bluuebunny/crossref_metadata_2025 huggingface dataset:
A nucleotide insertion and frameshift cause analbuminemia in an Italian family.
Authors: Watkins S , Madison J , Galliano M , Minchiotti L , Putnam F W
Published: May 2006

In analbuminemia, a very rare inherited syndrome, subjects produce little or no albumin (1/100th to 1/1000th normal), presumably because of a mutation in the albumin gene; yet, they have only moderate edema and few related symptoms owing to a compensatory increase in other plasma proteins. Because of the virtual absence of albumin the defect must be identified at the DNA level. In this study the mutation causing analbuminemia in an Italian family was investigated by analysis of DNA from a mother and her daughter. The mother was homozygous for the trait and had a serum albumin value of < 0.01 g/dl (about 1/500th normal); the daughter was heterozygous for the trait and had a nearly normal albumin value. Molecular cloning and sequence analysis of DNA from both mother and daughter showed that the mutation is caused by a nucleotide insertion in exon 8; this produces a frameshift leading to a premature stop, seven codons downstream. The methods of heteroduplex hybridization and single-strand conformation polymorphism were used to compare the DNA of the mother and daughter to the DNA of two unrelated analbuminemic individuals (one Italian and one American). This showed that all three analbuminemic individuals had different mutations; these also differed from the mutation in the only human case previously studied at the DNA level, which was a splicing defect affecting the ligation of the exon 6-exon 7 sequences. Thus, analbuminemia may result from a variety of mutations and is genetically heterogeneous.
Activation of JAK2 kinase mediated by the interleukin 6 signal transducer gp130.
Authors: Narazaki M , Witthuhn B A , Yoshida K , Silvennoinen O , Yasukawa K , Ihle J N , Kishimoto T , Taga T
Published: May 2006

The interleukin 6 receptor-associated signal transducer, gp130, is shared by receptor complexes for leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and interleukin 11. We show here that JAK2 kinase is rapidly tyrosine phosphorylated in mouse embryonic stem cells whose pluripotentiality is maintained only by gp130-sharing cytokines after stimulation that is known to induce gp130 homodimerization. JAK1 is also tyrosine phosphorylated, but to a lesser extent, under the same conditions. Comparable results are obtained with hemopoietic lineage cells such as myeloid leukemic M1 cells and pro-B-cell line-derived transfectants expressing gp130, the former of which differentiate into macrophages after stimulation of gp130 and the latter of which initiate DNA synthesis. gp130-dimerizing stimulus upregulates kinase activity of JAK2 as revealed by immunocomplex kinase assay. Deletion or point mutation in the membrane-proximal cytoplasmic motifs in gp130 that are conserved in the hemopoietic cytokine receptor family results in the loss of tyrosine phosphorylation of JAK2, which coincides with the lack of signal transducing capability of gp130 mutants.
Biochemical studies on capped RNA primers identify a class of oligonucleotide inhibitors of the influenza virus RNA polymerase.
Authors: Chung T D , Cianci C , Hagen M , Terry B , Matthews J T , Krystal M , Colonno R J
Published: May 2006

A synthetic 67-nt RNA substrate, containing a 32P-labeled cap-1 structure (m7G32pppGm) was specifically cleaved by the influenza virus RNA polymerase (EC 2.7.7.48) to yield a single capped 11-nt fragment capable of directly priming transcription. An analysis of systematic truncations of this RNA substrate demonstrated that an additional nucleotide beyond this cleavage site was required for cleavage. The minimal RNA chain length required for priming activity was found to be 9 nt, while in contrast an RNA chain length of at least 4 nt was required for efficient binding to the viral polymerase. On the basis of these chain length requirements we show that a pool of capped oligonucleotides too short to prime transcription, but long enough to bind with high affinity to the viral polymerase, are potent inhibitors of cap-dependent transcription in vitro.
Lyme disease: a growing threat to urban populations.
Authors: Steere A C
Published: May 2006

Lyme disease or Lyme borreliosis, which is caused by three groups of the spirochete Borrelia burgdorferi, is transmitted in North America, Europe, and Asia by ticks of the Ixodes ricinus complex. The primary areas around the world that are now affected by Lyme disease are near the terminal moraine of the glaciers 15,000 years ago. The emergence of Lyme disease in the United States in this century is thought to have occurred because of ecological conditions favorable for deer. From 1982 through 1991, 40,195 cases occurring in 47 states were reported to the Centers for Disease Control, but enzootic cycles of B. burgdorferi have been identified in only 19 states. During the last several decades, the disease has spread to new areas and has caused focal outbreaks, including locations near Boston, New York, and Philadelphia. Lyme disease is like syphilis in its multisystem involvement, occurrence in stages, and mimicry of other diseases. Diagnosis of late neurologic abnormalities of the disorder has created the most difficulty. A recent phenomenon is that a number of poorly understood conditions, such as chronic fatigue syndrome or fibromyalgia, are misdiagnosed as "chronic Lyme disease." Part of the reason for misdiagnosis is due to problems associated with diagnostic tests. The various manifestations of Lyme disease can usually be treated successfully with oral doxycycline or amoxicillin, except for objective neurologic manifestations, which seem to require intravenous therapy. Vector control of thick-borne diseases has been difficult and, therefore, reduction of the risk of infection has been limited primarily to personal protection measures.
Further perspective on the catalytic core and secondary structure of ribonuclease P RNA.
Authors: Haas E S , Brown J W , Pitulle C , Pace N R
Published: May 2006

