibody B3 is fused to a truncated form of Pseudomonas exotoxin (PE). The efficacy of monoclonal antibody B3 and B3 immunotoxins in cancer therapy and diagnosis may be limited by the human anti-mouse response. Here we describe the humanization of the Fv of B3(Fv)-PE38 by "framework exchange." The variable domains of the heavy (VH) and light (VL) chains were aligned with their best human homologs to identify framework residues that differ. Initially, 11 framework residues in VH and six in VL were changed by site-specific mutagenesis to human residues and introduced simultaneously into a preassembled single-chain Fv expression cassette. Six VH and five VL residues that differ were not changed because they were buried, in the interdomain interface, or previously found to result in decreased affinity when mutated. This basic design resulted in some 20-fold loss of activity. Changing VL residues at the interdomain interfacial position 100 and at the buried position 104 to the human sequence increased the activity 8-fold. Changing VH residue at position 82b from the human sequence back to that of the mouse restored the activity 2- to 3-fold to the full binding and cytotoxic activity of the mouse sequence. Humanized B3(Fv)-PE38 lost immunogenic epitopes recognized by sera from monkeys that had been immunized with B3(Fv)-PE38.
An anti-peptide antibody specific for the class A calcium channel alpha 1 subunit labels mammalian neuromuscular junction.
Authors: Ousley A H , Froehner S C
Published: May 2006

We have generated an anti-peptide antibody specific for the class A calcium channel alpha 1 subunit from rat brain. In immunoblots of the calcium channel complex partially purified from rat brain membranes, the antibody specifically recognized two doublets, one of apparent M(r) 210,000 and M(r) 180,000 and another of apparent M(r) 160,000 and M(r) 130,000. Immunofluorescent staining of sections of rat diaphragm showed that this antibody specifically recognizes antigens that are highly concentrated at neuromuscular synapses. Using this antibody, we also examined the distribution of the class A alpha 1 subunit in sagittal sections of rat cerebellum by immunoperoxidase staining. Specific immunoreactivity was localized in the granule cell layer, possibly to the cerebellar glomeruli, the unique structures in cerebellum where mossy fibers synapse with granule cell dendrites. Purkinje cell neurons were not stained specifically. These results indicate that the class A calcium channel alpha 1 subunit is highly concentrated at mammalian neuromuscular junction and has a restricted localization in cerebellum that does not include Purkinje cell soma or dendrites.
Expression of apple 1-aminocyclopropane-1-carboxylate synthase in Escherichia coli: kinetic characterization of wild-type and active-site mutant forms.
Authors: White M F , Vasquez J , Yang S F , Kirsch J F
Published: May 2006

The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) catalyzes the conversion of S-adenosylmethionine (AdoMet) to ACC and 5'-methylthioadenosine, the committed step in ethylene biosynthesis in plants. Apple ACC synthase was overexpressed in Escherichia coli (3 mg/liter) and purified to near homogeneity. A continuous assay was developed by coupling the ACC synthase reaction to the deamination of 5'-methylthioadenosine by adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Aspergillus oryzae. The enzyme is dimeric, with kcat = 9s-1 per monomer and Km = 12 microM for AdoMet. The pyridoxal phosphate-binding site of ACC synthase appears to be highly homologous to that of aspartate aminotransferase, suggesting similar roles for corresponding residues. Site-directed mutagenesis of Lys-273, Arg-407, and Tyr-233 (corresponding to residues 258, 386, and 225 in aspartate aminotransferase) and kinetic analyses of the mutants confirms their importance in the ACC synthase mechanism. The Lys-273 to Ala mutant has no detectable activity, supporting the identification of this residue as the base catalyzing C alpha proton abstraction. Mutation of Arg-407 to Lys results in a precipitous drop in kcat/Km and an increase in Km for AdoMet of at least 20-fold, in accordance with its proposed role as principal ligand for the substrate alpha-carboxylate group. Replacement of Tyr-233 with Phe causes a 24-fold increase in the Km for AdoMet and no change in kcat, suggesting that this residue plays a role in orienting the pyridoxal phosphate cofactor in the active site.
RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex.
Authors: Chiu M I , Katz H , Berlin V
Published: May 2006

