bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of residues in the Mu transposase essential for catalysis.
Authors: Baker T A , Luo L
Published: May 2006

A tetramer of Mu transposase (MuA) cleaves the phage Mu DNA and joins these ends to a target DNA to catalyze transposition. Substitution mutations at Asp-269 or Glu-392 within MuA destroy both the DNA cleavage and joining activities without blocking tetramer assembly, indicating that the mutations specifically affect catalysis. Although inactive under standard reaction conditions (10 mM Mg2+), the mutant proteins are partially resuscitated by 10-20 mM Mn2+, concentrations 5- to 10-fold higher than optimal for wild-type MuA. Amino acid sequence alignment and the similar effects of mutations suggests that Asp-269 and Glu-392 of MuA may be analogs of the first Asp and final Glu of a conserved triad of acidic amino acids present in many transposases and the retroviral integrases (the D-D-35-E motif). The higher Mn2+ optima observed with MuA derivatives altered at these positions supports a role for the conserved acidic amino acids in coordinating divalent metal ions in the active sites of transposases.
Morphological evolution through complex domains of fitness.
Authors: Niklas K J
Published: May 2006

Computer simulated phenotypic walks through multi-dimensional fitness-landscapes indicate that (i) the number of phenotypes capable of reconciling conflicting morphological requirements increases in proportion to the number of manifold functional obligations an organism must perform to grow, survive, and reproduce, and (ii) walks over multi-task fitness-landscapes require fewer but larger phenotypic transformations than those through single-task landscapes. These results were determined by (i) simulating a "morphospace" containing 200,000 phenotypes reminiscent of early Paleozoic vascular sporophytes, (ii) evaluating the capacity of each morphology to perform each of three tasks (light interception, mechanical support, and reproduction) as well as the ability to reconcile the conflicting morphological requirements for the four combinatorial permutations of these tasks, (iii) simulating the walks obtaining all phenotypic maxima or optima within the seven "fitness-landscapes," and (iv) computing the mean morphological variation attending these walks. The results of these simulations, whose credibility is discussed in the context of early vascular land-plant evolution, suggest that both the number and the accessibility of phenotypic optima increase as the number of functional obligations contributing to total fitness increases (i.e., as the complexity of optimal phenotypes increases, the fitnesses of optima fall closer to the mean fitness of all the phenotypes under selection).
Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters.
Authors: Kavanaugh M P , Miller D G , Zhang W , Law W , Kozak S L , Kabat D , Miller A D
Published: May 2006

Cell surface receptors for gibbon ape leukemia virus (Glvr-1) and murine amphotropic retrovirus (Ram-1) are distinct but related proteins having multiple membrane-spanning regions. Distant homology with a putative phosphate permease of Neurospora crassa suggested that these receptors might serve transport functions. By expression in Xenopus laevis oocytes and in mammalian cells, we have identified Glvr-1 and Ram-1 as sodium-dependent phosphate symporters. Two-electrode voltage-clamp analysis indicates net cation influx, suggesting that phosphate is transported with excess sodium ions. Phosphate uptake was reduced by > 50% in mouse fibroblasts expressing amphotropic envelope glycoprotein, which binds to Ram-1, indicating that Ram-1 is a major phosphate transporter in these cells. RNA analysis shows wide but distinct tissue distributions, with Glvr-1 expression being highest in bone marrow and Ram-1 in heart. Overexpression of Ram-1 severely repressed Glvr-1 synthesis in fibroblasts, suggesting that transporter expression may be controlled by net phosphate accumulation. Accordingly, depletion of extracellular phosphate increased Ram-1 and Glvr-1 expression 3- to 5-fold. These results suggest simple methods to modulate retroviral receptor expression, with possible applications to human gene therapy.
Linkage of mating-type loci distinguishes bipolar from tetrapolar mating in basidiomycetous smut fungi.
Authors: Bakkeren G , Kronstad J W
Published: May 2006

Sexual compatibility requires self vs. non-self recognition. Genetically, two compatibility or mating-type systems govern recognition in heterothallic basidiomycete fungi such as the edible and woodrotting mushrooms and the economically important rust and smut phytopathogens. A bipolar system is defined by a single genetic locus (MAT) that can have two or multiple alleles. A tetrapolar system has two loci, each with two or more specificities. We have employed two species from the genus Ustilago (smut fungi) to discover a molecular explanation for the genetic difference in mating systems. Ustilago maydis, a tetrapolar species, has two genetically unlinked loci that encode the distinct mating functions of cell fusion (a locus) and subsequent sexual development and pathogenicity (b locus). We have recently described a b locus in a bipolar species, Ustilago hordei, wherein the existence of an a locus has been suspected, but not demonstrated. We report here the cloning of an allele of the a locus (a1) from U. hordei and the discovery that physical linkage of the a and b loci in this bipolar fungus accounts for the distinct mating system. Linkage establishes a large complex MAT locus in U. hordei; this locus appears to be in a region suppressed for recombination.
Proposed three-dimensional structure for the cellular prion protein.
Authors: Huang Z , Gabriel J M , Baldwin M A , Fletterick R J , Prusiner S B , Cohen F E
Published: May 2006

