bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial genotype of a unisexual salamander of hybrid origin is unrelated to either of its nuclear haplotypes.
Authors: Kraus F , Miyamoto M M
Published: June 2006

We examined mitochondrial DNA (mtDNA), enzyme, and morphological variation among 17 unisexual Ambystoma of hybrid origin. Electrophoretic comparison of diagnostic enzymes indicates that these unisexuals are triploid with two nuclear genomes from the bisexual species Ambystoma laterale and one from Ambystoma jeffersonianum; however, according to restriction analysis, the mtDNAs of these specimens derive from a third species, Ambystoma texanum. This unusual situation is apparently due to a partially independent segregation event in an ancestor of these unisexuals. This situation highlights the potential importance of molecules with different inheritance patterns in elucidating complex cases of reticulate evolution.
Characterization of amplification core and esterase B1 gene responsible for insecticide resistance in Culex.
Authors: Mouches C , Pauplin Y , Agarwal M , Lemieux L , Herzog M , Abadon M , Beyssat-Arnaouty V , Hyrien O , de Saint Vincent B R , Georghiou G P
Published: June 2006

Organophosphorus insecticide (OP) resistance in several Culex species is associated with increased esterase activity resulting from amplification of the corresponding structural gene. In Culex pipiens quinquefasciatus, high levels of OP resistance (approximately 800 times) are due to the esterase B1 gene, which is amplified at least 250-fold. This gene has now been sequenced, and the structure of the amplification unit (amplicon) encompassing the structural gene has been partially characterized. The inferred amino acid sequence of the enzyme revealed regions of strong homology with other eukaryotic serine-esterases, such as cholinesterases, which are the target of OPs. The amplicon covers at least 30 kilobases and contains a constant and highly conserved "core" of 25 kilobases. This core carries a single copy of the esterase gene (2.8 kilobases) as well as other sequences that are present as single or low number copies in the genomes of mosquitoes lacking overproduction of the esterase B1 protein. In the amplicon, the esterase gene is framed by two DNA sequences that are repeated in other parts of the genome of resistant mosquitoes and found in the genome of susceptible mosquitoes but not near the esterase B1 gene. It is suggested that these repetitive sequences may have a role in the amplification process.
Site-directed mutagenesis of a plant viral satellite RNA changes its phenotype from ameliorative to necrogenic.
Authors: Sleat D E , Palukaitis P
Published: June 2006

Comparison of the nucleotide sequences of cucumber mosaic virus (CMV) satellite RNAs, which induce necrosis on tomatoes, reveals a highly conserved region within their 3' halves. The sequence of WL1 satellite (WL1-sat) RNA, which attenuates CMV symptoms on tomatoes, differs from all necrogenic satellite RNAs at three nucleotide positions within this conserved region. These nucleotides were progressively mutated to determine what sequence is required for the induction of necrosis in tomatoes. Infectious transcripts from a cDNA clone of WL1-sat RNA, and its mutated derivatives, were assayed on tomato. Three of the four mutant satellite RNAs, in which two of the three nucleotides correspond to those present in necrogenic satellite RNAs, and the rest of the molecule corresponded to WL1-sat RNA, attenuated CMV symptoms on tomatoes, and were phenotypically identical to the parental WL1-sat RNA. One other mutant satellite RNA, in which all three mutated nucleotides corresponded to the sequence of necrogenic satellite RNAs and the rest of the molecule corresponded to WL1-sat RNA, induced a lethal necrosis on tomatoes. Necrosis was also observed when the same mutant satellite RNA was associated with a second CMV strain as helper virus. These results indicate that a single nucleotide change at any of the three nucleotides examined distinguishes necrogenic from nonnecrogenic satellite RNAs. This necrosis was similar to that induced by a naturally necrogenic satellite RNA. The various mutations did not modify the effect of WL1-sat RNA on the symptoms induced by CMV on tobacco or squash.
Substrate-, hormone-, and cAMP-regulated cytochrome P450 degradation.
Authors: Eliasson E , Johansson I , Ingelman-Sundberg M
Published: June 2006