Phylogenetic comparative analyses of RNase P RNA-encoding gene sequences from Chlorobium limicola, Chlorobium tepidum, Bacteroides thetaiotaomicron, and Flavobacterium yabuuchiae refine the secondary structure model of the general (eu)bacterial RNase P RNA and show that a highly conserved feature of that RNA is not essential. Two helices, comprised of 2 base pairs each, are added to the secondary structure model and form part of a cruciform in the RNA. Novel sequence variations in the B. thetaiotaomicron and F. yabuuchiae RNA indicate the likelihood that all secondary structure resulting from canonical base-pairing has been detected: there are no remaining unpaired, contiguous, canonical complementarities in the structure model common to all bacterial RNase P RNAs. A nomenclature for the elements of the completed secondary structure model is proposed. The Chlorobium RNase P RNAs lack a stem-loop structure that is otherwise universally present and highly conserved in structure in other (eu)bacterial RNase P RNAs. The Chlorobium RNAs are nevertheless catalytic, with kinetic properties similar to those of RNase P RNAs of Escherichia coli and other Bacteria. Removal of this stem-loop structure from the E. coli RNA affects neither its affinity for nor its catalytic rate for cleavage of a precursor transfer RNA substrate. These results show that this structural element does not play a direct role in substrate binding or catalysis.
RanGAP1 induces GTPase activity of nuclear Ras-related Ran.
Authors: Bischoff F R , Klebe C , Kretschmer J , Wittinghofer A , Ponstingl H
Published: May 2006

The nuclear Ras-related protein Ran binds guanine nucleotide and is involved in cell cycle regulation. Models of the signal pathway predict Ran to be active as Ran.GTP at the initiation of S phase upon activation by the nucleotide exchange factor RCC1 and to be inactivated for the onset of mitosis by hydrolysis of bound GTP. Here a nuclear homodimeric 65-kDa protein, RanGAP1, is described, which we believe to be the immediate antagonist of RCC1. It was purified from HeLa cell lysates and induces GTPase activity of Ran, but not Ras, by more than 3 orders of magnitude. The Ran mutant Q69L, modeled after RasQ61L, which is unable to hydrolyze bound GTP, is insensitive to RanGAP1.
Heme oxygenase 1 mediates an adaptive response to oxidative stress in human skin fibroblasts.
Authors: Vile G F , Basu-Modak S , Waltner C , Tyrrell R M
Published: May 2006

Oxidative stress of human skin fibroblasts by treatment with ultraviolet A (UVA) radiation has been shown to lead to an increase in levels of the heme catabolizing enzyme heme oxygenase 1 [heme, hydrogen-donor:oxygen oxidoreductase (alpha-methene-oxidizing, hydroxylating), EC 1.14.99.3] and the iron storage protein ferritin. Here we show that human skin fibroblasts, preirradiated with UVA, sustain less membrane damage during a subsequent exposure to UVA radiation than cells that had not been preirradiated. Pretreating cells with heme oxygenase 1 antisense oligonucleotide inhibited the irradiation-dependent induction of both the heme oxygenase I enzyme and ferritin and abolished the protective effect of preirradiation. Inhibition of the UVA preirradiation-dependent increase in ferritin, but not heme oxygenase, with desferrioxamine also abolished the protection. This identifies heme oxygenase 1 as a crucial enzymatic intermediate in an oxidant stress-inducible antioxidant defense mechanism, involving ferritin, in human skin fibroblasts.
The human blue opsin promoter directs transgene expression in short-wave cones and bipolar cells in the mouse retina.
Authors: Chen J , Tucker C L , Woodford B , Szél A , Lem J , Gianella-Borradori A , Simon M I , Bogenmann E
Published: May 2006

Transgenic mouse lines were generated using either 3.8 or 1.1 kb of 5' upstream flanking sequence from the human blue opsin gene fused to the lacZ or human growth hormone reporter gene. Mice were analyzed for appropriate cell-specific and developmental expression patterns. In 13 independently derived lines of animals, transgene expression was limited to photoreceptor and inner nuclear layer cells. Photoreceptors were identified as cone cells based on morphological criteria and colocalization of transgene expression with the cone-associated marker, peanut agglutinin lectin. More specifically, transgene-positive photoreceptors were identified as short-wave cone cells (S-cones) by using the short-wave color opsin-specific antibody, OS-2. Reporter-gene-positive cells of the inner nuclear layer were identified as bipolar cells based on morphological criteria. Transgenes and the endogenous mouse short-wave opsin gene were transcriptionally coactivated at embryonic day 13. These results show that 3.8 or 1.1 kb of human blue opsin upstream flanking sequences are capable of directing expression in short-wave cone cells in a spatially and temporally appropriate fashion and that the human blue opsin gene is the homologue of the short-wave-sensitive pigment, S-opsin, in the short-wave cones of the mouse retina. Expression in the bipolar cells may reflect regulatory mechanisms that are common to these cells and to the cone photoreceptors.
Designing amino acids to determine the local conformations of peptides.
Authors: Burgess A W
Published: May 2006