Prion diseases are a group of neurodegenerative disorders in humans and animals that seem to result from a conformational change in the prion protein (PrP). Utilizing data obtained by circular dichroism and infrared spectroscopy, computational studies predicted the three-dimensional structure of the cellular form of PrP (PrPc). A heuristic approach consisting of the prediction of secondary structures and of an evaluation of the packing of secondary elements was used to search for plausible tertiary structures. After a series of experimental and theoretical constraints were applied, four structural models of four-helix bundles emerged. A group of amino acids within the four predicted helices were identified as important for tertiary interactions between helices. These amino acids could be essential for maintaining a stable tertiary structure of PrPc. Among four plausible structural models for PrPc, the X-bundle model seemed to correlate best with 5 of 11 known point mutations that segregate with the inherited prion diseases. These 5 mutations cluster around a central hydrophobic core in the X-bundle structure. Furthermore, these mutations occur at or near those amino acids which are predicted to be important for helix-helix interactions. The three-dimensional structure of PrPc proposed here may not only provide a basis for rationalizing mutations of the PrP gene in the inherited prion diseases but also guide design of genetically engineered PrP molecules for further experimental studies.
A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in vitro.
Authors: Ellison V , Brown P O
Published: May 2006

Retroviral replication depends on integration of the viral genome into a chromosome of the host cell. The steps in this process are orchestrated in vivo by a large nucleoprotein complex and are catalyzed by the retroviral enzyme integrase. Several biochemical properties of the in vivo nucleoprotein complex were reproduced in vitro with purified integrase of human immunodeficiency virus type 1 and model viral DNA substrates. A stable complex between integrase and viral DNA was detected as an early intermediate in the integration reaction. After formation of this initial complex, the enzyme processively catalyzed the 3' end processing and strand transfer steps in the reaction. Complexes containing only purified integrase and the model viral DNA end were stable under a variety of conditions and efficiently used nonviral DNA molecules as integration targets. These complexes required a divalent cation for their formation, and their stability was highly dependent on the 5'-terminal dinucleotide of the viral DNA, for which no functional role has previously been defined. Thus, interactions between integrase and the extreme ends of the viral DNA molecule may be sufficient to account for the stability of the in vivo integration complex.
Protein-tyrosine-phosphatase SHPTP2 couples platelet-derived growth factor receptor beta to Ras.
Authors: Bennett A M , Tang T L , Sugimoto S , Walsh C T , Neel B G
Published: May 2006

Protein-tyrosine-phosphatase SHPTP2 (Syp/PTP-1D/PTP2C) is the homologue of the Drosophila corkscrew (csw) gene product, which transmits positive signals from receptor tyrosine kinases. Likewise, SHPTP2 has been implicated in positive signaling from platelet-derived growth factor receptor beta (PDGFR). Upon PDGF stimulation, SHPTP2 binds to the PDGFR and becomes tyrosine-phosphorylated. We have identified tyrosine-542 (pY542TNI) as the major in vivo site of SHPTP2 tyrosine phosphorylation. The pY542TNI sequence conforms to the consensus binding site for the SH2 domain of Grb2, which, by association with Sos1, couples some growth factor receptors to Ras. Following PDGF stimulation, Grb2 binds tyrosine-phosphorylated SHPTP2. Moreover, a mutant PDGFR lacking its SHPTP2 binding site displays markedly reduced Grb2 binding. These data indicate that phosphorylation of SHPTP2 couples Grb2 to PDGFR in vivo, providing a mechanism for Ras activation by PDGFR and for positive signaling via SHPTP2 and Csw.
Expression of an activated erythropoietin or a colony-stimulating factor 1 receptor by pluripotent progenitors enhances colony formation but does not induce differentiation.
Authors: Pharr P N , Ogawa M , Hofbauer A , Longmore G D
Published: May 2006

Whether the presence of specific receptors on the surface of developing cells is the cause or consequence of lineage restriction is not known. If activation of specific receptors is the driving event in differentiation, the premature expression of specific receptors would promote differentiation along that pathway. In this study pluripotent progenitors, obtained from blast cell colonies (pooled or individual) of 5-fluorouracil-treated mice, were infected with retroviral vectors containing either an activated receptor for erythropoietin (EPO), an erythroid progenitor growth factor, or the receptor for colony-stimulating factor 1 (CSF-1), a macrophage growth factor. These receptors exhibit expression patterns restricted to committed progenitors. The developmental potential of infected pluripotent progenitors was not changed, although they expressed the exogenous genes, suggesting that in these cells activation of lineage-specific receptors does not induce differentiation. Acquisition of a constitutively activated EPO receptor allowed erythroid development in mixed colonies in the absence of EPO, as expected. Infection of progenitors with a virus containing the CSF-1 receptor promoted the development of granulocyte/macrophage (GM) colonies but did not alter the differentiation potential of either colony-forming unit (CFU)-GM or CFU-mix.
Prevention of hepatitis C virus infection in chimpanzees after antibody-mediated in vitro neutralization.
Authors: Farci P , Alter H J , Wong D C , Miller R H , Govindarajan S , Engle R , Shapiro M , Purcell R H
Published: May 2006