The hepatic cytochrome P450 system, with numerous different P450 enzymes, is characterized by its inducibility by a variety of endogenous and exogenous compounds. Specific forms of P450, exhibiting distinct but partially overlapping substrate specificities, are increased in response to a given chemical. Consequently, the rate of elimination of the inducing compound is often enhanced and the system is in this respect adaptive to changes in the environment. Transcriptional activation mechanisms for the endo- or xenobiotically controlled P450 synthesis are well documented. Here we describe a mechanism for posttranslational ligand-dependent stabilization of ethanol-inducible P450IIE1 in hepatocyte cultures. Glucagon or 8-bromoadenosine 3',5'-cyclic monophosphate causes an enhanced rate of P450IIE1 degradation in the hepatocytes as well as phosphorylation on Ser-129, a reaction which denatures the protein under in vitro conditions. Substrates for the enzyme, such as ethanol and imidazole, protect the enzyme from phosphorylation and degradation in hepatocytes but do not influence phosphorylation or degradation of phenobarbital-inducible P450IIB1. Our proposed mechanism, which remains to be shown under in vivo conditions, describes the P450 molecules as receptors for the compounds in question and might provide a way by which endo- and xenobiotics regulate their own rate of metabolism.
Two types of abnormal genes for plasminogen in families with a predisposition for thrombosis.
Authors: Ichinose A , Espling E S , Takamatsu J , Saito H , Shinmyozu K , Maruyama I , Petersen T E , Davie E W
Published: June 2006

The gene coding for plasminogen has been compared with several abnormal genes from Japanese patients by the polymerase chain reaction and DNA sequence analysis. Two types of abnormal genes coding for plasminogen were identified in these patients. In the type I mutation, a guanosine in GCT coding for Ala-601 near the active-site histidine was replaced by an adenosine resulting in ACT coding for threonine. This mutation was also shown by the loss of a cleavage site for Fnu4HI endonuclease, a restriction enzyme that recognizes GCTGC but not ACTGC. In the type II mutation, a guanosine in GTC coding for Val-355 was replaced by a thymidine resulting in TTC coding for phenylalanine. This change was readily shown by digestion with Ava II endonuclease, a restriction enzyme that recognizes GGTCC and not GTTCC. The type I mutation has been found to be identical to a plasminogen variant identified in Japanese patients by amino acid sequence analysis and also detected by isoelectric focusing, whereas the type II mutation is a unique amino acid substitution in the connecting region between the third and fourth kringles in plasminogen. DNA sequence analysis also revealed that the abnormal genes carry several silent nucleotide substitutions located primarily within introns and 5' and 3' flanking regions.
Activated alpha subunit of Go protein induces oocyte maturation.
Authors: Kroll S D , Omri G , Landau E M , Iyengar R
Published: June 2006

The capability of various activated guanine nucleotide binding regulatory protein (G protein) alpha subunits to induce meiotic maturation was studied. Activated Go protein alpha subunit (alpha o*) but not the three inhibitory G protein alpha subunits triggered meiotic maturation in Xenopus oocytes. The effect was concentration dependent with a half-maximal effect in the 100-200 pM range. Injection of alpha o* stimulated protein kinase C activity. Coinjection of the peptide containing residues 19-36 of protein kinase C [PKC-(19-36)], a specific protein kinase C inhibitor, blocked the alpha o*- but not progesterone-induced maturation. Cycloheximide and the injection of antisense oligonucleotides specific to the c-mos transcript blocked alpha o-induced maturation. Immunoprecipitation with a mos protein-specific monoclonal antibody showed that alpha o-injected oocytes had phosphorylated mos protein. When PKC-(19-36) was coinjected with alpha o*, phosphorylated mos protein was not observed. These observations indicate that alpha o*, through protein kinase C and the translation of c-mos, can trigger meiotic division of Xenopus oocytes. Our results raise the possibility that persistently activated G proteins through cellular protooncogenes may regulate cell-cycle resumption.
Molecular cloning and transformation of cyclodiene resistance in Drosophila: an invertebrate gamma-aminobutyric acid subtype A receptor locus.
Authors: Ffrench-Constant R H , Mortlock D P , Shaffer C D , MacIntyre R J , Roush R T
Published: June 2006