The local conformations of proteins and peptides are determined by the amino acid sequence. However, the 20 amino acids encoded by the genome allow the peptide backbone to fold into many conformations, so that even for a small peptide it becomes very difficult to predict the three-dimensional structure. By using empirical conformational energy calculations, a set of amino acids has been designed that would be expected to constrain the conformation of a peptide or a protein to one or two local minima. Most of these amino acids are based on asymmetric substitutions at the C alpha atom of each residue. The H alpha atom of alanine was replaced by various groups: -OCH3, -NCH3, -SCH3, -CONH2, -CONHCH3, -CON(CH3)2, -NH.CO.CH3, -phenyl, or -o-(OCH3)phenyl. Several of these new amino acids are predicted to fold into unique peptide conformations such as right-handed alpha-helical, left-handed alpha-helical, or extended. In an attempt to produce an amino acid that favored the C(eq)7 conformation (torsion angles: phi = -70 degrees and psi = +70 degrees), an extra amide group was added to the C beta atom of the asparagine side chain. Conformationally restricted amino acids of this type could prove useful for developing new peptide pharmaceuticals, catalysts, or polymers.
Chromophore-assisted laser inactivation of patched protein switches cell fate in the larval visual system of Drosophila.
Authors: Schmucker D , Su A L , Beermann A , Jäckle H , Jay D G
Published: May 2006

The Drosophila segment-polarity gene patched (ptc) is an integral component of the segmentation gene cascade acting in the early embryo. At later stages of embryogenesis, ptc is expressed in the primordia of epithelial placodes of a specific portion of the brain, the optic lobes. Mutant analysis shows that the lack of ptc activity alters the fate of optic-lobe primordia precursors. In ptc mutants they give rise to supernumerary neurons in the larval light-sensory system, termed Bolwig organ, which is derived from precursor cells next to the optic-lobe anlagen. We specifically eliminated ptc protein by chromophore-assisted laser inactivation (CALI) in late wild-type embryos. Such embryos show a normal segment pattern, but they develop phenocopies equivalent to the phenotype of ptc mutant Bolwig organs. Our results demonstrate that the CALI technique can be applied to separate genetic functions at different developmental stages of a living organism and that the segment-polarity gene ptc is redeployed to functionally discriminate between distinct developmental pathways in adjacent pools of precursor cells.
Fatal familial insomnia and familial Creutzfeldt-Jakob disease: different prion proteins determined by a DNA polymorphism.
Authors: Monari L , Chen S G , Brown P , Parchi P , Petersen R B , Mikol J , Gray F , Cortelli P , Montagna P , Ghetti B
Published: May 2006

Fatal familial insomnia and a subtype of Creutzfeldt-Jakob disease, two clinically and pathologically distinct diseases, are linked to the same mutation at codon 178 (Asp-178-->Asn) but segregate with different genotypes determined by this mutation and the methionine-valine polymorphism at codon 129 of the prion protein gene. The abnormal isoforms of the prion protein in these two diseases were found to differ both in the relative abundance of glycosylated forms and in the size of the protease-resistant fragments. The size difference was consistent with a different protease cleavage site, suggesting a different conformation of the protease-resistant prion protein present in the two diseases. These differences are likely to be responsible for the type and location of the lesions that characterize these two diseases. Therefore, the combination of the mutation at codon 178 and the polymorphism at codon 129 determines the disease phenotype by producing two altered conformations of the prion protein.
Invasion and maintenance of a gene duplication.
Authors: Clark A G
Published: May 2006

The ubiquity of multigene families is evidence for the frequent occurrence of gene duplication, but the origin of multigene families from a single gene remains a little-studied aspect of genome evolution. Although it is clear that a duplication can arise and become fixed in a population purely by random genetic drift and that the rate of fixation is accelerated if the duplication is directly advantageous, the nature of gene duplication suggests that other factors may influence the fate of a novel duplication. In the face of disadvantageous loss-of-function mutations, duplication of a functional gene may provide a buffer against such mutations. Here the conditions for invasion of a rare duplication starting from a mutation-selection balance are derived with formal population genetic models in both haploids and diploids. Recurrent duplication protects the duplicated chromosome from loss and can be very effective in increasing its frequency in a population. In the absence of recurrent duplication, one might suppose that a duplication would be favored by natural selection because it can mask the effects of deleterious mutations. However, the models show that a duplication can invade only if it provides a direct advantage to the organism. This result is closely related to recent theoretical work on the evolution of ploidy.
Adhesion of hard spheres under the influence of double-layer, van der Waals, and gravitational potentials at a solid/liquid interface.
Authors: Senger B , Schaaf P , Bafaluy F J , Cuisinier F J , Talbot J , Voegel J C
Published: May 2006

The deposition process of colloidal particles or microorganisms on flat surfaces is analyzed by means of computer simulations. Interparticle interactions (double layer and van der Waals) and weak gravitational forces are taken into account; hydrodynamic interactions, on the other hand, are neglected. In particular, the deposition probability as a function of the deposition location of a particle in the presence of one or two identical fixed particles is discussed. It is shown, in particular, that the ratio of the adhesion probabilities at a given location r, for particles subject to weak gravitation, in the presence and in the absence of the interparticle interaction U(r) follows approximately a Boltzmann law exp[-U(r)/kT], even though the adsorption process is fully irreversible. This result validates, as far as the distribution function of particles on a surface is concerned, Adamczyk's assumption [Adamczyk, Z., Zembala, M., Siwek, B. & Warszynski, P. (1990) J. Colloid Interface Sci. 140, 123-137] that the adhesion process of Brownian particles can be modeled by a random sequential adsorption model with an adsorption probability equal to exp[-U(r)/kT].
Molecular cloning, sequencing, and functional expression of a cDNA encoding human coproporphyrinogen oxidase.
Authors: Martasek P , Camadro J M , Delfau-Larue M H , Dumas J B , Montagne J J , de Verneuil H , Labbe P , Grandchamp B
Published: May 2006