Hepatitis C virus (HCV) is the most important etiologic agent of non-A, non-B hepatitis and is a major cause of chronic liver disease and hepatocellular carcinoma. Development of an effective vaccine would be the most practical method for prevention of the infection, but whether infection with HCV elicits protective immunity in the host is unclear. Neutralization of HCV in vitro was attempted with plasma of a chronically infected patient, and the residual infectivity was evaluated by inoculation of eight seronegative chimpanzees. The source of HCV was plasma obtained from a patient during the acute phase of posttransfusion non-A, non-B hepatitis, which had previously been titered for infectivity in chimpanzees. Neutralization was achieved with plasma obtained from the same patient 2 yr after the onset of primary infection but not with plasma obtained 11 yr later, although both plasmas contained antibodies against nonstructural and structural (including envelope) HCV proteins. Analysis of sequential viral isolates from the same patient revealed significant genetic divergence as early as 2 yr after infection. However, the HCV recovered from the patient 2 yr after the infection had a striking sequence similarity with the HCV recovered from one of the chimpanzees inoculated with the acute-phase virus, suggesting that the progenitor of the new strain was already present 2 yr earlier. This evidence, together with the different sequences of HCV recovered from the chimpanzees that received the same inoculum, confirms that HCV is present in vivo as a quasispecies. These results provide experimental evidence in vivo that HCV infection elicits a neutralizing antibody response in humans but suggest that such antibodies are isolate-specific. This result raises concerns for the development of a broadly reactive vaccine against HCV.
Formation of receptive fields in realistic visual environments according to the Bienenstock, Cooper, and Munro (BCM) theory.
Authors: Law C C , Cooper L N
Published: May 2006

The Bienenstock, Cooper, and Munro (BCM) theory of synaptic plasticity has successfully reproduced the development of orientation selectivity and ocular dominance in kitten visual cortex in normal, as well as deprived, visual environments. To better compare the consequences of this theory with experiment, previous abstractions of the visual environment are replaced in this work by real visual images with retinal processing. The visual environment is represented by 24 gray-scale natural images that are shifted across retinal fields. In this environment, the BCM neuron develops receptive fields similar to the fields of simple cells found in kitten striate cortex. These fields display adjacent excitatory and inhibitory bands when tested with spot stimuli, orientation selectivity when tested with bar stimuli, and spatial-frequency selectivity when tested with sinusoidal gratings. In addition, their development in various deprived visual environments agrees with experimental results.
Testosterone increases the recruitment and/or survival of new high vocal center neurons in adult female canaries.
Authors: Rasika S , Nottebohm F , Alvarez-Buylla A
Published: May 2006

New neurons are added to the high vocal center (HVC) of adult male and female canaries. Exogenous testosterone induces a marked increase in HVC size in adult female canaries, though the mechanisms responsible for this increase remain unknown. To understand the mechanisms, we analyzed the effects of testosterone on neuronal recruitment in the female HVC. Intact adult female canaries received Silastic implants that were empty or filled with testosterone. Birds in the short-survival group received the Silastic implant, followed by a single injection of [3H]thymidine 2 days later, and were killed on the following day. Birds in the long-survival group were injected once a day for 5 days with [3H]thymidine and received the Silastic implant 20 and 40 days later. These birds were killed 60 days after the first injection of [3H]thymidine. The number of 3H-labeled ventricular zone cells above, rostral, or caudal to HVC was not affected by the hormone treatment in the short-survival birds, suggesting that testosterone did not affect neuronal production. However, the number of 3H-labeled HVC neurons that projected to robust nucleus of the archistriatum (RA) in the long-survival birds was three times greater in the hormone-treated than in the control group, though the total number of RA-projecting cells did not change significantly. Testosterone also induced an increase in the size of the HVC cells that project to RA. Thus, these experiments suggest that testosterone affects the recruitment and/or survival of newly generated RA-projecting HVC neurons but does not affect their production.
Immunotherapy with anti-CD3 monoclonal antibodies and recombinant interleukin 2: stimulation of molecular programs of cytotoxic killer cells and induction of tumor regression.
Authors: Nakajima F , Khanna A , Xu G , Lagman M , Haschemeyer R , Mouradian J , Wang J C , Stenzel K H , Rubin A L , Suthanthiran M
Published: May 2006

Adoptive cellular immunotherapy, infusions of interleukin 2 (IL-2) in conjunction with in vitro-activated killer cells, has brought new hope to patients with cancer. The broad application of this strategy, however, is constrained by the need for repeated leukapheresis and by the labor-intensive process of in vitro activation of cells. Also, current protocols generally use nonphysiological and toxic concentrations of IL-2. Identification of an in vivo stimulant that renders T cells responsive to physiologic concentrations of IL-2 represents a potential improvement over existing approaches. We have determined whether in vivo administration of monoclonal antibodies (mAbs) directed at the T-cell surface protein CD3 induces T-cell responsiveness to IL-2, stimulates cytolytic molecular programs of natural killer cells and cytotoxic T cells, and induces tumor regression. These hypotheses were explored in a murine hepatic MCA-102 fibrosarcoma model. We report that in vivo administration of anti-CD3 mAbs plus IL-2 results in intrahepatic expression of mRNA-encoding perforin, cytotoxic T-cell-specific serine esterase, and tumor necrosis factor alpha. Anti-CD3 mAbs alone or IL-2 alone failed to induce or induced minimal expression of these molecular mediators of cytotoxicity. The anti-CD3 mAbs plus IL-2 regimen also resulted in a significantly smaller number of hepatic metastases and a significantly longer survival time of tumor-bearing mice, compared to treatment with anti-CD3 mAbs alone or IL-2 alone. Our findings suggest that a regimen of anti-CD3 mAbs plus IL-2 is a more effective antitumor regimen compared with anti-CD3 mAbs alone or IL-2 alone and advance an alternative immunotherapy strategy of potential value for the treatment of cancer in humans.
gp160, a tissue-specific marker for insulin-activated glucose transport.
Authors: Kandror K V , Pilch P F
Published: May 2006