Cyclodiene resistance represents 60% of the reported cases of insecticide resistance and is also present in vertebrates. Resistance is due to insensitivity of the cyclodiene/picrotoxinin binding site on the gamma-aminobutyric acid subtype A (GABAA) receptor-chloride ionophore complex. Following isolation of cyclodiene-resistant Drosophila mutants, we report the cloning of the locus conferring resistance via a "chromosomal walk" and rescue of the susceptible phenotype by P-element-mediated germ-line transformation. Amino acid sequence analysis of a cDNA from the locus reveals homology with vertebrate GABAA subunits. To our knowledge, this represents the first cloning of an invertebrate GABA receptor and also allows us to manipulate the resistance status of an insect via germ-line transformation. This gene may be useful as a selectable marker in other insect systems.
Physiological and anatomical evidence for a magnocellular defect in developmental dyslexia.
Authors: Livingstone M S , Rosen G D , Drislane F W , Galaburda A M
Published: June 2006

Several behavioral studies have shown that developmental dyslexics do poorly in tests requiring rapid visual processing. In primates fast, low-contrast visual information is carried by the magnocellular subdivision of the visual pathway, and slow, high-contrast information is carried by the parvocellular division. In this study, we found that dyslexic subjects showed diminished visually evoked potentials to rapid, low-contrast stimuli but normal responses to slow or high-contrast stimuli. The abnormalities in the dyslexic subjects' evoked potentials were consistent with a defect in the magnocellular pathway at the level of visual area 1 or earlier. We then compared the lateral geniculate nuclei from five dyslexic brains to five control brains and found abnormalities in the magnocellular, but not the parvocellular, layers. Studies using auditory and somatosensory tests have shown that dyslexics do poorly in these modalities only when the tests require rapid discriminations. We therefore hypothesize that many cortical systems are similarly divided into a fast and a slow subdivision and that dyslexia specifically affects the fast subdivisions.
C-terminal processing of barley alpha-amylase 1 in malt, aleurone protoplasts, and yeast.
Authors: Søgaard M , Olsen F L , Svensson B
Published: June 2006

C-terminal processing of low pI barley alpha-amylase (AMY1) results in multiple forms in malt, aleurone protoplasts, and transformed yeast. Expression of an AMY1 cDNA in yeast thus leads to four secreted forms with distinct pI values between 4.7 and 5.1 and essentially identical Mr. AMY1-1 and AMY1-2 lacking the C-terminal Arg-Ser are generated by carboxypeptidase in vitro from AMY1-3 and AMY1-4, respectively. In vivo processing is due to the KEX1-encoded yeast carboxypeptidase. AMY1-2 and AMY1-4 are fully active, whereas AMY+-1 and AMY1-3 retain 3-4% activity toward p-nitrophenyl maltoheptaoside and have one fewer SH group, due to reaction with glutathione. AMY1-1-AMY1-4 are indistinguishable from malt AMY1 with respect to Ca(2+)-, substrate-, and beta-cyclodextrin-binding as well as recognition by three monoclonal antibodies and limited proteolysis by proteinase K. Transient AMY1 precursors present in barley aleurone protoplasts were trapped by addition of serine carboxypeptidase inhibitors, indicating that endogenous carboxypeptidase participates in the maturation of AMY1 during germination. Three pairs of precursor/mature AMY1 forms are recognized, presumably corresponding to the three genes encoding AMY1. Malt carboxypeptidase II can convert in vitro the precursors isolated from protoplasts into processed enzyme, and AMY1 from malt accordingly lacks the C-terminal heptapeptide. This report thus demonstrates posttranslational protein modification by carboxypeptidase in higher plants.
Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage.
Authors: Barrio L C , Suchyna T , Bargiello T , Xu L X , Roginski R S , Bennett M V , Nicholson B J
Published: June 2006

