bluuebunny/crossref_metadata_2025 huggingface dataset:
Relative binding affinities of distamycin and its analog to d(CGCAAGTTGGC).d(GCCAACTTGCG): comparison of simulation results with experiment.
Authors: Singh S B , Wemmer D E , Kollman P A
Published: May 2006

We report here an effort to use molecular dynamics/free energy perturbation methodology to calculate relative binding affinities of two related drugs to DNA. Specifically, we focus on the relative binding free energies of distamycin (Dst) and its analog, 2-imidazoledistamycin (2-ImD), to d(CGCAAGTTGGC).d(GCCAACTTGCG). The pyrrole (Dst) and the imidazole variant (2-ImD) differ only in that the C-H is substituted by an N in the central ring. The starting conformation for these calculations was the previously determined solution structure of two 2-ImD molecules in the minor groove of the above 11-residue DNA. In this complex both the ligands have the imidazole nitrogen (N3) oriented toward the amino group of G6. However only ligand 1 (site I) has N3 within the hydrogen bonding distance from N2 amino proton of G6. We have calculated the difference in free energy of binding of 2-ImD versus Dst in three different cases by mutating 2-ImD-->Dst reversibly. In the first case ligand 1 (site I) is mutated, in the second case ligand 2 (site II) is mutated, and in the third case both the ligands are mutated. These calculations show that at site I Dst has weaker binding affinity than 2-ImD by 0.7 kcal/mol, at site II Dst has stronger binding affinity than 2-ImD by 2.9 kcal/mol, and when occupying both site I and site II, Dst binds with greater affinity than 2-ImD by 1.8 kcal/mol. Recent experimental findings agree semiquantitatively (within 1 kcal/mol) with the calculations presented here. Hence the methodology presented here can be used to predict relative binding energies of two or more closely related molecules to DNA.
Iron reverses impermeable chelator inhibition of DNA synthesis in CCl 39 cells.
Authors: Alcain F J , Löw H , Crane F L
Published: May 2006

Treatment of Chinese hamster lung fibroblasts (CCl 39 cells) with the impermeable iron(II) chelator bathophenanthroline disulfonate (BPS) inhibits DNA synthesis when cell growth is initiated with growth factors including epidermal growth factor plus insulin, thrombin, or ceruloplasmin, but not with 10% fetal calf serum. The BPS treatment inhibits transplasma membrane electron transport. The treatment leads to release of iron from the cells as determined by BPS iron(II) complex formation over 90 min. Growth factor stimulation of DNA synthesis and electron transport are restored by addition of di- or trivalent iron to the cells in the form of ferric ammonium citrate, ferrous ammonium sulfate, or diferric transferrin. The effect with BPS differs from the inhibition of growth by hydroxyurea, which acts on the ribonucleotide reductase, or diethylenetriaminepentaacetic acid, which is another impermeable chelating agent, in that these agents inhibit growth in 10% fetal calf serum. The BPS effect is consistent with removal of iron from a site on the cell surface that controls DNA synthesis.
Epistatic interaction and functional compensation between the two tissue- and cell-specific sucrose synthase genes in maize.
Authors: Chourey P S , Taliercio E W
Published: May 2006

A tissue-specific epistatic mode of gene interaction was observed between molecularly homologous genes Sh1 and Sus1 (hereafter, Sh and Sus), encoding the sucrose synthase (SS) isozymes, SS1 and SS2, respectively. In Sh Sus genotype, both SS genes were expressed simultaneously and approximately equally in young seedlings; however, only the Sus-encoded SS2 protein was seen in the developing embryos. By contrast, the mutant sus genotype, lacking detectable levels of the SS2 protein in various tissues tested, showed expression of the Sh locus as judged by the detection of the SS1 protein in such embryos. Ectopic expression in embryos was seen from two separate Sh alleles, Sh-W22 and Sh'-5 (a revertant allele derived upon Ds excision from sh-m5933). In each case, the Sh expression at the protein level in embryos was unique to genotypes with the mutant sus gene. Based on the observed lack of phenotypic change in the sus mutant, we suggest that the ectopic expression of the Sh in otherwise Sus-specific tissues leads to functional compensation. There was no epistatic interaction of Sh and Sus at the RNA level as SS1 transcripts were detectable in both Sus and sus embryos. Thus, embryo specificity between the two SS genes was determined at posttranscriptional or at translational level of control. We surmise on the basis of these data that metabolic regulatory controls seem to override the normal constraints of tissue and cell specificity of the nonallelic isozyme genes to maintain efficient use of the pathways.
Localization of Fanconi anemia C protein to the cytoplasm of mammalian cells.
Authors: Youssoufian H
Published: May 2006

Features of chromosomal aberrations, hypersensitivity to DNA crosslinking agents, and predisposition to malignancy have suggested a fundamental anomaly of DNA repair in Fanconi anemia. The function of the recently isolated FACC (Fanconi anemia group C complementing) gene for a subset of this disorder is not yet known. The notion that FACC plays a direct role in DNA repair would predict that the polypeptide should reside in the nucleus. In this study, a polyclonal antiserum raised against FACC was used to determine the subcellular location of the polypeptide. Immunofluorescence and subcellular fractionation studies of human cell lines as well as COS-7 cells transiently expressing human FACC showed that the protein was localized primarily to the cytoplasm under steady-state conditions, transit through the cell cycle, and exposure to crosslinking or cytotoxic agents. However, placement of a nuclear localization signal from the simian virus 40 large tumor antigen at the amino terminus of FACC directed the hybrid protein to the nuclei of transfected COS-7 cells. These observations suggest an indirect role for FACC in regulating DNA repair in this group of Fanconi anemia.
Hypothesis: disseminated intravascular inflammation as the inflammatory counterpart to disseminated intravascular coagulation.
Authors: Bull B S , Bull M H
Published: May 2006