We have isolated and partially sequenced a M(r) 160,000 glycoprotein whose rate of cycling to and from the adipocyte cell surface is enhanced by insulin in a manner apparently identical to the effect of insulin on GLUT4 cycling. Based on the protein sequence, we have prepared an antipeptide antibody against this protein, gp160. The antibody recognizes a M(r) 160,000 protein whose subcellular distribution is identical to that of GLUT4. This was determined by three separate criteria: (i) Western blotting of fractionated adipocyte membranes from cells exposed to insulin or not, (ii) adsorption of vesicles with anti-GLUT4 antibodies followed by Western blotting, and (iii) separation of microsomal vesicles by sucrose velocity and density gradients. By all three criteria, GLUT4 and gp160 are completely colocalized in rat fat cells. Moreover, gp160 can be detected by Western blot only in fat and cardiac and skeletal muscles and was absent from all other tissues tested. Thus, gp160 is an additional marker for physiologically important, insulin-sensitive glucose transport. Its further study at the protein and DNA level may reveal information about the mechanistic details of insulin-activated GLUT4 translocation as well as information concerning the tissue-specific expression of GLUT4 and gp160.
Linkage of a human brain malformation, familial holoprosencephaly, to chromosome 7 and evidence for genetic heterogeneity.
Authors: Muenke M , Gurrieri F , Bay C , Yi D H , Collins A L , Johnson V P , Hennekam R C , Schaefer G B , Weik L , Lubinsky M S
Published: May 2006

Holoprosencephaly (HPE) is a common malformation of the developing forebrain and midface characterized by incomplete penetrance and variable expressivity. Familial HPE has been reported in many families with autosomal dominant inheritance in some and apparent autosomal recessive inheritance in others. We have examined 125 individuals from nine families with autosomal dominant HPE. Expression in gene carriers varied from alobar HPE and cyclopia through microforms such as microcephaly or single central incisor to normal phenotype. We performed linkage studies by either Southern blot or polymerase chain reaction analyses with DNA markers (D7S22, D7S550, and D7S483) that are deleted from some patients with sporadic HPE and flank a translocation breakpoint in 7q36 associated with HPE. The strongest support for linkage was with D7S22, which was linked with no recombination to autosomal dominant HPE in eight of nine families with a combined logarithm of odds score of 6.4 with an affected-only model-free analysis and 8.2 with a reduced-penetrance model and all phenotypes. Close linkage to this region could be excluded in one family, and there was significant evidence of genetic heterogeneity. These results show that a gene for autosomal dominant HPE is located in a chromosomal region (7q36) known to be involved in sporadic HPE with visible cytogenetic deletions. They also demonstrate genetic heterogeneity in familial HPE. We hypothesize that mutations of a gene in 7q36, designated HPE3, are responsible for both sporadic HPE and a majority of families with autosomal dominant HPE.
Apoptosis in substantia nigra following developmental striatal excitotoxic injury.
Authors: Macaya A , Munell F , Gubits R M , Burke R E
Published: May 2006

We have previously observed that an axon-sparing injury to the developing striatum induced by the excitotoxin quinolinate results in a decrease in dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the adult. This decrease occurs in the absence of direct injury to the SNpc. As the striatum is a major target for the SNpc dopaminergic system, we have hypothesized that a decrease in the size of the striatal target during development may result in an induced regressive event in the SNpc, similar to what has been described for many developing neural systems with peripheral targets. We have examined by morphologic and biochemical means the time course and character of cell death in SN following a unilateral striatal lesion with quinolinate in immature rats. The striatal lesion is associated with an induced cell death event in the ipsilateral SN, observed first in SNpc and then in SN pars reticulata. The morphologic characteristics of the dying cells were typical of apoptosis. Immunostaining for tyrosine hydroxylase indicated that some of the apoptotic cells in the SNpc were dopaminergic. We conclude that developmental striatal excitotoxic injury is associated with induced apoptotic cell death in SN.
A functional anatomical study of associative learning in humans.
Authors: Molchan S E , Sunderland T , McIntosh A R , Herscovitch P , Schreurs B G
Published: May 2006