Gap junctions are formed by a family of homologous proteins termed connexins. Their channels are dodecamers, and homomeric forms differ in their properties with respect to control by voltage and other gating stimuli. We report here the properties of coupling from expression of connexin complementary RNAs (cRNAs; sense to mRNA, antisense to cDNA) in Xenopus oocyte pairs in which endogenous coupling was blocked by injection of DNA oligonucleotides antisense to the mRNA of Cx38, the principal endogenous connexin. We found that a connexin recently sequenced from rat liver, Cx26, formed functional gap junctions whose conductance exhibited voltage dependence with unusual characteristics suggestive of two gating mechanisms. Junctional conductance (gj) was increased to a small degree by depolarization and decreased by hyperpolarization of either cell in a coupled pair, indicating dependence on the potential between the inside and outside of the cells (Vi-o). These changes were fast compared with the resolution of their measurement (ca. 10 ms). On a slower timescale, large transjunctional potentials (Vj) of either sign caused a more substantial decrease in conductance similar to that previously reported for several other gap junctions. Homotypic junctions formed of another connexin, Cx32, exhibited a similar slow dependence on Vj but no dependence on Vi-o. In contrast, heterotypic junctions between an oocyte expressing Cx26 and one expressing Cx32 were electrically asymmetric; they exhibited a greater fast change in gj, which depended, however, on Vj, such that gj increased with relative positivity on the Cx26 side and decreased with relative negativity on the Cx26 side. There was also a large slow decrease in gj in response to Vj for relative positivity on the Cx26 side but not for Vj of the opposite sign. These data indicate that properties of the hemichannels contributed by the two connexins in the heterotypic case were changed from their properties in homotypic junctions. The fast change in gj may involve a mechanism analogous to that at fast rectifying electrical synapses. Experiments in which oocytes expressing Cx32 were paired with oocytes expressing both Cx26 and Cx32 demonstrated that asymmetric junctions would form between oocytes expressing both connexins, thereby confirming their potential relevance in vivo, where the same coupled cells are known to express both proteins.
N-(fluorenyl-9-methoxycarbonyl) amino acids, a class of antiinflammatory agents with a different mechanism of action.
Authors: Burch R M , Weitzberg M , Blok N , Muhlhauser R , Martin D , Farmer S G , Bator J M , Connor J R , Green M , Ko C
Published: June 2006

Several members of a series of N-(fluorenyl-9-methoxycarbonyl) amino acids were found to possess a broad spectrum of antiinflammatory activity. The compounds were active against oxazolone dermatitis in mice and adjuvant arthritis in rats, models in which activated T lymphocytes are implicated. The compounds also inhibited T-lymphocyte activation in vitro, assessed by using the mixed lymphocyte reaction. The compounds inhibited the reversed passive Arthus reaction in rats and arachidonic acid-induced dermatitis in mice, models in which leukocyte infiltration is responsible for the inflammatory reaction. More complete evaluation was made of one compound, N-(fluorenyl-9-methoxycarbonyl)leucine (NPC 15199). On histologic examination after arachidonic acid administration, NPC 15199 was found to block recruitment of neutrophils into the inflammatory site. The compound was not a general myelotoxin. Prolonged treatment of animals did not alter bone-marrow progenitor number or the numbers of circulating white blood cells. Further, several white cell functions were not inhibited in vitro, including neutrophil respiratory burst and macrophage phagocytosis. NPC 15199 was effective in blocking antigen arthritis in rabbits and was effective in a therapeutic protocol, reversing oxazolone edema. These studies suggest that N-(fluorenyl-9-methoxycarbonyl) amino acids may be valuable therapeutic agents for inflammatory diseases.
Ultradian oscillations in somatostatin and growth hormone-releasing hormone mRNAs in the brains of adult male rats.
Authors: Zeitler P , Tannenbaum G S , Clifton D K , Steiner R A
Published: June 2006