Coproporphyrinogen oxidase (EC 1.3.3.3) catalyzes the sixth step in the heme biosynthetic pathway, the oxidation of coproporphyrinogen III to protoporphyrinogen IX. The activity of this enzyme is deficient in the disease hereditary coproporphyria. The sequence of the cDNA and predicted amino acid sequence of the human coproporphyrinogen oxidase are presented. The human protein sequence contains a region completely homologous to that we obtained by sequencing an 11-amino acid peptide fragment from purified murine liver coproporphyrinogen oxidase. Results of Southern blotting were consistent with the presence of a single human coproporphyrinogen oxidase gene, and Northern blotting demonstrated one transcript of similar size in erythroid and nonerythroid cell lines. Expression of the cDNA coding for the putative mature human coproporphyrinogen oxidase in Escherichia coli resulted in a 17-fold increase in coproporphyrinogen activity over endogenous activity.
Na3[B20H17NH3]: synthesis and liposomal delivery to murine tumors.
Authors: Feakes D A , Shelly K , Knobler C B , Hawthorne M F
Published: May 2006

The polyhedral borane ion [n-B20H18]2- reacts with liquid ammonia in the presence of a suitable base to produce an apical-equatorial (ae) isomer of the [B20H17NH3]3- ion, [1-(2'-B10H9)-2-NH3B10H8]3-. The structure of this product has been confirmed by 11B NMR spectroscopy and x-ray crystallography. This species undergoes acid-catalyzed rearrangement to an apical-apical (a2) isomer, [1-(1'-B10H9)-2-NH3B10H8]3-, whose structure has been determined by 11B NMR spectroscopy. The sodium salts of both the ae and the a2 isomers of [B20H17NH3]3- have been encapsulated within small unilamellar liposomes, composed of distearoyl phosphatidylcholine/cholesterol (1:1), and investigated as boron-delivery agents for boron neutron capture therapy (BNCT) of cancer. The biodistribution of boron was determined after the injection of liposomal suspensions into BALB/c mice bearing EMT6 tumors. Both [B20H17NH3]3- isomers exhibited excellent tumor uptake and selectivity at very low injected doses, achieving peak tumor boron concentrations of 30-40 micrograms of B/g of tissue and tumor/blood boron ratios of approximately 5. The enhanced retention of the [B20H17NH3]3- isomers by EMT6 tumors may be attributed to their facile intracellular oxidation to an extremely reactive NH3-substituted [n-B20H18]2- ion, the electrophilic [B20H17NH3]- ion. Both isomers of [B20H17NH3]3- are at least 0.5 V more easily oxidized than other previously investigated species containing 20 boron atoms. In another experiment, [ae-B20H17NH3]3- was encapsulated in liposomes prepared with 5% PEG-2000-distearoyl phosphatidylethanolamine in the liposome membrane. As expected, these liposomes exhibited a longer circulation lifetime in the biodistribution experiment, resulting in the continued accumulation of boron in the tumor over the entire 48-hr experiment and reaching a maximum of 47 micrograms of B/g of tumor.
Vertical versus planar neural induction in Rana pipiens embryos.
Authors: Saint-Jeannet J P , Dawid I B
Published: May 2006

The neural plate in the amphibian embryo is induced in the ectoderm by signals from the dorsal mesoderm. In the extensively studied species Xenopus laevis, such signals are believed to proceed along two alternate pathways, defined as vertical and planar induction. We have studied the relative importance of these pathways in Rana pipiens. In the embryo of this frog, dorsal mesoderm involution can be diverted from its normal course by injection of peptides that inhibit interaction of fibronectin with its receptor. In such embryos, dorsal mesoderm failed to migrate across the blastocoel roof but moved bilaterally along the equator, leading to the formation of two notochords. Neural tissue differentiation occurred in close association with each notochord, but no neural tissue formed along the dorsal midline as might have been predicted by a predominantly planar induction model. While in X. laevis planar induction has been reported to be a major pathway in neuralizing the ecoderm, the results presented here indicate that vertical induction predominates in initiating neural development in R. pipiens embryos.
Neurotrophin 3 stimulates the differentiation of motoneurons from avian neural tube progenitor cells.
Authors: Averbuch-Heller L , Pruginin M , Kahane N , Tsoulfas P , Parada L , Rosenthal A , Kalcheim C
Published: May 2006

Neurotrophin 3 (NT-3) promotes differentiation of neural tube progenitors into motoneurons expressing the BEN/SC1 and islet-1 epitopes. A 1.75- to 6.7-fold increase in BEN-positive motoneurons was obtained when quail neural tube cells were cultured with NT-3 at 0.1-10 ng/ml, respectively. In contrast, the overall number of cells, as well as the proportion of motoneurons that developed from cycling precursors, did not change. Addition of NT-3 at 1 ng/ml to cells obtained from ventral half-neural tubes promoted a 2.5-fold stimulation in motoneuron number, confirming the specificity of the effect. Moreover, NT-3 had no significant effect on survival of differentiated avian motoneurons. The distribution of trkC mRNA, which encodes the high-affinity receptor for NT-3, is consistent with these findings. trkC expression is homogeneous in the embryonic day 2 (E2) neural tube, becomes restricted to the mantle layer on E3, where differentiation occurs, and disappears from the ventral third of the E4-E5 spinal cord right before the onset of normal motoneuron death. These results suggest that NT-3 and trkC regulate early neurogenesis in the avian central nervous system.
PP1 gamma 2, a testis-specific protein-serine/threonine-phosphatase type 1 catalytic subunit, is associated with a protein having high sequence homology with the 78-kDa glucose-regulated protein, a member of the 70-kDa heat shock protein family.
Authors: Chun Y S , Shima H , Nagasaki K , Sugimura T , Nagao M
Published: May 2006