We have identified a leukocyte activation syndrome that is occasionally associated with the transfusion of intraoperatively recovered erythrocytes. This syndrome appears to result from intravascular damage caused by leukocytes activated during the erythrocyte salvage process. We hypothesize that this syndrome is part of a larger disease grouping: disseminated intravascular inflammation (DII). DII is the analog of the coagulation disorder disseminated intravascular coagulation. In disseminated intravascular coagulation, the organ damage results from uncontrolled activation of the clotting pathway; in DII the damage is caused by leukocytes that have become activated by direct contact with bacteria or in rare instances--such as erythrocyte salvage--in the absence of bacteria and bacterial products. Recent studies of the hazards associated with intraoperative blood salvage indicate that activation of leukocytes can be achieved by exposure to activated platelets alone. If such activated leukocytes are reinfused along with the washed erythrocytes, widespread organ damage may result. The lung is the organ most severely affected by activated leukocytes. Adult respiratory distress syndrome is one outcome. It is likely that DII is a presently unrecognized pathophysiological process that complicates a variety of primary disease states and increases their lethality.
Selection of catalytic antibodies for a biosynthetic reaction from a combinatorial cDNA library by complementation of an auxotrophic Escherichia coli: antibodies for orotate decarboxylation.
Authors: Smiley J A , Benkovic S J
Published: May 2006

Antibodies capable of decarboxylating orotate were sought by immunization with a hapten designed to elicit antibodies with combining sites that resemble the orotate-binding and catalytic portion of the active site of the enzyme orotidine 5'-monophosphate (OMP) decarboxylase (orotidine-5'-monophosphate carboxy-lyase, EC 4.1.1.23). Active recombinant antibody fragments (Fabs) were selected from a combinatorial cDNA library by complementation of a pyrF strain of Escherichia coli and growth of the library-expressing cells on pyrimidine-free medium. In this biological screen, a sufficiently active antibody from the library would decarboxylate orotate to produce uracil, a pyrimidine source for the auxotroph, and would provide the cells with a growth advantage compared to cells without an active antibody. Six recombinant Fabs yielded identifiable colonies in a screen of 16,000 transformants. To enhance its stability and expression level, one of the six positive fragments was converted into single-chain form. In this form, the antibody fragment conferred a definite growth advantage to the auxotroph that was eliminated when the hapten was included in the medium. The purified single-chain antibody displayed orotate decarboxylase activity in vitro, as determined by a 14CO2 displacement assay. The specific activity of the antibody is approximately 10(-7) times that of naturally occurring OMP decarboxylase, but this antibody-catalyzed rate is estimated to be 10(8) times the background rate. The results offer the potential to use these methods to obtain catalytic antibodies for other biosynthetic reactions as well as to assess the effectiveness of the hapten transition state or active site analog in eliciting antibody catalysts.
Nephrogenic diabetes insipidus: an X chromosome-linked dominant inheritance pattern with a vasopressin type 2 receptor gene that is structurally normal.
Authors: Friedman E , Bale A E , Carson E , Boson W L , Nordenskjöld M , Ritzén M , Ferreira P C , Jammal A , De Marco L
Published: May 2006

Nephrogenic diabetes insipidus is a rare hereditary disorder, most commonly transmitted in an X chromosome-linked recessive manner and characterized by the lack of renal response to the action of antidiuretic hormone [Arg8]vasopressin. The vasopressin type 2 receptor (V2R) has been suggested to be the gene that causes the disease, and its role in disease pathogenesis is supported by mutations within this gene in affected individuals. Using the PCR, denaturing gradient gel electrophoresis, and direct DNA sequencing, we examined the V2R gene in four unrelated kindreds. In addition, linkage analysis with chromosome Xq28 markers was done in one large Brazilian kindred with an apparent unusual X chromosome-linked dominant inheritance pattern. In one family, a mutation in codon 280, causing a Tyr-->Cys substitution in the sixth transmembrane domain of the receptor, was found. In the other three additional families with nephrogenic diabetes insipidus, the V2R-coding region was normal in sequence. In one large Brazilian kindred displaying an unusual X chromosome-linked dominant mode of inheritance, the disease-related gene was localized to the same region of the X chromosome as the V2R, but no mutations were found, thus raising the possibility that this disease is caused by a gene other than V2R.
Cloning of casbene synthase cDNA: evidence for conserved structural features among terpenoid cyclases in plants.
Authors: Mau C J , West C A
Published: May 2006