In the adult male rat, growth hormone (GH) secretion is characterized by an ultradian rhythm generated by the rhythmic interplay of the stimulatory effects of GH-releasing hormone (GHRH) and the inhibitory effects of somatostatin (Ss). Although considerable evidence indicates that GHRH and Ss are secreted in reciprocal 3- to 4-hr rhythms, the mechanism underlying the rhythmic secretion of these two neuropeptides is unknown. We tested the hypothesis that the rhythmic and reciprocal oscillations in secretion of Ss and GHRH are associated with parallel changes in synthesis and that this would be reflected by coincident oscillations in levels of the respective mRNAs. In the first experiment, Ss mRNA was significantly greater in the periventricular nucleus of animals sacrificed at the time of a presumed peak in the GH rhythm than in animals sacrificed at the time of a presumed trough; this variation was limited to the anterior third of this nucleus. Conversely, GHRH mRNA content throughout the arcuate nucleus was significantly greater at the time of a GH trough. In the second experiment, groups of animals were sacrificed during two consecutive cycles. In this set of animals, Ss mRNA content was 40% greater (P less than 0.005) during peak GH concentrations, whereas GHRH mRNA content was 42% greater (P less than 0.005) during the GH trough. This difference persisted when the two cycles were analyzed separately. The findings that the cellular mRNA content for Ss and GHRH varies in a reciprocal manner with the presumed secretion of these neuropeptides suggest that, like secretion, the synthesis of Ss and GHRH also varies rhythmically. The occurrence of this rhythm suggests a model for a transcriptional oscillator that may subserve the generation of this and possibly other neuroendocrine rhythms.
A search for protein structural changes accompanying the contractile interaction.
Authors: Johnson W C , Bivin D B , Ue K , Morales M F
Published: June 2006

It appears that small movements (detected hitherto only by fluorescence resonance energy transfer measurements and crosslinking studies) in a region of the myosin S-1 particle may mediate chemomechanical energy transduction in the contractile system. Here we find under conditions of high precision at 10 degrees C and 20 degrees C that ATP binding to S-1 causes small (0.4%) changes in CD signal, delta epsilon 222, as do temperature changes in the regime below 16 degrees C. ATP binding perturbs tryptophan residues that we now think are in the mobile region, and we find here that temperature affects tryptophan fluorescence in much the same way that it affects the CD signal, so we believe that the CD signal reports transduction-related movements in S-1. If S-1 is exposed to the range 16-30 degrees C, CD signal falls with temperature; ATP counteracts this fall. Analysis of vacuum-UV CD spectra yields 42% alpha-helix, 9% antiparallel beta-sheet, 7% parallel beta-sheet, 14% beta-turns, and 29% other structures.
Tat transactivation of the human immunodeficiency virus type 1 promoter is influenced by basal promoter activity and the simian virus 40 origin of DNA replication.
Authors: Kessler M , Mathews M B
Published: June 2006

We examined the activation of transcription from the human immunodeficiency virus type 1 (HIV-1) promoter by the viral Tat protein in a transient expression system. Plasmids contained a HIV-reporter gene cassette and a simian virus 40 origin of DNA replication. Run-on assays of transcription complex distribution and analysis of cytoplasmic RNA accumulation confirmed that Tat is able to activate transcription by two mechanisms: by increasing the rate of transcriptional initiation and the efficiency of transcriptional elongation. The degree to which Tat stimulated initiation is determined by the basal level of HIV-directed transcription, which is influenced by the presence [corrected] of the simian virus 40 replication origin. Tat functions primarily to increase the efficiency of elongation when the origin is present and the basal level of transcription is high [corrected]. On the other hand, Tat functions primarily to increase the rate of initiation when the origin is absent [corrected] and the basal level of transcription is 10-fold lower. These studies suggest that the site of integration of the virus into the cellular genome may significantly affect the level of expression from the HIV promoter and consequently the pathobiology of the virus.
Lysosomal hydrolases of different classes are abnormally distributed in brains of patients with Alzheimer disease.
Authors: Cataldo A M , Paskevich P A , Kominami E , Nixon R A
Published: June 2006