Protein phosphatase 1 gamma 2 (PP1 gamma 2) is a testis-specific isotype of the protein-serine/threonine-phosphatase type 1 catalytic subunit. Three native forms of PP1 gamma 2 were detected in a crude fraction of rat testis by electrophoresis in a nondenaturing polyacrylamide gel. We purified a major native form of PP1 gamma 2 to homogeneity by successive column chromatography on Mono Q-Sepharose, EAH-agarose, protamine-agarose, and G3000SW and by electrophoresis in a nondenaturing polyacrylamide gel. The G3000SW-purified PP1 gamma 2 native form had an apparent molecular mass of 170 kDa. The purified holoenzyme from nondenaturing polyacrylamide gel was composed of the catalytic subunit and two noncatalytic subunits, of 78 kDa and 55 kDa. Partial amino acid sequence analysis of the 78-kDa protein suggested that it is the 78-kDa glucose-regulated protein, a member of the 70-kDa heat shock protein family. The 78-kDa protein may possibly function as a chaperone or by confining substrate specificity of PP1 gamma 2.
Mast cells with gonadotropin-releasing hormone-like immunoreactivity in the brain of doves.
Authors: Silverman A J , Millar R P , King J A , Zhuang X , Silver R
Published: May 2006

Using an antiserum (LR-1) raised against mammalian gonadotropin-releasing hormone (GnRH), we previously identified a nonneuronal cell that was more numerous in the medial habenula (MH) of courting ring doves than in individuals housed in visual isolation. The current studies suggest that they are mast cells. Both acidic toluidine blue and toluidine blue dissolved in water/butanediol revealed metachromatic cells with a distribution and morphology similar to that obtained by immunostaining with the GnRH antiserum in the MH. Some cells had granules reactive to safranin in the presence of alcian blue, indicative of a highly sulfated proteoglycan of the heparin family. Immunocytochemical studies demonstrated that all MH cells containing GnRH-like immunoreactivity contained histamine, another mast cell marker. The GnRH-immunoreactive cells had a unilobular, ovoid nucleus. Secretory granules within the cells were electron dense and displayed a variety of internal structures. Fine filamentous processes appeared evenly distributed on the cell surface whether cells were located on the pial surface or within the brain parenchyma. All of these features are characteristic of mast cells. To test whether the epitope recognized by the GnRH antiserum was produced by the mast cells or endocytosed from the cerebrospinal fluid, an iodinated GnRH analog was injected intracerebroventricularly at the initiation of courtship. Radioautography revealed no radioactive cells in the brain, indicating that the GnRH antibody recognized a molecule synthesized by the nonneuronal cells rather than internalized by a receptor-mediated mechanism. These observations suggest an interaction between a component of the immune network and specific regions of the central nervous system.
Mutants of Escherichia coli K-12 that are resistant to a selenium analog of lipoic acid identify unknown genes in lipoate metabolism.
Authors: Reed K E , Morris T W , Cronan J E
Published: May 2006

Lipoic acid is a disulfide-containing cofactor required for the reactions catalyzed by alpha-ketoacid dehydrogenase enzyme complexes. We report the chemical synthesis and biological properties of lipoic acid analogs in which one or both sulfur atoms were replaced by selenium. Replacement of either the C-6 or the C-8 sulfur atom with selenium results in lipoic acid derivatives with apparently unaltered biological properties. However, simultaneous replacement of both sulfur atoms gave an analog (selenolipoic acid) that inhibited growth of wild-type Escherichia coli when present in minimal glucose medium at 50 ng/ml. This growth inhibition was reversed by the addition of either excess lipoic acid or acetate plus succinate. Labeling experiments with [75Se]selenolipoic acid showed that this compound was efficiently incorporated into the alpha-ketoacid dehydrogenase complexes of growing cells. Spontaneously arising selenolipoic acid-resistant (slr) mutants were isolated. Two of these isolates resistant to high levels of selenolipoic acid were studied in detail. The slr-1 mutation, which was mapped to min 99.6 of the E. coli chromosome, increased the lipoate requirement of lipA strains by 4-fold and appeared to define a gene encoding a lipoate-protein ligase. The slr-7 mutation, which was mapped to min 15.25 of the chromosome, completely suppressed the lipoate requirement of lipA strains and defined a gene of unknown function in the synthesis of lipoic acid.
Discrimination of odortypes determined by the major histocompatibility complex among outbred mice.
Authors: Yamazaki K , Beauchamp G K , Shen F W , Bard J , Boyse E A
Published: May 2006