A near-full-length casbene synthase cDNA clone, pCS7, was isolated by using a partial cDNA clone, pCS4, to probe a lambda gt10 library constructed from poly(A)+ RNA from elicited castor bean seedlings. The cDNA insert had a length of 1983 bases with a polyadenylate tail of 19 bases. Translation of the cDNA sequence revealed an open reading frame encoding a 601-aa protein with a predicted M(r) of 68,960. Search of the GenBank data base with the deduced translation product revealed 42% identity and 65% similarity with 5-epi-aristolochene synthase from tobacco and 31% identity and 53% similarity with limonene synthase from spearmint. Each of the three proteins catalyzes an intramolecular cyclization of a prenyl diphosphate substrate to a specific cyclic terpenoid hydrocarbon product. The proposed reaction mechanisms for the three catalytic processes share common chemical features, even though the products being formed are members of three different classes of terpenoid compounds. Analysis of the alignment of the three proteins suggests that both primary and secondary structural elements are conserved. These similarities suggest that the genes that encode terpenoid cyclization enzymes of this type in angiosperms have undergone divergent evolution from an ancestral progenitor gene. In support of this proposition, the locations of five of the six introns in the casbene synthase gene align very closely with those of the five introns in the 5-epi-aristolochene synthase gene.
Reversal of spatial memory impairments in aged rats by nerve growth factor and neurotrophins 3 and 4/5 but not by brain-derived neurotrophic factor.
Authors: Fischer W , Sirevaag A , Wiegand S J , Lindsay R M , Björklund A
Published: May 2006

Aged rats, displaying impairments in spatial learning and memory associated with marked cellular atrophy of forebrain cholinergic neurons, received intracerebroventricular infusions of one of the four neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), or neurotrophin 4/5 (NT-4/5), or a combination of NGF and BDNF, or vehicle. During the 4-week infusion period rats receiving NGF, NT-3, or NT-4/5 showed improved acquisition and retention of spatial memory. With NGF and NT-3, but not NT-4/5, this was accompanied by a significant reduction in cholinergic neuron atrophy in septum, nucleus basalis, and striatum. BDNF, in contrast, was without effect either alone or in combination with NGF. These results show that memory deficits associated with aging can be reversed by several members of the neurotrophin family and that this effect may be mediated through activation of multiple neurotrophin receptors associated with cholinergic and possibly noncholinergic systems in the brain.
Dimorphic transition in Escherichia coli and Salmonella typhimurium: surface-induced differentiation into hyperflagellate swarmer cells.
Authors: Harshey R M , Matsuyama T
Published: May 2006

We describe a new behavioral response in Escherichia coli and Salmonella typhimurium in which the bacteria differentiate into filamentous, multinucleate, hyperflagellate cells that navigate the surface of solid media by means of coordinated swarming motility. The cue for differentiation into swarmer cells is provided by the concentration and composition of the agar. Examination of the behavior of various mutants shows that the flagellar apparatus used for swimming motility and the chemotaxis system are indispensable for swarming motility.
X-ray structures of isoforms of the actin-binding protein profilin that differ in their affinity for phosphatidylinositol phosphates.
Authors: Fedorov A A , Magnus K A , Graupe M H , Lattman E E , Pollard T D , Almo S C
Published: May 2006

We determined the structures of Acanthamoeba profilin I and profilin II by x-ray crystallography at resolutions of 2.0 and 2.8 A, respectively. The polypeptide folds and the actin-binding surfaces of the amoeba profilins are very similar to those of bovine and human profilins. The electrostatic potential surfaces of the two Acanthamoeba isoforms differ. Two areas of high positive potential on the surface of profilin II are candidate binding sites for phosphatidylinositol phosphates. The proximity of these sites to the actin binding site provides an explanation for the competition between actin and lipids for binding profilin.
Autoantigenic proteins that bind recombinogenic sequences in Epstein-Barr virus and cellular DNA.
Authors: Sun R , Spain T A , Lin S F , Miller G
Published: May 2006

We have identified conserved autoantigenic cellular proteins that bind to G-rich sequence motifs in recombinogenic regions of Epstein-Barr virus (EBV) DNA. This binding activity, called TRBP, recognizes the EBV terminal repeats, a locus responsible for interconversion of linear and circular EBV DNA. We found that TRBP also binds to EBV DNA sequences involved in deletion of EBNA2, a gene product required for immortalization. We show that TRBP binds sequences present in repetitive cellular DNA, such as variable-number tandem repeats (VNTR) and immunoglobulin heavy-chain class switch regions. We propose that EBV utilizes cellular DNA recombination systems to mediate several types of viral genome alterations. These findings may lead to an understanding of the mechanism of rearrangements of EBV DNA that are a central feature of the biology of the virus.
Overlapping neural networks for multiple motor engrams.
Authors: Lukashin A V , Wilcox G L , Georgopoulos A P
Published: May 2006

The hypothesis was tested that learned movement trajectories of different shapes can be stored in, and generated by, largely overlapping neural networks. Indeed, it was possible to train a massively interconnected neural network to generate different shapes of internally stored, dynamically evolving movement trajectories using a general-purpose core part, common to all networks, and a special-purpose part, specific for a particular trajectory. The weights of connections between the core units do not carry any information about trajectories. The core network alone could generate externally instructed trajectories but not internally stored ones, for which both the core and the trajectory-specific part were needed. All information about the movements is stored in the weights of connections between the core part and the specialized units and between the specialized units themselves. Due to these connections the core part reveals specific dynamical behavior for a particular trajectory and, as the result, discriminates different tasks. The percentage of trajectory-specific units needed to generate a certain trajectory was small (2-5%), and the total output of the network is almost entirely provided by the core part, whereas the role of the small specialized parts is to drive the dynamical behavior. These results suggest an efficient and effective mechanism for storing learned motor patterns in, and reproducing them by, overlapping neural networks and are in accord with neurophysiological findings of trajectory-specific cells and with neurological observations of loss of specific motor skills in the presence of otherwise intact motor control.
Deficient induction of human hsp70 heat shock gene transcription in Y79 retinoblastoma cells despite activation of heat shock factor 1.
Authors: Mathur S K , Sistonen L , Brown I R , Murphy S P , Sarge K D , Morimoto R I
Published: May 2006