The purpose of the study was to map the functional neuroanatomy of simple associative learning in humans. Eyeblink conditioning was studied in eight normal volunteers using positron emission tomography and H215O. Regional cerebral blood flow was assessed during three sequential phases: (i) explicitly unpaired presentations of the unconditioned stimulus (air puff to the right eye) and conditioned stimulus (binaural tone), (ii) paired presentations of the two stimuli (associative learning), and (iii) presentation of the conditioned stimulus alone. During associative learning, relative to the unpaired phase, blood flow was significantly increased in primary auditory and left posterior cingulate cortices and significantly decreased in areas of the right cerebellar, right prefrontal, right parietal, and insular cortices and right neostriatum. The lateralization of the changes may relate to the functional organization of memory and learning processes in the brain. The activation in primary auditory cortex is an example, using a neuroimaging technique, of a learning-related change in primary sensory cortex in humans. The changes in areas such as the cerebellum, prefrontal cortex, and neostriatum provide support for their roles in associative learning as proposed by animal models. Moreover, these findings show that in humans, even simple classical conditioning involves distributed changes in multiple neural systems.
DNA double helix promotes a linkage isomerization reaction in trans-diamminedichloroplatinum(II)-modified DNA.
Authors: Dalbiès R , Payet D , Leng M
Published: May 2006

In the reaction between trans-diamminedichloroplatinum(II) and a single-stranded pyrimidin-rich oligodeoxyribonucleotide (22-mer) containing the central sequence TGAGT, the 1,3-trans-[Pt(NH3)2[d(GAG)]] cross-link is formed. The 1,3-intrastrand cross-link is inert within the single-stranded oligonucleotide. In contrast, it rearranges to an interstrand cross-link when the platinated oligonucleotide is paired with its complementary deoxyribo- or ribonucleotide strand. The half-life of the 1,3-intrastrand cross-link, approximately 6 h at 37 degrees C, is independent of the nature and concentration of the salt (NaCl or NaClO4). It is not dramatically affected when the intervening adenine residue between the chelated guanine residues is replaced by a cytosine or a thymine residue or when the T.A base pair adjacent to the 5' or 3' side of the adduct is replaced by a C.G base pair. On the other hand, a mismatch on the 3' or 5' side of the adduct prevents the rearrangement. We propose that the linkage isomerization reaction results from a direct nucleophilic attack of the cytosine residue complementary to the platinated 5' guanine residue on the platinum residue. Among others, the potential use of the DNA.RNA-promoted reaction is discussed in the context of the antisense strategy to irreversibly cross-link the antisense oligonucleotides to their targets.
Gene therapy of rat 9L gliosarcoma tumors by transduction with selectable genes does not require drug selection.
Authors: Tapscott S J , Miller A D , Olson J M , Berger M S , Groudine M , Spence A M
Published: May 2006

9L rat glioma cells have been used as a model for brain tumor therapies. It has been reported that in vivo infection of 9L cells with a replication-defective retrovirus expressing the herpes simplex thymidine kinase gene resulted in decreased tumor formation following treatment with the antiviral drug ganciclovir. In the study reported here, rats were injected either intracerebrally or subcutaneously with 9L glioma cells expressing a chimeric hygromycin phosphotransferase-thymidine kinase fusion protein or with unmodified 9L cells. Tumor formation was decreased in the rats receiving modified cells, even in the absence of treatment with ganciclovir. Suppression of tumor growth was also observed with cells modified to express the intracellular selectable marker neomycin phosphotransferase. These results indicate that an intracellular selectable marker, in the absence of pharmacologic selection, can inhibit tumor growth of 9L cells. The demonstration that intracellular marker genes can negatively influence the survival of transplanted cells has important implications for in vivo studies that use genetically modified cells.
The conserved core domain of the human TATA binding protein is sufficient to assemble the multisubunit RNA polymerase I-specific transcription factor SL1.
Authors: Rudloff U , Eberhard D , Grummt I
Published: May 2006

The human ribosomal RNA polymerase (Pol) I promoter selectivity factor SL1 is a complex consisting of the TATA binding protein (TBP) and three TBP-associated factors (TAFs). We have investigated which elements of TBP are involved in the assembly of Pol I-specific TBP-TAF complexes by comparing SL1 isolated from two human cell lines, one expressing epitope-tagged full-length TBP and another expressing a deletion of nearly the entire N-terminal domain (e delta NTBP). We have immunopurified epitope-tagged full-length TBP- and e delta NTBP-TAF complexes and show that e delta NTBP reconstitutes SL1 activity almost as well as full-length TBP. Moreover, e delta NTBP is shown to be associated with all three Pol I-specific TAFs. Thus, the core of TBP alone is sufficient for the correct assembly of the Pol I-specific TBP-TAF complex, and the variable N-terminal region of human TBP is not required for transcriptional activity. We also demonstrate by an in vitro protein-protein interaction assay that TBP directly interacts with the smallest TAF, TAFI48.
Characterization of glucose transport and cloning of a hexose transporter gene in Trypanosoma cruzi.
Authors: Tetaud E , Bringaud F , Chabas S , Barrett M P , Baltz T
Published: May 2006

A gene from Trypanosoma cruzi, TcrHT1, which encodes a member of the glucose transporter superfamily has been cloned. The gene is similar in sequence to the T. brucei hexose transporter THT1 and the Leishmania transporter Pro-1 and is present in the T. cruzi genome as a cluster of at least eight tandemly reiterated copies. Northern blot analysis revealed two mRNA transcripts which differ in size with respect to their 3' untranslated regions. When injected with in vitro transcribed TcrHT1 mRNA, Xenopus oocytes express a hexose transporter with properties similar to those of T. cruzi. Glucose transport in T. cruzi is mediated via a carrier with unique properties when compared with the other glucose transporters already characterized among the Kinetoplastida. It is a facilitated transporter with a high affinity for D-glucose (Km = 84.1 +/- 7.9 microM and Vmax = 46 +/- 9.4 nmol/min per mg of protein) that shares with other kinetoplastid hexose transporters the ability to recognize D-fructose, which distinguishes these carriers from the human erythrocyte glucose transporter GLUT1.
Phosphorylation of the p34 subunit of human single-stranded-DNA-binding protein in cyclin A-activated G1 extracts is catalyzed by cdk-cyclin A complex and DNA-dependent protein kinase.
Authors: Pan Z Q , Amin A A , Gibbs E , Niu H , Hurwitz J
Published: May 2006