Rapamycin is a potent immunosuppressant that blocks the G1/S transition in antigen-activated T cells and in yeast. The similar effects of rapamycin in animal cells and yeast suggest that the biochemical steps affected by rapamycin are conserved. Using a two-hybrid system we isolated mammalian clones that interact with the human FK506/rapamycin-binding protein (FKBP12) in the presence of rapamycin. Specific interactors, designated RAPT1, encode overlapping sequences homologous to yeast Tor, a putative novel phosphatidylinositol 3-kinase. A region of 133 amino acids of RAPT1 is sufficient for binding to the FKBP12/rapamycin complex. The corresponding region in yeast Tor contains the serine residue that when mutated to arginine confers resistance to rapamycin. Introduction of this mutation into RAPT1 abolishes its interaction with the FKBP12/rapamycin complex.
Physiological cytosolic Ca2+ transients evoke concurrent mitochondrial depolarizations.
Authors: Loew L M , Carrington W , Tuft R A , Fay F S
Published: May 2006

Calcium, a ubiquitous second messenger, stimulates the activity of several mitochondrial dehydrogenases. This has led to the suggestion that the same messenger that signals cell activation could also activate mitochondrial electron/proton transport, thereby meeting demands for increased cellular energy. To test this in live cells, quantitative three-dimensional microscopy and ratio imaging were used to measure membrane potential of individual mitochondria and cytosolic calcium distribution. Mitochondria reversibly depolarized as cytosolic calcium rose and then fell following physiological stimulation. Thus, the dominant response of the mitochondrion to a rise in cytosolic [Ca2+] is to draw on the electrochemical potential, possibly to accelerate processes directly involved in ATP synthesis and calcium homeostasis.
Homeotic transformation of cervical vertebrae in Hoxa-4 mutant mice.
Authors: Horan G S , Wu K , Wolgemuth D J , Behringer R R
Published: May 2006

Hoxa-4 (previously known as Hox-1.4) is a mouse homeobox-containing gene that is expressed in the presumptive hindbrain and spinal cord, prevertebrae, and other tissues during embryogenesis. To understand the role of Hoxa-4 during development, we generated Hoxa-4 mutant mice. Homozygous mutants were viable and fertile. Analysis of neonatal skeletons revealed the development of ribs on the seventh cervical vertebra at variable penetrance and expressivity. A low frequency of alterations in sternal morphogenesis was also observed. In addition, we analyzed the skeletons of transgenic mice that overexpress Hoxa-4 and found that the formation of the small rib anlagen that often develop on the seventh cervical vertebra was suppressed. Analysis of adult homozygous mutant skeletons revealed that the dorsal process normally associated with the second cervical vertebra was also found on the third cervical vertebra. These results demonstrate that Hoxa-4 plays a role in conferring positional information along the anteroposterior axis to specify the identity of the third and the seventh cervical vertebrae.
Phosphorylation negatively regulates the function of coactivator PC4.
Authors: Ge H , Zhao Y , Chait B T , Roeder R G
Published: May 2006

Human positive cofactor 4 (PC4) mediates activator-dependent transcription by RNA polymerase II, apparently through interactions with transcriptional activators and the basal transcription machinery. We report here that PC4 function is modulated by in vivo phosphorylation. Protein-protein interaction studies and in vitro transcription assays demonstrate that only the nonphosphorylated form of PC4 is functionally active. Although recombinant PC4 can be phosphorylated by casein kinase II and protein kinase C in vitro, mutational and mass spectrometric analyses suggest that the in vivo hyperphosphorylation of PC4 is mediated mainly by casein kinase II and restricted to an N-terminal serine-rich region. These observations provide one example of a transcriptional cofactor that is negatively regulated by casein kinase II phosphorylation.
Allele-specific quantification of Drosophila engrailed and invected transcripts.
Authors: Goldsborough A S , Kornberg T B
Published: May 2006