One of the basic features of the inducible heat shock response is the activation of heat shock factor which results in the rapid transcriptional induction of the heat shock genes. Although it is widely considered that the heat shock response is ubiquitous, several reports have indicated that the transcriptional response can vary in both intensity and kinetics and often in a tissue-specific manner. Of interest have been studies on the expression of heat shock genes in the brain, particularly observations that certain cultured neuronal cells exhibit a diminished heat shock response. We demonstrate that transcription of the gene encoding a 70-kDa heat shock protein (hsp70) is diminished upon heat shock in Y79 human retinoblastoma cells (which are of neuronal origin) despite both the activation of heat shock factor 1 and induced transcription of another heat shock gene, hsp90 alpha. This uncoupling of stress-induced transcription of the hsp70 and hsp90 alpha genes, which are typically coordinately regulated in response to stress, appears to be due to the selective inability of trans-acting factors, including heat shock factor 1, to bind in vivo to the hsp70 promoter as the result of a chromatin-mediated effect.
The physical state of the negative strand of hepatitis C virus RNA in serum of patients with chronic hepatitis C.
Authors: Shindo M , Di Bisceglie A M , Akatsuka T , Fong T L , Scaglione L , Donets M , Hoofnagle J H , Feinstone S M
Published: May 2006

Negative strands of the hepatitis C virus (HCV) genome (a positive-stranded RNA virus) have been found in a nuclease-resistant form in the serum of patients with HCV infections. We determined whether a complete negative-strand copy is present in the serum, whether the negative strand is particle-associated, and finally, whether it is virion-associated and encapsidated like the positive (genomic) strand. Isopyknic sucrose and cesium chloride density ultracentrifugation followed by a strand-specific reverse transcription-polymerase chain reaction on the collected fractions was performed to determine whether both positive and negative strands were associated with similar particles. Both strands comigrated to approximately the same density (1.11-1.16 g/cm3) in sucrose. After treatment of the plasma with detergent (0.1% Nonidet P-40) to remove the viral envelope and centrifugation on cesium chloride gradients, the positive strands shifted to a density of 1.35 g/cm3, and the negative strands were not detected. By using antibodies specific for the HCV core or envelope glycoproteins E1 or E2 coated onto the wells of a microtiter plate, it was possible to specifically bind HCV or viral cores to the solid phase. Pelleted virus particles were resuspended in either PBS or PBS with 0.1% Nonidet P-40 to expose the core. These pellets were then incubated in antibody-coated microtiter wells. RNA extracted from the bound and unbound fractions was tested for HCV RNA. The anti-core antibody was able to bind positive strands but not negative strands only in detergent-treated samples. In the nondetergent-treated pellets, the anti-E1 and -E2 bound the positive strand, but only anti-E1 bound the negative strands. These findings indicate that while both strands of HCV RNA can be detected in serum, the positive strand is encapsidated within the enveloped core, and the negative strand appears to be in a membrane particle associated with the viral envelope protein E1 but does not appear to be within the HCV core of circulating virions.
Overexpression of hepatic lipase in transgenic rabbits leads to a marked reduction of plasma high density lipoproteins and intermediate density lipoproteins.
Authors: Fan J , Wang J , Bensadoun A , Lauer S J , Dang Q , Mahley R W , Taylor J M
Published: May 2006

To elucidate the precise metabolic roles of hepatic lipase (HL), a human HL cDNA in a liver-specific expression vector was used to generate transgenic lines in the rabbit, an animal that normally expresses low levels of this enzyme. HL was detected in the plasma of all rabbits only after the administration of heparin; HL activity in transgenic rabbits was found at levels up to 80-fold greater than that in nontransgenic littermates. This increase in enzyme activity was associated with as much as a 5-fold decrease in total plasma cholesterol levels. Expression of the transgene resulted in a dramatic reduction in the level of large high density lipoproteins (HDL1 and HDL2) as well as dense HDL3. A reduction in the quantity of intermediate density lipoproteins (IDL) was also observed. These results demonstrate that HL functions in the metabolism of HDL and IDL, thereby playing a key role in plasma cholesterol homeostasis.
The development of mitochondrial medicine.
Authors: Luft R
Published: May 2006