The human single-stranded-DNA-binding protein (HSSB, also called RP-A) is a trimeric complex (p70, p34, and p14) required for multiple functions in DNA transactions. We report here that the p34 subunit of HSSB was hyperphosphorylated by kinase activities present in G1 extract (obtained from HeLa cells in G1 phase) preincubated with human cyclin A. This hyperphosphorylated HSSB product included at least four species of p34 that migrated more slowly through denaturing polyacrylamide gels than the hypophosphorylated form. Fractionation of cyclin A-activated G1 extract identified two kinases involved in the hyperphosphorylation of HSSB p34: cdk-cyclin A complex and DNA-dependent p350 protein kinase (DNA-PK). Kinetic analysis revealed that in cyclin A-activated G1 extract, p34 was first phosphorylated by cdk-cyclin A prior to the action of DNA-PK. Addition of p21cip1, a specific inhibitor of cdk-cyclin A but not DNA-PK, nearly abolished the hyperphosphorylation of HSSB p34 in G1 extract preincubated with cyclin A. This suggests a requirement of the cdk-cyclin A activity for the phosphorylation of p34 by DNA-PK in G1 extract.
Recombinant human IgA expressed in insect cells.
Authors: Carayannopoulos L , Max E E , Capra J D
Published: May 2006

IgA serves as the first line of humoral defense at all mucosal surfaces and is present in large quantities in serum. To map the sites of interaction of immune effector molecules with the IgA constant region (C alpha), we have expressed soluble, chimeric human IgA in insect cells using recombinant baculoviruses. This antibody is correctly assembled into heavy chain/light chain heterodimers, N-glycosylated, and secreted by the insect cells; further, when coexpressed with a human J chain, the antibodies can assemble into dimers. The recombinant protein is authentic by a number of criteria, including antigen-binding, recognition by monoclonal antibodies, complement fixation via the alternative pathway, and specific binding to the monocyte IgA Fc receptor. We have also constructed viruses which encode structurally altered IgA heavy chains. Using one of these variant viruses, we have shown that glycosylation of the second domain of C alpha is required for interaction with the monocyte IgA Fc receptor. This system should prove useful in further characterization of the structure-function relationships in human C alpha.
Relative abundance of Alzheimer A beta amyloid peptide variants in Alzheimer disease and normal aging.
Authors: Näslund J , Schierhorn A , Hellman U , Lannfelt L , Roses A D , Tjernberg L O , Silberring J , Gandy S E , Winblad B , Greengard P
Published: May 2006

The Alzheimer A beta amyloid peptide (A beta) is the principal proteinaceous component of amyloid associated with Alzheimer disease (AD). We have determined the relative abundance of A beta structural variants present in amyloid from brains of 10 individuals with sporadic AD, 2 individuals with familial AD carrying specific mutations in the Alzheimer amyloid precursor protein gene, and 5 nondemented elderly controls. A procedure of isolation based on the extreme insolubility of A beta amyloid was used. The purified, nondigested A beta was analyzed by N-terminal sequencing and electrospray-ionization mass spectrometry. Three principal A beta variants were detected--A beta-(1-40), A beta-(1-42), and A beta-(11-42)--in all brains analyzed. The predominant variant in sporadic AD was A beta-(1-40), whereas the principal A beta variant in nondemented elderly controls was A beta-(1-42). The ratio A beta-(1-40)/A beta-(1-42) differed by 10-fold between brains from nondemented controls and those with sporadic AD.
Statistical analysis of nucleotide sequences of the hemagglutinin gene of human influenza A viruses.
Authors: Ina Y , Gojobori T
Published: May 2006

To examine whether positive selection operates on the hemagglutinin 1 (HA1) gene of human influenza A viruses (H1 subtype), 21 nucleotide sequences of the HA1 gene were statistically analyzed. The nucleotide sequences were divided into antigenic and nonantigenic sites. The nucleotide diversities for antigenic and nonantigenic sites of the HA1 gene were computed at synonymous and nonsynonymous sites separately. For nonantigenic sites, the nucleotide diversities were larger at synonymous sites than at nonsynonymous sites. This is consistent with the neutral theory of molecular evolution. For antigenic sites, however, the nucleotide diversities at nonsynonymous sites were larger than those at synonymous sites. These results suggest that positive selection operates on antigenic sites of the HA1 gene of human influenza A viruses (H1 subtype).
Inhibition of ribonucleotide reductase by 2'-substituted deoxycytidine analogs: possible application in AIDS treatment.
Authors: Bianchi V , Borella S , Calderazzo F , Ferraro P , Chieco Bianchi L , Reichard P
Published: May 2006