beta-Amyloid formation requires multiple abnormal proteolytic cleavages of amyloid precursor protein (APP), including one within its intramembrane domain. Lysosomes, which contain a wide variety of proteases (cathepsins) and other acid hydrolases, are major sites for the turnover of membrane proteins and other cell constituents. Using immunocytochemistry, immunoelectron microscopy, and enzyme histochemistry, we studied the expression and cellular distributions of 10 lysosomal hydrolases, including 4 cathepsins, in neocortex from patients with Alzheimer disease and control (non-Alzheimer-disease) individuals. In control brains, acid hydrolases were localized exclusively to intracellular lysosome-related compartments, and 8 of the 10 enzymes predominated in neurons. In Alzheimer disease brains, strongly immunoreactive lysosomes and lipofuscin granules accumulated markedly in the perikarya and proximal dendrites of many cortical neurons, some of which were undergoing degeneration. More strikingly, these same hydrolases were present in equally high or higher levels in senile plaques in Alzheimer disease, but they were not found extracellularly in control brains, including those from Parkinson or Huntington disease patients. At the ultrastructural level, hydrolase immunoreactivity in senile plaques was localized to extracellular lipofuscin granules similar in morphology to those within degenerating neurons. Two cathepsins that were undetectable in neurons were absent from senile plaques. These results show that lysosome function is altered in cortical neurons in Alzheimer disease. The presence of a broad spectrum of acid hydrolases in senile plaques indicates that lysosomes and their contents may be liberated from cells, principally neurons and their processes, as they degenerate. Because cathepsins can cleave polypeptide sites on APP relevant for beta-amyloid formation, their abnormal extracellular localization and dysregulation in Alzheimer disease can account for the multiple hydrolytic events in beta-amyloid formation. The actions of membrane-degrading acid hydrolases could also explain how the intramembrane portion of APP containing the C terminus of beta-amyloid becomes accessible to proteases.
Prohormone processing in Xenopus oocytes: characterization of cleavage signals and cleavage enzymes.
Authors: Korner J , Chun J , O'Bryan L , Axel R
Published: June 2006

In this study, we characterize the sequences required for the cleavage of prohormones in Xenopus oocytes. We demonstrate that the yeast alpha-factor and the Aplysia egg-laying hormone (ELH) precursors are not cleaved in oocytes following simple pairs of basic residues, such as Lys-Arg, but that the ELH precursor is cleaved following the consensus sequence Arg-Xaa-(Lys/Arg)-Arg. This motif is conserved among precursors that are cleaved in virtually all mammalian cell types. Mutations that generate this sequence in the alpha-factor prohormone also result in efficient processing within oocytes. Cleavage at this consensus sequence may be due to the action of the Xenopus homologues of mammalian furin.
Application of crystallographic and modeling methods in the design of purine nucleoside phosphorylase inhibitors.
Authors: Ealick S E , Babu Y S , Bugg C E , Erion M D , Guida W C , Montgomery J A , Secrist J A
Published: June 2006

Competitive inhibitors of the salvage pathway enzyme purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) have been designed by using the three-dimensional structure of the enzyme as determined by x-ray crystallography. The process was an iterative one that utilized interactive computer graphics, Monte Carlo-based conformational searching, energy minimization, and x-ray crystallography. The proposed compounds were synthesized and tested by an in vitro assay. Among the compounds designed and synthesized are the most potent competitive inhibitors of purine nucleoside phosphorylase thus far reported.
In vitro neurons in mammalian cortical layer 4 exhibit intrinsic oscillatory activity in the 10- to 50-Hz frequency range.
Authors: Llinás R R , Grace A A , Yarom Y
Published: June 2006