Primary defects in mitochondrial function are implicated in over 100 diseases, and the list continues to grow. Yet the first mitochondrial defect--a myopathy--was demonstrated only 35 years ago. The field's dramatic expansion reflects growth of knowledge in three areas: (i) characterization of mitochondrial structure and function, (ii) elucidation of the steps involved in mitochondrial biosynthesis, and (iii) discovery of specific mitochondrial DNA. Many mitochondrial diseases are accompanied by mutations in this DNA. Inheritance is by maternal transmission. The metabolic defects encompass the electron transport complexes, intermediates of the tricarboxylic acid cycle, and substrate transport. The clinical manifestations are protean, most often involving skeletal muscle and the central nervous system. In addition to being a primary cause of disease, mitochondrial DNA mutations and impaired oxidation have now been found to occur as secondary phenomena in aging as well as in age-related degenerative diseases such as Parkinson, Alzheimer, and Huntington diseases, amyotrophic lateral sclerosis and cardiomyopathies, atherosclerosis, and diabetes mellitus. Manifestations of both the primary and secondary mitochondrial diseases are thought to result from the production of oxygen free radicals. With increased understanding of the mechanisms underlying the mitochondrial dysfunctions has come the beginnings of therapeutic strategies, based mostly on the administration of antioxidants, replacement of cofactors, and provision of nutrients. At the present accelerating pace of development of what may be called mitochondrial medicine, much more is likely to be achieved within the next few years.
Genetic changes in the transforming growth factor beta (TGF-beta) type II receptor gene in human gastric cancer cells: correlation with sensitivity to growth inhibition by TGF-beta.
Authors: Park K , Kim S J , Bang Y J , Park J G , Kim N K , Roberts A B , Sporn M B
Published: May 2006

We have found several genetic changes in the TGF-beta-type II receptor gene in human gastric cancer cell lines resistant to the growth inhibitory effect of TGF-beta. Southern blot analysis showed deletion of the type II receptor gene in two of eight cell lines and amplification in another two lines. The single cell line we studied that is sensitive to growth inhibition by TGF-beta showed no structural abnormalities of the type II receptor gene. Some of the gastric cancer cells resistant to the growth inhibitory effect of TGF-beta express either truncated or no detectable TGF-beta type II receptor mRNAs, whereas the one that retains responsiveness to the growth inhibitory effect of TGF-beta expresses a full-size type II receptor mRNA. Immunoprecipitation followed by Western blot analysis showed parallel changes in TGF-beta type II receptor expression. Our results suggest that one of the possible mechanisms of escape from autocrine or paracrine growth control by TGF-beta during carcinogenesis could involve genetic changes in the TGF-beta type II receptor gene itself or altered expression of its mRNA.
The structure of a complex of hexameric insulin and 4'-hydroxyacetanilide.
Authors: Smith G D , Ciszak E
Published: May 2006

X-ray crystallographic studies have been carried out on human insulin crystals grown in the presence of 4'-hydroxyacetanilide (Tylenol) and show that this nontoxic phenolic derivative can induce the T-->R transition, producing a T3R3 hexamer. Two different crystals, grown under different conditions, are rhombohedral, space group R3, with cell constants a = 81.11, c = 37.97 and a = 80.88, c = 37.60 A. The T3R3 hexamer is symmetric, resulting from the presence of a crystallographic threefold axis, and the asymmetric unit consists of a TR dimer. Data to a resolution of 1.9 A were measured on a crystal from each of the two crystallizations and the structures have been refined to residuals of 0.168 and 0.173. The guest molecule is bound by the R-state monomer through the formation of two hydrogen bonds from the hydroxy group of Tylenol to the carbonyl oxygen and the nitrogen of A6 Cys and A11 Cys, respectively. Due to steric constraints of the phenolic binding site, the acetamide group of Tylenol is rotated approximately 50 degrees out of the plane of the phenyl group and the methyl group is cis; no hydrogen bonds exist between the acetamide group and the hexamer. Although the zinc ion, which is bound to the R-state trimer, has tetrahedral coordination in both structures, the T-state zinc is observed to have octahedral coordination in one structure but tetrahedral coordination in the other. The side chain of A10 Ile in the R-state monomer adopts a high-energy conformation as a result of close contact to a residue in an adjacent dimer and may explain in part the differences between therapeutic preparations of beef insulin, for which A10 is a Val residue, and human insulin.
The bovine papillomavirus E2 protein modulates the assembly of but is not stably maintained in a replication-competent multimeric E1-replication origin complex.
Authors: Lusky M , Hurwitz J , Seo Y S
Published: May 2006

Initiation of bovine papillomavirus (BPV) DNA synthesis in vivo and in vitro depends on the interaction of the viral initiator protein E1 with the replication origin (ori+ DNA). The viral E2 protein assists this interaction, resulting in a cooperative assembly of both proteins on the replication origin. Using gel mobility-shift experiments, we demonstrate that in the presence of both E1 and E2 proteins two classes of ori+ DNA complexes were formed: complex 1 (c1) and complex 2 (c2). Formation of c1 depended on both the E1 and E2 proteins and both proteins were contained within c1. The generation of c2 was dependent on the E1 protein and could be enhanced by E2, but the E2 protein was not detected within c2. At high E2/E1 ratios, c1 was the dominant complex formed. Under these conditions, E1-dependent BPV DNA synthesis in vitro was inhibited. At low E2/E1 ratios, the stimulation of c2 was correlated with the stimulation of BPV DNA replication by E2 in vitro. These data suggest that E2 assists E1 in the formation of an intermediate c1 complex, which is replication inactive. The c1 complex is converted in turn to the replication-active c2 complex, which contains E1 but lacks E2. We propose that the ratios of c1 and c2 formed in response to the levels of E1 and E2 protein determine the potential for BPV DNA synthesis in vitro and in vivo and may contribute to copy number regulation of BPV plasmids within the cell.
A mismatch recognition defect in colon carcinoma confers DNA microsatellite instability and a mutator phenotype.
Authors: Aquilina G , Hess P , Branch P , MacGeoch C , Casciano I , Karran P , Bignami M
Published: May 2006