Genetically determined body odors that distinguish one mouse from another are termed odortypes. The best known odortypes, highly expressed in urine, are those specified by H-2, the major histocompatibility complex of the mouse, but other odortypes originate from unidentified loci in the rest of the genome, including both sex chromosomes. The definition of H-2 odortypes and evidence that their perception affects reproductive behavior have so far depended on studies with inbred mouse strains whose genetic differences are confined to the H-2 complex of genes. To simulate feral conditions more closely, a freely segregating population was bred from crosses involving four unrelated inbred strains contributing four different H-2 haplotypes. After H-2 typing, this outbred population was divided into four groups of freely segregating mice, comprising the four distinct H-2 genotypes represented, to serve as conventional donors of urine for evaluation in the standard Y-maze system used in the training and testing of mice for H-2 odortype discrimination. With respect to utility in training mice for H-2 odortype discrimination, and to degrees of concordance attained in the Y-maze by trained mice, these urinary H-2 odortype sources from outbred mice were no less effective than urines customarily obtained for those purposes from nonsegregating inbred donors. We conclude that discrimination of H-2 odortypes is not appreciably affected or impaired by the usual concurrent segregation within the genome as a whole.
Cloning mammalian genes by expression selection of genetic suppressor elements: association of kinesin with drug resistance and cell immortalization.
Authors: Gudkov A V , Kazarov A R , Thimmapaya R , Axenovich S A , Mazo I A , Roninson I B
Published: May 2006

We describe a general strategy for cloning mammalian genes whose downregulation results in a selectable phenotype. This strategy is based on expression selection of genetic suppressor elements (GSEs), cDNA fragments encoding either specific peptides that act as dominant inhibitors of protein function or antisense RNA segments that efficiently inhibit gene expression. Since GSEs counteract the gene from which they are derived, they can be used as dominant selectable markers for the phenotype associated with downregulation of the corresponding gene. A retroviral library containing random fragments of normalized (uniform abundance) cDNA expressed in mouse NIH 3T3 cells was used to select for GSEs inducing resistance to the anticancer drug etoposide. Three GSEs were isolated, two of which are derived from unknown genes and the third encodes antisense RNA for the heavy chain of a motor protein kinesin. The kinesin-derived GSE induces resistance to several DNA-damaging drugs and immortalizes senescent mouse embryo fibroblasts, indicating that kinesin is involved in the mechanisms of drug sensitivity and in vitro senescence. Expression of the human kinesin heavy-chain gene was decreased in four of four etoposide-resistant HeLa cell lines, derived by conventional drug selection, indicating that downregulation of kinesin represents a natural mechanism of drug resistance in mammalian cells.
Synapsin IIa accelerates functional development of neuromuscular synapses.
Authors: Schaeffer E , Alder J , Greengard P , Poo M M
Published: May 2006

We have investigated the possible involvement of the synaptic vesicle protein synapsin IIa in synapse development. Synapsin IIa was introduced into Xenopus embryonic spinal neurons by early blastomere injection, and nerve-muscle cultures were prepared. Synaptic currents were measured by comparing synapses in which the presynaptic neuron either contained [syn IIa (+)] or lacked (control) exogenous synapsin IIa. Syn IIa (+) synapses had a 3.6-fold increase in the frequency and a 2.1-fold increase in the amplitude of spontaneous synaptic currents, compared to controls, after 2 days in culture. Synapsin IIa also increased the amplitude of evoked synaptic currents by 2.3-fold in 2-day cultures. The evoked synaptic current amplitudes of syn IIa (+) synapses had a lower coefficient of variation indicating a more stable evoked response. These enhanced synaptic activities were independent of the presence or absence of the protein in the postsynaptic muscle cell. The findings indicate a role for synapsin IIa in synapse maturation.
Peptide nucleic acid.DNA strand displacement loops as artificial transcription promoters.
Authors: Møllegaard N E , Buchardt O , Egholm M , Nielsen P E
Published: May 2006

Homopyrimidine peptide nucleic acids (PNAs) form loop structures when binding to complementary double-stranded DNA by strand displacement, and we now show that RNA polymerase recognizes these and initiates RNA transcription from PNA/double-stranded DNA strand displacement complexes at an efficiency comparable to that of the strong Escherichia coli lacUV5 promoter. Thus PNA targets can be considered as artificial promoters controlled positively by the corresponding PNA as a transcription factor. Our results have implications for the mechanism of action of RNA polymerase and suggest the use of PNA as specific gene activating reagents and drugs.
Organization of pioneer retinal axons within the optic tract of the rhesus monkey.
Authors: Meissirel C , Chalupa L M
Published: May 2006

Retinal ganglion cell axons must make a decision at the embryonic optic chiasm to grow into the appropriate optic tract. To gain insight into the cues that play a role in sorting out the crossed from the uncrossed optic axons, we investigated the sequence of their initial ingrowth in rhesus monkey embryos. Two carbocyanine dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate and 4-(4-dihexadecylaminostyryl)-N-methylpyridinium iodide, were placed, respectively, into the left and right retinas to identify the course of uncrossed and crossed retinal axons through the optic chiasm and tract. Our results show that at embryonic day 36 the most advanced retinal projections are uncrossed. At this age the leading crossed axons are just reaching the chiasmatic midline, whereas the uncrossed fibers have already entered the optic tract. This indicates that the pathfinding of these pioneer uncrossed fibers does not require the presence of retinal axons from the opposite eye. At subsequent stages of development (embryonic days 40 and 42) there is a clear partial segregation of the uncrossed and crossed retinal axons within the optic tract: the uncrossed-component course is in the deeper portion of the optic tract, whereas the crossed component lies in a more superficial region. Thus, the spatial organization of retinal axons within the primordial optic tract reflects the sequential addition of the uncrossed and crossed retinal fibers. The orderly and sequential ingrowth of these pioneer retinal axons indicates that specific chiasmatic cues are expressed early in development and that such pioneer fibers may serve as guides for the later-arriving retinal fibers.
Structure of the binding site for nonnucleoside inhibitors of the reverse transcriptase of human immunodeficiency virus type 1.
Authors: Smerdon S J , Jäger J , Wang J , Kohlstaedt L A , Chirino A J , Friedman J M , Rice P A , Steitz T A
Published: May 2006