We report here the presence of fast subthreshold oscillatory potentials recorded in vitro from neurons within layer 4 of the guinea pig frontal cortex. Two types of oscillatory neurons were recorded: (i) One type exhibited subthreshold oscillations whose frequency increased with membrane depolarization and encompassed a range of 10-45 Hz. Action potentials in this type of neuron demonstrated clear after-hyperpolarizations. (ii) The second type of neuron was characterized by narrow-frequency oscillations near 35-50 Hz. These oscillations often outlasted the initiating depolarizing stimulus. No calcium component could be identified in their action potential. In both types of cell the subthreshold oscillations were tetrodotoxin-sensitive, indicating that the depolarizing phase of the oscillation was generated by a voltage-dependent sodium conductance. The initial depolarizing phase was followed by a potassium conductance responsible for the falling phase of the oscillatory wave. In both types of cell, the subthreshold oscillation could trigger spikes at the oscillatory frequency, if the membrane was sufficiently depolarized. Combining intracellular recordings with Lucifer yellow staining showed that the narrow-frequency oscillatory activity was produced by a sparsely spinous interneuron located in layer 4 of the cortex. This neuron has extensive local axonal collaterals that ramify in layers 3 and 4 such that they may contribute to the columnar synchronization of activity in the 40- to 50-Hz range. Cortical activity in this frequency range has been proposed as the basis for the "conjunctive properties" of central nervous system networks.
Pollen-mediated introgression and hybrid speciation in Louisiana irises.
Authors: Arnold M L , Buckner C M , Robinson J J
Published: June 2006

Populations of the "Louisiana iris" species Iris fulva, I. hexagona, and I. nelsonii were examined genetically to test for interspecific gene flow between I. fulva and I. hexagona, for pollen- versus seed-mediated introgression between these species, and for the presumed hybrid origin of I. nelsonii. Genetic markers were identified by using both a polymerase chain reaction-like method that allows the identification of random, nuclear markers and standard polymerase chain reaction experiments involving specific chloroplast DNA (cpDNA) oligonucleotides. Restriction endonuclease digestions of the cpDNA amplification products resolved diagnostic restriction site differences for I. fulva and I. hexagona. The distribution of the species-specific nuclear markers supports a hypothesis of bidirectional introgression between I. fulva and I. hexagona. Thus, individuals analyzed from a contemporary hybrid population demonstrate multilocus genotypes that are indicative of advanced-generation hybrid individuals. Furthermore, several markers from the alternate species were present in low frequency in one allopatric population each of I. fulva and I. hexagona. Data from the nuclear analysis also support the hypothesized hybrid origin of I. nelsonii from the interaction of I. fulva and I. hexagona. Finally, cpDNA data support the hypothesis that the localized and the dispersed introgression are largely due to pollen transfer. In addition to the biological implications, this study demonstrates the power of the polymerase chain reaction methodology for the rapid identification of random and specific genetic markers for testing evolutionary genetic hypotheses.
An enhancer variant of Moloney murine leukemia virus defective in leukemogenesis does not generate detectable mink cell focus-inducing virus in vivo.
Authors: Brightman B K , Rein A , Trepp D J , Fan H
Published: June 2006

Moloney murine leukemia virus (Mo-MuLV) induces T-cell lymphoma when inoculated into neonatal mice. This is a multistep process. Early events observed in infected mice include generalized hematopoietic hyperplasia in the spleen and appearance of mink cell focus-inducing (MCF) recombinants; end-stage tumors are characterized by insertional proviral activation of protooncogenes. We previously showed that an Mo-MuLV enhancer variant, Mo+PyF101 Mo-MuLV, has greatly reduced leukemogenicity and is deficient in induction of preleukemic hyperplasia. In this report, we have examined Mo+PyF101 Mo-MuLV-inoculated mice for the presence of MCF recombinants. In contrast to wild-type Mo-MuLV-inoculated mice, Mo+PyF101 Mo-MuLV-inoculated mice did not generate detectable MCF recombinants. This failure was at least partly due to an inability of the MCF virus to propagate in vivo, since a molecularly cloned infectious Mo+PyF101 MCF virus did not replicate, even when inoculated as a Mo+PyF101 Mo-MuLV pseudotype. These results show that the leukemogenic defect of Mo+PyF101 Mo-MuLV is associated with its inability to generate MCF recombinants capable of replication in vivo. This, in turn, is consistent with the view that MCF recombinants play a significant role in Mo-MuLV-induced disease and, in particular, may play a role early in the disease process.
Protein turnover as a component in the light/dark regulation of phosphoenolpyruvate carboxylase protein-serine kinase activity in C4 plants.
Authors: Jiao J , Echevarría C , Vidal J , Chollet R
Published: June 2006