We have analyzed spontaneous mutations in the adenine phosphoribosyltransferase gene of Chinese hamster clone B cells that exhibit a mutator phenotype because of defective mismatch binding. The mutator phenotype conferred increases in a limited number of mutational classes. The rates of transitions and most transversions were not significantly increased. The rates of A to T transversions and -2 frameshifts were strikingly elevated. These mutations were in repeated elements and 5 of 9 of the frameshifts were dinucleotide deletions in DNA sequences resembling microsatellites. The mismatch binding protein that is defective in the mutator line is a G-T mismatch recognition factor. Band-shift analysis indicated that the preferred substrate for the mismatch recognition protein is duplex DNA containing an extrahelical mono- or dinucleotide within repeated sequences. In agreement with a role in preventing minus frameshifts, a defective binding protein conferred an instability in clone B microsatellite DNA. A mismatch binding defect was also detected in Lo Vo, a human colorectal carcinoma cell line. Extracts of clone B or a second mismatch binding-deficient line, Raji-F12, did not complement Lo Vo extracts, indicating that these lines share a common defect. Our data provide a mechanistic explanation for the relation between defective mismatch recognition and the microsatellite instability of human colon cancer.
Specific sequences from the carboxyl terminus of human p53 gene product form anti-parallel tetramers in solution.
Authors: Sakamoto H , Lewis M S , Kodama H , Appella E , Sakaguchi K
Published: May 2006

Human p53 is a tumor-suppressor gene product associated with control of the cell cycle and with growth suppression, and it is known to form homotetramers in solution. To investigate the relationship of structure to tetramerization, nine peptides corresponding to carboxyl-terminal sequences in human p53 were chemically synthesized, and their equilibrium associative properties were determined by analytical ultracentrifugation. Secondary structure, as determined by circular dichroism measurements, was correlated with oligomerization properties of each peptide. The sedimentation profiles of peptides 319-393 and 319-360 fit a two-state model of peptide monomers in equilibrium with peptide tetramers. Successive deletion of amino- and carboxyl-terminal residues from 319-360 reduced tetramer formation. Further, substitution of alanine for Leu-323, Tyr-327, and Leu-330 abolished tetramerization. Circular dichroism studies showed that peptide 319-351 had the highest alpha-helix content, while the other peptides that did not form tetramers had low helical structure. These studies define a minimal region and identify certain critical residues involved in tetramerization. Cross-linking studies between monomer units in the tetramer suggest that the helices adopt an anti-parallel arrangement. We propose that conformational shifts in the helical structure of the p53 tetramerization domain result in a repositioning of subunits relative to one another. This repositioning provides an explanation relating conformational changes at the carboxyl terminus with changes in sequence-specific DNA binding by the highly conserved central domain.
The transcription factor TFIIS zinc ribbon dipeptide Asp-Glu is critical for stimulation of elongation and RNA cleavage by RNA polymerase II.
Authors: Jeon C , Yoon H , Agarwal K
Published: May 2006

The eukaryotic transcription factor TFIIS enhances elongation and nascent transcript cleavage activities of RNA polymerase II in a stalled elongation complex. By site-directed mutagenesis, we have demonstrated that invariant residues Asp-261 and Glu-262 of the nucleic acid-binding TFIIS Zn ribbon are critical for stimulation of both elongation and RNA cleavage activities of RNA polymerase II. Substitution of either of these residues inactivates both TFIIS functions, suggesting a related role in both activities. These acidic residues may participate in phosphoryl transfer reactions by a two-metal-ion mechanism in a manner analogous to Klenow fragment. The RNA polymerase II itself may contain a Zn ribbon, in as much as the polymerase's 15-kDa subunit contains a sequence that aligns well with the TFIIS Zn ribbon sequence, including a similarly placed pair of acidic residues.
Direct isolation of genes encoded within a homogeneously staining region by chromosome microdissection.
Authors: Su Y A , Trent J M , Guan X Y , Meltzer P S
Published: May 2006

Identification of genes involved in recurring chromosome rearrangements has provided significant insight into the molecular basis of malignancy. We describe here a strategy combining chromosome microdissection and hybrid selection for the direct isolation of chromosome region-specific genes. We modeled this strategy by using sequences recovered from the microdissection of a homogeneously staining region to allow isolation of genes that were overexpressed and present at high copy number within the homogeneously staining region, including the direct isolation of two genes encoded within a 12q homogeneously staining region found in the osteosarcoma cell line OsA-CL. Although first applied to amplified genes, this strategy should be applicable to the isolation of cDNAs from any chromosomal region.
Serial transmission in rodents of neurodegeneration from transgenic mice expressing mutant prion protein.
Authors: Hsiao K K , Groth D , Scott M , Yang S L , Serban H , Rapp D , Foster D , Torchia M , Dearmond S J , Prusiner S B
Published: May 2006