The dipyridodiazepinone Nevirapine is a potent and highly specific inhibitor of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1). It is a member of an important class of nonnucleoside drugs that appear to share part or all of the same binding site on the enzyme but are susceptible to a variety of spontaneous drug-resistance mutations. The co-crystal-structure of HIV-1 RT and Nevirapine has been solved previously at 3.5-A resolution and now is partially refined against data extending to 2.9-A spacing. The drug is bound in a hydrophobic pocket and in contact with some 38 protein atoms from the p66 palm and thumb subdomains. Most, but not all, nonnucleoside drug-resistance mutations map to residues in close contact with Nevirapine. The major effects of these mutations are to introduce steric clashes with the drug molecule or to remove favorable protein-drug contacts. Additionally, four residues (Phe-227, Trp-229, Leu-234, and Tyr-319) in contact with Nevirapine have not been selected as sites of drug-resistance mutations, implying that there may be limitations on the number and types of resistance mutations that yield viable virus. Strategies of inhibitor design that target interactions with these conserved residues may yield drugs that are less vulnerable to escape mutations.
Analysis of the binding of the Src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-Src.
Authors: Sabe H , Hata A , Okada M , Nakagawa H , Hanafusa H
Published: May 2006

Csk (C-terminal Src kinase), a protein-tyrosine kinase, bearing the Src homology 2 and 3 (SH2 and SH3) domains, has been implicated in phosphorylation of c-Src Tyr-527, resulting in suppression of c-Src kinase activity. We found that mutations in the SH2 or SH3 domain of Csk, though they did not affect its kinase activity, resulted in a loss of suppression of c-Src activity in fibroblasts. In normal fibroblasts, tyrosine-phosphorylated paxillin and focal adhesion kinase pp125FAK, which colocalize at focal adhesion plaques, were the major proteins to which the Csk SH2 domain bound. Loss of binding to these proteins by the Csk SH2 mutants correlated with loss of the activity to suppress c-Src. Consistent with this observation, the levels of tyrosine phosphorylation of paxillin and pp125FAK were greatly reduced during mitosis, whereas the kinase activity of c-Src was elevated. We suggest that the SH2 domain is required for Csk to suppress c-Src, perhaps in combination with the SH3 domain, by anchoring Csk to a particular subcellular location where c-Src may exist. Our data also indicate that a certain fraction of the Csk and Src family kinases function at the focal adhesion plaques. The activity of the c-Src kinase localized at the focal adhesion plaques appears to be regulated by cell adhesion to the extracellular matrix.
Avian erythroblastosis virus E26: only one (myb) of two cell-derived coding regions is necessary for oncogenicity.
Authors: Wu Y , Duesberg P
Published: May 2006

The oncogene hypothesis postulates that mutated cellular genes, termed proto-onc genes, function as cancer genes because they are related to retroviral onc genes. However, in contrast to retroviral onc genes, mutated proto-onc genes from cancers are not sufficient for carcinogenesis. Therefore, it has been proposed that mutated proto-onc genes depend on other proto-onc genes for carcinogenesis. Since the oncogene of the avian leukemia virus E26 includes coding regions derived from two cellular proto-onc genes, proto-myb and proto-ets, this hybrid gene has been proposed to be a model for two-gene-carcinogenesis. Here we set out to test this proposal. For this purpose myb and ets deletion mutants of cloned E26 provirus were prepared, and the corresponding viruses, produced by transfected primary chicken embryo cells, were tested for leukemogenicity in newborn chickens. It was found that an ets deletion mutant was just as leukemogenic as the wild-type virus and that a myb deletion mutant lacked leukemogenicity completely. To eliminate the possibility that our E26 myb deletion mutant failed to be leukemogenic because it failed to replicate, the virus was titered by a quantitative polymerase chain reaction (PCR) method. By this method, E26 from the plasma of infected chickens was first allowed to reverse-transcribe viral RNA to cDNA in vitro, and then the cDNA concentration was determined from the lowest dilution that gave a positive signal after amplification of E26 cDNA by the PCR method. Virus titers of about 10(5) per ml were found for wild type and for myb and ets deletion mutants of E26. It is concluded that the ets region is not essential for carcinogenesis, and E26 derives transforming function from overexpression of its proto-myb coding region via the retroviral promoter. Thus, E26 is a single-hit carcinogen and, like all other oncogenic retroviruses, is not a model for two-gene-carcinogenesis. Viral ets probably reflects a genetic accident that transduced sequences of proto-ets together with proto-myb in generating E26.
Mutations in the pre-core region of hepatitis B virus serve to enhance the stability of the secondary structure of the pre-genome encapsidation signal.
Authors: Lok A S , Akarca U , Greene S
Published: May 2006