After phosphorylation to the corresponding diphosphates, 2'-azido-2'-deoxycytidine and 2'-difluorocytidine act as powerful inhibitors of ribonucleotide reductase. Phosphorylation requires deoxycytidine kinase, an enzyme with particularly high activity in lymphoid cells. Therefore, the deoxycytidine analogs can be expected to inhibit the reductase with some specificity for the lymphoid system. Pretreatment of human CEM lymphoblasts with the analogs considerably increased the phosphorylation of 3'-deoxy-3'-azidothymidine (AzT). The increased phosphorylation of AzT is caused by a prolongation of the S phase of the cell cycle. Our results suggest the possibility of a combination of 2'-substituted deoxycytidine analogs with AzT in the treatment of AIDS. Gao et al. [Gao, W.-Y., Cara, A., Gallo, R. C. & Lori, F. (1993) Proc. Natl. Acad. Sci. USA 90, 8925-8928] have suggested the use of the ribonucleotide reductase inhibitor hydroxyurea for this purpose, since the resulting decrease in the size of deoxyribonucleotide pools decreases the processivity of the HIV reverse transcriptase. From our results it would appear that the 2'-substituted deoxycytidine analogs might be preferable to hydroxyurea.
Transcript cleavage by RNA polymerase II arrested by a cyclobutane pyrimidine dimer in the DNA template.
Authors: Donahue B A , Yin S , Taylor J S , Reines D , Hanawalt P C
Published: May 2006

A current model for transcription-coupled DNA repair is that RNA polymerase, arrested at a DNA lesion, directs the repair machinery to the transcribed strand of an active gene. To help elucidate this role of RNA polymerase, we constructed DNA templates containing the major late promoter of adenovirus and a cyclobutane pyrimidine dimer (CPD) at a specific site. CPDs, the predominant DNA lesions formed by ultraviolet radiation, are good substrates for transcription-coupled repair. A CPD located on the transcribed strand of the template was a strong block to polymerase movement, whereas a CPD located on the nontranscribed strand had no effect on transcription. Furthermore, the arrested polymerase shielded the CPD from recognition by photolyase, a bacterial DNA repair protein. Transcription elongation factor SII (also called TFIIS) facilitates read-through of a variety of transcriptional pause sites by a process in which RNA polymerase II cleaves the nascent transcript before elongation resumes. We show that SII induces nascent transcript cleavage by RNA polymerase II stalled at a CPD. However, this cleavage does not remove the arrested polymerase from the site of the DNA lesion, nor does it facilitate translesional bypass by the polymerase. The arrested ternary complex is stable and competent to resume elongation, demonstrating that neither the polymerase nor the RNA product dissociates from the DNA template.
Redundant functions of the genes knirps and knirps-related for the establishment of anterior Drosophila head structures.
Authors: González-Gaitán M , Rothe M , Wimmer E A , Taubert H , Jäckle H
Published: May 2006

Developmental gene functions of Drosophila are typically characterized by a recognizable mutant phenotype. When molecular probes of such genes were used to isolate homologues, distinct spatially and temporally restricted expression patterns were observed in vertebrates as well. However, corresponding "gene knock-outs" often revealed subtle or no scorable phenotypes, a phenomenon attributed to redundant gene functions. We found that the evolutionarily related genes knirps (kni) and knirps-related (knrl) contribute to a similar phenomenon in Drosophila. The two closely situated genes show identical expression patterns in the developing embryo, including the posterior and anterior expression domains in the blastoderm. Here we show that the two biochemically equivalent gene products are both functional in the head anlage and that the lack of one gene activity can be overcome by the activity of the other. Whereas kni is also required for abdominal segmentation, knrl is nonfunctional in its posterior expression domain. Thus, the kni/knrl pair of genes provides a region-specific buffering system, rather than a case of global functional redundancy.
Expression of a specific marker of avian programmed cell death in both apoptosis and necrosis.
Authors: Fernandez P A , Rotello R J , Rangini Z , Doupe A , Drexler H C , Yuan J
Published: May 2006

Apoptosis and necrosis are two types of cell death with different morphologic features. We report here the isolation of a monoclonal antibody, BV2, that specifically recognizes cells undergoing developmental programmed cell death in different tissues of the chicken and zebra-finch embryos. The antigen recognized by BV2 monoclonal antibody is detected in vitro in primary chicken embryonic fibroblasts induced to die by actinomycin D, as well as fibroblasts induced to die by chemical anoxia. The expression of this specific antigen during necrosis appears to require active protein synthesis. These findings provide evidence that cells from different embryonic tissues undergoing programmed cell death during vertebrate development express similar antigens and indicate that apoptosis and necrosis may share similar biochemical features.
Photolabile precursors of glutamate: synthesis, photochemical properties, and activation of glutamate receptors on a microsecond time scale.
Authors: Wieboldt R , Gee K R , Niu L , Ramesh D , Carpenter B K , Hess G P
Published: May 2006