Two lines of transgenic (Tg) mice expressing high (H) levels of the mutant P101L prion protein (PrP) developed a neurologic illness and central nervous system pathology indistinguishable from experimental murine scrapie; these mice were designated Tg(MoPrP-P101L)H. Brain homogenates from Tg(MoPrP-P101L)H mice were inoculated intracerebrally into CD-1 Swiss mice, Syrian hamsters, and Tg196 mice, Tg mice expressing the MoPrP-P101L transgene at low levels. None of the CD-1 mice developed central nervous system dysfunction, whereas approximately 10% of hamsters and approximately 40% of the Tg196 mice manifested neurologic signs between 117 and 639 days after inoculation. Serial transmission of neurodegeneration in Tg196 mice and Syrian hamsters was initiated with brain extracts, producing incubation times of approximately 400 and approximately 75 days, respectively. Although the Tg(MoPrP-P101L)H mice appear to accumulate only low levels of infections prions in their brains, the serial transmission of disease to inoculated recipients argues that prion formation occurs de novo in the brains of these uninoculated animals. These Tg mouse studies, taken together with similar findings in humans dying of inherited prion diseases, provide additional evidence that prions lack a foreign nucleic acid.
Negative regulation of early B lymphopoiesis by interleukin 3 and interleukin 1 alpha.
Authors: Hirayama F , Clark S C , Ogawa M
Published: May 2006

We recently developed a two-step methyl cellulose culture system for murine lymphohemopoietic progenitors that are capable of differentiation along the myeloid and B-lymphoid lineages. In this system, two-factor combinations, which include steel factor plus interleukin (IL) 6, IL-11, or granulocyte colony-stimulating factor effectively supported the lymphomyeloid potential of primary colonies. Interestingly, IL-3 could neither replace nor act synergistically with steel factor in maintaining the B-lymphoid potential of the primary colonies although the frequency of colony formation was the same with IL-3 and steel factor. We now report that addition of IL-3 or IL-1 alpha to a permissive system suppresses the B-lymphoid potential of primitive progenitor cells in primary culture in dose-dependent fashion. In vivo transfer of the primary colonies to scid mice confirmed the suppressive effects of IL-3 and IL-1 alpha. In addition, IL-1 alpha inhibited pre-B-cell colony formation in the secondary culture. Once pre-B-cell colonies had formed in secondary culture, neither factor affected the proliferation of the pre-B cells. These results suggest negative regulatory roles for IL-3 and IL-1 alpha in early stages of B lymphopoiesis.
Envelope glycoprotein gp120 of human immunodeficiency virus type 1 alters ion transport in astrocytes: implications for AIDS dementia complex.
Authors: Benos D J , Hahn B H , Bubien J K , Ghosh S K , Mashburn N A , Chaikin M A , Shaw G M , Benveniste E N
Published: May 2006

Infection by human immunodeficiency virus type 1 (HIV-1) is often complicated by a variety of neurological abnormalities. The most common clinical syndrome, termed acquired immunodeficiency syndrome (AIDS) dementia complex, presents as a subcortical dementia with cognitive, motor, and behavioral disturbances and is unique to HIV-1 infection. The pathogenesis of this syndrome is poorly understood but is believed to involve interactions among virally infected macrophages/microglia, astrocytes, and neurons. In this study, we show that exposure of primary rat and human astrocytes to heat-activated HIV-1 virions, or to eukaryotically expressed HIV-1 and HIV-2 envelope glycoproteins (gp120) stimulates amiloride-sensitive Na+/H+ antiport, potassium conductance, and glutamate efflux. These effects are blocked specifically by amiloride, an inhibitor of Na+/H+ antiport and by the selective removal of gp120 with immobilized monoclonal antibody. As a result of modulation of astrocytic function by gp120, the ensuing neuronal depolarization and glutamate exposure could activate both voltage-gated and N-methyl-D-aspartate-regulated Ca2+ channels, leading to increases in intraneuronal Ca2+ and neuronal death. These findings implicate the astrocyte directly in the pathogenesis of AIDS dementia complex.
Diversity and virulence thresholds in AIDS.
Authors: de Boer R J , Boerlijst M C
Published: May 2006

We propose a model for the interaction between human immunodeficiency virus and the immune system. Two differential equations describe the interactions between one strain of virus and one clone of T lymphocytes. We use the model to generalize earlier results pertaining to the AIDS diversity threshold [Nowak, M. A., Anderson, R. M., McLean, A. R., Wolfs, T. F. W., Goudsmit, J. and May, R. M. (1991) Science 254, 963-969]. Our model has (i) a stable steady state corresponding to the "controlled" persistence of the virus and (ii) a region corresponding to AIDS. The separatrix between the two regimes is formed by the stable manifold of a saddle point. We define a dimensionless "virulence" parameter which combines the infectivity and antigenicity of a virus strain. We derive analytically two parameter conditions involving virulence. The first corresponds to a saddle-node bifurcation which causes AIDS due to the loss of the stable equilibrium. The second corresponds to a global bifurcation which causes AIDS due to a change in the basins of attraction. Incorporating diversity into the model, we derive a diversity threshold corresponding to the saddle-node bifurcation. In this threshold condition diversity and virulence have an equivalent effect. By studying the effect of diversity on the critical virulence that is required for a new mutant to cause AIDS, we again establish that diversity and virulence are equivalent parameters. Because in our model increasing diversity decreases the critical virulence, the strain that eventually causes AIDS need not be a virulent one.
Function of major histocompatibility complex class II promoters requires cooperative binding between factors RFX and NF-Y.
Authors: Reith W , Siegrist C A , Durand B , Barras E , Mach B
Published: May 2006