We conducted a large-scale survey to determine the frequency and clinical significance of mutations in the pre-core region of hepatitis B virus (HBV). Sera from 263 patients with chronic HBV infection were analyzed by direct sequencing of PCR-amplified HBV DNA. Four major missense/nonsense mutations (M) were found: (M1) C-->T at nucleotide position 1856, Pro-->Ser at codon 15; (M2) G-->A at position 1896, Trp-->stop at codon 28; (M3) G-->A at position 1898, Gly-->Ser at codon 29; and (M4) G-->A at position 1899, Gly-->Asp at codon 29. The commonest conserved mutation was M0: T-->C at position 1858, Pro-->Pro at codon 15. We found that M1 and M2 were mutually exclusive, M3 was only found in association with M1, and M4 was predominantly found in association with M2. All patients with M1 but none of those with M2 had M0. The invariable coexistence of certain mutations in codon 15 and codons 28 and 29 and the mutual exclusion of other mutations in these two noncontiguous regions is related to the stem-loop structure of the pre-genome encapsidation sequence located in the precore/core region. M2 and M4 enhance the stability of the stem by providing two additional paired sites. M1 destroys an existing base pair. However, M1 only occurred in the presence of M0, which provides an extra paired site, and 50% of patients with M1 had M3, a compensatory mutation that restores base pairing at this site. Our data support the proposed secondary structure of the pre-genome encapsidation sequence. The primary function of the mutations in the pre-core region is to enhance stability of this secondary structure to ensure perpetuation of viral replication.
Cleavage of supercoiled plasmid DNA by autoantibody Fab fragment: application of the flow linear dichroism technique.
Authors: Gololobov G V , Chernova E A , Schourov D V , Smirnov I V , Kudelina I A , Gabibov A G
Published: May 2006

A highly effective method consisting of two affinity chromatography steps and ion-exchange and gel-filtration chromatography steps was developed for purification of autoantibodies from human sera with DNA-hydrolyzing activity. Antibody Fab fragment, which had been purified 130-fold, was shown to catalyze plasmid DNA cleavage. The flow linear dichroism technique was used for quantitative and qualitative studying of supercoiled plasmid DNA cleavage by these autoantibodies in comparison with DNase I and EcoRI restriction endonuclease. The DNA autoantibody Fab fragment was shown to hydrolyze plasmid DNA by Mg(2+)-dependent single-strand multiple nicking of the substrate. Kinetic properties of the DNA autoantibody Fab fragment were evaluated from the flow linear dichroism and agarose gel electrophoresis data and revealed a high affinity (Kobsm = 43 nM) and considerable catalytic efficiency (kappcat/Kobsm = 0.32 min-1.nM-1) of the reaction.
The chemistry of sexual selection.
Authors: Eisner T , Meinwald J
Published: May 2006

The moth Utetheisa ornatrix (Lepidoptera: Arctiidae) is protected against predation by pyrrolizidine alkaloids that it sequesters as a larva from its foodplants. At mating, the male transfers alkaloid to the female with the spermatophore, a gift that the female supplements with alkaloid of her own and transmits to the eggs. Eggs are protected as a result. The male produces a pheromone, hydroxydanaidal, that he derives from the alkaloid and emits from a pair of extrusible brushes (coremata) during precopulatory interaction with the female. Males rendered experimentally alkaloid-free fail to produce the pheromone and are less successful in courtship. The male produces the pheromone in proportion both to his alkaloid load and to the amount of alkaloid he transfers to the female. The pheromone could thus serve as an indication of male "worth" and provide a basis for female choice. Utetheisa females are promiscuous and therefore are able to accrue multiple nuptial gifts (alkaloid and nutrient, both transmitted with the spermatophore). They use sperm selectively, favoring those of larger males. Larger males in nature are also richer in alkaloid. Females therefore reinforce after copulation the choice mechanism they already exercise during courtship.
Jasmonic acid distribution and action in plants: regulation during development and response to biotic and abiotic stress.
Authors: Creelman R A , Mullet J E
Published: May 2006

Jasmonic acid (JA) is a naturally occurring growth regulator found in higher plants. Several physiological roles have been described for this compound (or a related compound, methyl jasmonate) during plant development and in response to biotic and abiotic stress. To accurately determine JA levels in plant tissue, we have synthesized JA containing 13C for use as an internal standard with an isotopic composition of [225]:[224] 0.98:0.02 compared with [225]:[224] 0.15:0.85 for natural material. GC analysis (flame ionization detection and MS) indicate that the internal standard is composed of 92% 2-(+/-)-[13C]JA and 8% 2-(+/-)-7-iso-[13C]JA. In soybean plants, JA levels were highest in young leaves, flowers, and fruit (highest in the pericarp). In soybean seeds and seedlings, JA levels were highest in the youngest organs including the hypocotyl hook, plumule, and 12-h axis. In soybean leaves that had been dehydrated to cause a 15% decrease in fresh weight, JA levels increased approximately 5-fold within 2 h and declined to approximately control levels by 4 h. In contrast, a lag time of 1-2 h occurred before abscisic acid accumulation reached a maximum. These results will be discussed in the context of multiple pathways for JA biosynthesis and the role of JA in plant development and responses to environmental signals.
Contrasting complexity of two rust resistance loci in flax.
Authors: Ellis J G , Lawrence G J , Finnegan E J , Anderson P A
Published: May 2006

DNA probes from the L6 rust resistance gene of flax (Linum usitatissimum) hybridize to resistance genes at the unlinked M locus, indicating sequence similarities between genes at the two loci. Genetic and molecular data indicate that the L locus is simple and contains a single gene with 13 alleles and that the M locus is complex and contains a tandem array of genes of similar sequence. Thus the evolution of these two related loci has been different. The consequence of the contrasting structures of the L and M loci on the evolution of different rust resistance specificities can now be investigated at the molecular level