Changes in levels of transcription can be difficult to gauge in animals with lethal mutations. For example, mutations in a regulatory region of an essential gene can have secondary consequences that complicate attempts to quantify the transcripts produced by the mutant gene. We describe a method that circumvents this problem by revealing the relative amount of transcript produced from each allele in a heterozygote. With this method, recessive lethal mutations can be analyzed in animals that are phenotypically wild type. We used this technique to analyze mutations in the regulatory region of the Drosophila engrailed gene and found that truncations reduce transcription to levels that depend both upon the tissue and upon the location of the chromosomal break. We also found that these mutations affect expression of the linked invected gene, suggesting that engrailed and invected share a complex set of regulatory elements that operate over at least 85 kb. We suggest that this technique will have general utility for the quantitation of allele-specific transcripts, even when amounts of tissue are limiting.
Molecular evidence for two forms of Crohn disease.
Authors: Gilberts E C , Greenstein A J , Katsel P , Harpaz N , Greenstein R J
Published: May 2006

Recent epidemiological evidence suggests that there are two forms of Crohn disease (CD): perforating and nonperforating. We hypothesized that, just as with tuberculoid and lepromatous leprosy, differences in the two forms of CD would be both identified and determined by differences in the host immune response. Resected intestinal tissue from control patients as well as perforating and nonperforating CD patients was evaluated for mRNA levels. We employed 32P PCR amplification with published or custom-designed primers of a housekeeping gene (beta-actin); a human T-cell marker (CD3-delta); and the cytokines tumor necrosis factor alpha, transforming growth factor beta, granulocyte/macrophage colony-stimulating factor, interleukin (IL) 1 beta, IL-1ra, and IL-6. Differences were identified with IL-1 beta (control = 162 +/- 57 vs. perforating = 464 +/- 154 vs. nonperforating = 12,582 +/- 4733; P < or = 0.02) and IL-1ra (control = 1337 +/- 622 vs. perforating = 2194 +/- 775 vs. nonperforating = 9715 +/- 2988; P < or = 0.02). These data corroborate the epidemiological observation that there are two forms of CD. Nonperforating CD, the more benign form, is associated with increased IL-1 beta and IL-1ra mRNA expression. We conclude that it is the host immune response that determines which form of CD becomes manifest in any given individual and discuss the investigative, diagnostic, and therapeutic implications of these observations.
An alternative Fc gamma-receptor ligand: potential role in T-cell development.
Authors: Sandor M , Galon J , Takacs L , Tatsumi Y , Mueller A L , Sautes C , Lynch R G
Published: May 2006

Fetal pre-T cells express low-affinity receptors for IgG (Fc gamma R) at a developmental stage prior to the rearrangement and expression of immunoglobulin genes. The present studies investigated the possible functional significance of Fc gamma R on fetal pre-T cells. Between 13 and 17 days of fetal development a subpopulation of T-cell receptor-, Thy-1+ thymocytes express for gamma R. The same cells contain mRNA for several forms of Fc gamma R (Fc gamma RII beta 1, beta 2, and Fc gamma RIII). Concurrently, a Pgp-1-, Thy-1-, surface-immunoglobulin- fetal thymic cell binds recombinant soluble Fc gamma R. In principle this cell can interact with the pre-T cells through this counter-receptor. To test this possibility anti-Fc gamma RII/III antibody (2.4G2) was injected into pregnant mice and then into their offspring for 6 wk postpartum. The injected antibody induced a slight increase in the proportion of CD4 or CD8 single-positive, alpha/beta T cells in the thymus. However, in fetal thymic cultures in the presence of 2.4G2 or the recombinant soluble Fc gamma R there was an accelerated differentiation of thymocytes to single-positive, CD3-bright, heat-stable antigen-dull, alpha/beta T cells. These experiments show that Fc gamma Rs are present on pre-T cells during early fetal thymic development, and that a non-IgG ligand of the Fc gamma R is expressed concurrently on Thy- fetal thymocytes. Furthermore, the presumed interaction of Fc gamma R and the alternative ligand(s) influences T-cell development. IgG binding could be an adapted function of Fc gamma Rs, and, as shown for many members of the Ig super family, these receptors may have originally served as cell-cell recognition/interaction molecules required for hematopoietic development.
Kinetics of T-cell receptor binding to peptide/I-Ek complexes: correlation of the dissociation rate with T-cell responsiveness.
Authors: Matsui K , Boniface J J , Steffner P , Reay P A , Davis M M
Published: May 2006