Transcription of major histocompatibility complex (MHC) class II genes is controlled largely by the conserved promoter elements called the X and Y boxes. We show here that RFX, the X box-binding protein deficient in certain MHC class II-deficient immunodeficiency patients (CID), and the Y box-binding protein NF-Y bind cooperatively. Functional relevance of this protein-protein interaction is suggested by the fact that promoter activity correlates with cooperative binding of RFX and NF-Y rather than with binding of RFX or NF-Y alone. Stability of the RFX/NF-Y complex is affected by alterations in X-Y box spacing. These results are consistent with the fact that MHC class II promoter function is dependent on correct stereospecific alignment of the X and Y boxes. Cooperative binding involving RFX, NF-Y, and perhaps other MHC class II promoter-binding proteins may explain why the highly specific defect in binding of RFX observed in CID cells is associated in vivo with a bare promoter in which all of the cis-acting elements, including the X and Y boxes, are unoccupied.
Plant mitochondrial nucleic acid sequences as a tool for phylogenetic analysis.
Authors: Hiesel R , von Haeseler A , Brennicke A
Published: May 2006

To evaluate the potential of mitochondrial nucleic acid sequences as a phylogenetic tool, we have analyzed cytochrome oxidase subunit III (coxIII) coding sequences in representatives of the major groups of land plants. The phylogenetic tree derived from these mitochondrial sequences confirms the monophyletic origin of land plant mitochondria with the general order and descent of land plants deduced by other molecular, physiological, and morphological traits. The mitochondrial sequences strongly suggest a close phylogenetic relationship between Bryophyta and Lycopodiatae, whereas Psilophytatae cluster with the other vascular plants. In addition to the high sequence similarity, both Hepaticophytina and Lycopodiatae contain a related intron in the coxIII gene that, to our knowledge, is not found in any other plant species. The slowly evolving mitochondrial sequences of plants are shown to provide a useful phylogenetic tool to evaluate distant evolutionary relationships within this kingdom.
Photoaffinity labeling of Arabidopsis thaliana plasma membrane vesicles by 5-azido-[7-3H]indole-3-acetic acid: identification of a glutathione S-transferase.
Authors: Zettl R , Schell J , Palme K
Published: May 2006

We used 5-azido-[7-3H]indole-3-acetic acid (5-azido-[7-3H]IAA), a photoaffinity analogue of the plant hormone indole-3-acetic acid (IAA), to search for auxin-binding proteins in Arabidopsis thaliana membranes. We identified an auxin-binding protein with a molecular mass of 24 kDa (Atpm24) in microsomes as well as in plasma membrane vesicles. Atpm24 was solubilized by 1% Triton X-100 and partially purified. A cDNA clone (Atpm24.1) corresponding to Atpm24 was isolated. The amino acid sequence predicted from the Atpm24.1 cDNA contains 212 amino acid residues with a relative molecular mass of 24,128 Da. Data base searches revealed that the predicted protein has homology to glutathione S-transferases (GSTs; EC 2.5.1.18). When Atpm24.1 was expressed in Escherichia coli, we found a high level of GST activity in the bacterial extracts. We have analyzed the substrate specificity of this protein and found that cumene hydroperoxide and trans-stilbene oxide but not trans-cinnamic acid or IAA-CoA were substrates. A role for this GST in physiological processes of plants is discussed.
An integral membrane protein (LMP2) blocks reactivation of Epstein-Barr virus from latency following surface immunoglobulin crosslinking.
Authors: Miller C L , Lee J H , Kieff E , Longnecker R
Published: May 2006

The role of latent membrane protein 2 (LMP2) in Epstein-Barr virus (EBV) infection was evaluated by using latently infected primary B lymphocytes that had been growth transformed by wild-type or specifically mutated EBV recombinants. LMP2 null mutant recombinant EBV-infected cells were similar to normal B lymphocytes in their rapid increase in intracellular free calcium after surface immunoglobulin crosslinking. These cells also became more permissive for lytic EBV replication. In sharp contrast, wild-type control infected cells had little or no increase in intracellular free calcium or in permissivity for EBV replication. The block to surface immunoglobulin crosslinking-induced permissivity in cells expressing wild-type LMP2 could be bypassed by raising intracellular free calcium levels with an ionophore and by activating protein kinase C with phorbol 12-myristate 13-acetate. LMP2A, not LMP2B, mediates this effect on calcium mobilization. Genetic and biochemical data are consistent with these effects being due to the interaction of the LMP2A N-terminal cytoplasmic domain with B lymphocyte src family tyrosine kinases.
Coaxial stacking of helixes enhances binding of oligoribonucleotides and improves predictions of RNA folding.
Authors: Walter A E , Turner D H , Kim J , Lyttle M H , Müller P , Mathews D H , Zuker M
Published: May 2006

An RNA model system consisting of an oligomer binding to a 4-nt overhang at the 5' end of a hairpin stem provides thermodynamic parameters for helix-helix interfaces. In a sequence-dependent manner, oligomers bind up to 1000-fold more tightly adjacent to the hairpin stem than predicted for binding to a free tetramer at 37 degrees C. For the interface (/) in [formula: see text] additional free energy change, delta delta G 37 degrees, for binding is roughly the nearest-neighbor delta G 37 degrees for propagation of an uninterrupted helix of equivalent sequence, CGGC. When X and Z are omitted, the delta delta 37 degrees is even more favorable by approximately 1 kcal/mol (1 cal = 4.184J). On average, predictions of 11 RNA secondary structures improve from 67 to 74% accuracy by inclusion of similar stacking contributions.