Newly synthesized photolabile derivatives of glutamate, caged glutamate, that release free glutamate on a microsecond time scale after a pulse of UV laser light are described. 2-Nitrobenzyl derivatives were attached to the amino or carboxyl groups of glutamate. Substitution with a -CO2- group at the benzylic carbon accelerates the photolysis reaction when compared to -H and -CH3 substituents. gamma-O-(alpha-Carboxy-2-nitrobenzyl)glutamate is stable at neutral pH. In 100 mM phosphate buffer at pH 7.0, the compound is photolyzed at 308 nm with a quantum product yield of 0.14. The half-life of the major component of the photolytic reaction, as judged by the transient absorbance change at 430 nm, is 21 microseconds (approximately 90%); the half-life of a minor component (approximately 10%) is 0.2 ms. The amino-linked derivatives have half-lives in the millisecond region and a 4-fold lower quantum yield. The potential of the newly synthesized compound for use in rapid chemical kinetic investigations of glutamate receptors is demonstrated. (i) The caged glutamate at 1 mM concentration does not desensitize glutamate receptors in rat hippocampal neurons. (ii) Caged glutamate (1 mM) does not inhibit activation of the receptors by 50 microM glutamate. (iii) Photolysis of the compound induces rapid onset of transmembrane currents in rat hippocampal neurons.
Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53.
Authors: Scheffner M , Huibregtse J M , Howley P M
Published: May 2006

The E6 protein of the oncogenic human papillomavirus types 16 and 18 facilitates the rapid degradation of the tumor-suppressor protein p53 via the ubiquitin-dependent proteolytic pathway. The E6 protein binds to a cellular protein of 100 kDa termed E6-AP. The complex of E6 and E6-AP specifically interacts with p53 and induces the ubiquitination of p53 in a reaction which requires the ubiquitin-activating enzyme (E1) and a cellular fraction thought to contain a mammalian ubiquitin-conjugating enzyme (E2). This mammalian E2 activity could be replaced with bacterially expressed UBC8 from Arabidopsis thaliana, which belongs to a subfamily of E2s including yeast UBC4 and UBC5 which are highly conserved at the amino acid level. In this paper we describe the cloning of a human cDNA encoding a human E2 that we have designated UbcH5 and that is related to Arabidopsis UBC8 and the other members of this subfamily. We demonstrate that UbcH5 can function in the E6/E6-AP-induced ubiquitination of p53.
Development of cancer cachexia-like syndrome and adrenal tumors in inhibin-deficient mice.
Authors: Matzuk M M , Finegold M J , Mather J P , Krummen L , Lu H , Bradley A
Published: May 2006

Activins and inhibins, members of the type beta transforming growth factor superfamily of growth regulatory proteins, are produced in multiple tissues and affect diverse physiologic processes. Using embryonic stem cell technology, we previously demonstrated that inhibin can function as a gonadal tumor suppressor. In this study, we show that development of gonadal tumors is rapidly followed by a cancer cachexia-like wasting syndrome. Cachectic inhibin-deficient mice develop hepatocellular necrosis around the central vein and parietal cell depletion and mucosal atrophy in the glandular stomach, are anemic, and demonstrate severe weight loss. The liver pathology is consistent with studies demonstrating an effect of elevated activins on rat hepatocytes. In inhibin-deficient mice with tumors, activins are > 10-fold elevated in the serum and are likely causing some of the cachexia symptoms. In contrast, inhibin-deficient mice gonadectomized at an early age do not develop this wasting syndrome. However, these gonadectomized, inhibin-deficient mice eventually develop adrenal cortical sex steroidogenic tumors with nearly 100% penetrance, demonstrating that inhibin is also a tumor suppressor for the adrenal gland.
Synergy in the spectral tuning of retinal pigments: complete accounting of the opsin shift in bacteriorhodopsin.
Authors: Hu J , Griffin R G , Herzfeld J
Published: May 2006

UV-visible and solid-state NMR studies of a series of 6-s-trans protonated Schiff bases of retinal with aniline show that the bathochromic shift induced by weakening the imine counterion is significantly greater in the 6-s-trans conformation than in the 6-s-cis conformation. Based on the observed magnitude of this coupling between the electronic effects of 6-s isomerization and imine counterion strength in the model compounds, the large opsin shift and unusual chemical shifts in light-adapted bacteriorhodopsin can be fully explained. These phenomena therefore do not require a negative point charge or polarizability effects in the chromophore binding pocket. The results are consistent with an effective center-to-center distance between the Schiff base and its counterion of about 4 A in light-adapted bacteriorhodopsin.
Long-term culture and functional characterization of follicular cells from adult normal human thyroids.
Authors: Curcio F , Ambesi-Impiombato F S , Perrella G , Coon H G
Published: May 2006

We have obtained long-term cultures of differentiated proliferating follicular cells from normal adult human thyroid glands. In vitro growth of such human cells has been sustained by a modified F-12 medium, supplemented with bovine hypothalamus and pituitary extracts and no added thyrotropin. Cultures have been expanded, cloned, frozen, successfully retrieved, and characterized. Functional characterization of these cells shows constitutive thyroglobulin production and release and thyrotropin-dependent adenosine 3',5'-cyclic monophosphate production, the latter apparently not associated with significant increases in DNA synthesis or cell proliferation. Genetic characterization of these cells by chromosome counting showed the normal diploid chromosome number. The ability to cultivate differentiated human thyroid follicular cells in long-term culture opens possibilities for investigating the transduction pathways of thyrotropin stimulation in normal and pathological human tissues, developing clinically relevant in vitro assays, and considering cellular and molecular therapies.