Recognition by T-cell antigen receptors (TCRs) of processed peptides bound to major histocompatibility complex (MHC) molecules is required for the initiation of most T-lymphocyte responses. Despite the availability of soluble forms of TCRs and MHC heterodimers, this interaction has proven difficult to study directly due to the very low affinity. We report here on the kinetics of TCR binding to peptide/MHC complexes in a cell-free system using surface plasmon resonance. The apparent association rates for the interactions of related peptide/MHC complexes to one such TCR are relatively slow (900-3000 M-1.s-1) and dissociation rates are very fast (0.3-0.06 s-1) with t1/2 of 2-12 s at 25 degrees C. The calculated affinity of the engineered soluble molecules compares well with previously reported competition data for native TCRs or competition data reported here for native peptide/MHC complexes, indicating that these soluble heterodimers bind in the same manner as the original molecules expressed on cells. We also find that the peptide variants which give weaker T-cell stimulatory responses have similar affinities but distinctly faster dissociation rates compared with the original peptide (when loaded onto the MHC molecule) and that this later property may be responsible for their lower activity. This has implications for both downstream signaling events and models of TCR-peptide antagonists.
A putative fourth Na+,K(+)-ATPase alpha-subunit gene is expressed in testis.
Authors: Shamraj O I , Lingrel J B
Published: May 2006

The Na+,K(+)-ATPase alpha subunit has three known isoforms, alpha 1, alpha 2 and alpha 3, each encoded by a separate gene. This study was undertaken to determine the functional status of a fourth human alpha-like gene, ATP1AL2. Partial genomic sequence analysis revealed regions exhibiting sequence similarity with exons 3-6 of the Na+,K(+)-ATPase alpha isoform genes. ATP1AL2 cDNAs spanning the coding sequence of a novel P-type ATPase alpha subunit were isolated from a rat testis library. The predicted polypeptide is 1028 amino acids long and exhibits 76-78% identity with the rat Na+,K(+)-ATPase alpha 1, alpha 2 and alpha 3 isoforms, indicating that ATP1AL2 may encode a fourth Na+,K(+)-ATPase alpha isoform. A 3.9-kb mRNA is expressed abundantly in human and rat testis.
Thermodynamic procedure to synthesize heteropolymers that can renature to recognize a given target molecule.
Authors: Pande V S , Grosberg A Y , Tanaka T
Published: May 2006

We suggest a procedure to synthesize polymers with characteristics similar to those observed in globular proteins: renaturability and the existence of an "active site" capable of specifically recognizing a given target molecule. This procedure is investigated by computer simulation, which finds a yield of up to 65%. We believe that, in principle, this scheme can be realized in vitro. The applicability of this approach as a model of prebiotic synthesis in vivo is also discussed.
Cloning and expression of a cytoskeleton-associated diacylglycerol kinase that is dominantly expressed in cerebellum.
Authors: Goto K , Funayama M , Kondo H
Published: May 2006

A third species of diacylglycerol kinase (EC 2.7.1.107) cDNA was cloned from a rat brain cDNA library. The isolated cDNA encoded a 788-amino acid, 88-kDa polypeptide. This isozyme shared 58% identity with the previously isolated rat 80-kDa and 90-kDa diacylglycerol kinases. EF hand motifs, cysteine-rich zinc finger-like sequences, and putative ATP-binding site were all conserved among these isozymes. The 88-kDa diacylglycerol kinase was expressed specifically in brain and localized predominantly in cerebellar Purkinje cells. This isozyme was associated equally with particulate and supernatant fractions in cDNA-transfected COS-7 cells and dominantly with the particulate fraction in the brain. After Triton X-100 extraction, this isozyme remained in the detergent-insoluble cytoskeletal fraction of the brain and transfected COS-7 cells.