Maize leaf phosphoenolpyruvate carboxylase [PEPC; orthophosphate:oxaloacetate carboxy-lyase (phosphorylating), EC 4.1.1.31] protein-serine kinase (PEPC-PK) phosphorylates serine-15 of its target enzyme, thus leading to an increase in catalytic activity and a concomitant decrease in malate sensitivity of this cytoplasmic C4 photosynthesis enzyme in the light. We have recently demonstrated that the PEPC-PK activity in maize leaves is slowly, but strikingly, increased in the light and decreased in darkness. In this report, we provide evidence that cycloheximide, an inhibitor of cytoplasmic protein synthesis, when fed to detached leaves of C4 monocots (maize, sorghum) and dicots (Portulaca oleracea) in the dark or light, completely prevents the in vivo light activation of PEPC-PK activity regardless of whether the protein kinase activity is assessed in vivo or in vitro. In contrast, chloramphenicol, an inhibitor of protein synthesis in chloroplasts, has no effect on the light activation of maize PEPC-PK. Similarly, treatment with cycloheximide did not influence the light activation of other photosynthesis-related enzymes in maize, including cytoplasmic sucrose-phosphate synthase and chloroplast stromal NADPH-malate dehydrogenase and pyruvate, Pi dikinase. These and related results, in which detached maize leaves were treated simultaneously with cycloheximide and microcystin-LR, a potent in vivo and in vitro inhibitor of the PEPC type 2A protein phosphatase, indicate that short-term protein turnover of the PEPC-PK itself or some other essential component(s) (e.g., a putative protein that modifies this kinase activity) is one of the primary levels in the complex and unique regulatory cascade effecting the reversible light activation/seryl phosphorylation of PEPC in the mesophyll cytoplasm of C4 plants.
Expression of the terminal protein region of hepatitis B virus inhibits cellular responses to interferons alpha and gamma and double-stranded RNA.
Authors: Foster G R , Ackrill A M , Goldin R D , Kerr I M , Thomas H C , Stark G R
Published: June 2006

Constructs expressing the core, surface, X, or polymerase proteins of hepatitis B virus were transfected into human cells. In transient assays, only the polymerase inhibited the responses to interferons alpha and gamma (IFN-alpha and -gamma). Stable expression of the polymerase was achieved in the cell line 2fTGH, which carries an IFN-inducible marker gene, by growth under conditions that select for inhibition of the response to IFN-alpha, but the clones grew poorly. When expressed alone, the terminal protein domain of the polymerase gene inhibited the response to IFN-alpha and the reverse transcriptase plus RNase H domains appeared to be toxic. Clones of cells expressing terminal protein alone, selected for the loss of response to IFN-alpha, grew normally and had no detectable response to IFN-alpha, IFN-gamma, or double-stranded RNA. Binding of IFN-alpha to these cells was not impaired but did not lead to activation of the E alpha subunit of the IFN-induced transcription factor E. These observations are of potential importance in relation to the pathogenesis of chronic hepatitis B virus infection and the resistance of such infection to IFN-alpha therapy.
Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin.
Authors: Pai L H , Batra J K , FitzGerald D J , Willingham M C , Pastan I
Published: June 2006

B3 is a monoclonal antibody that reacts with a carbohydrate epitope present on a variety of proteins located on the surface of many cancer cells and a limited number of normal tissues. We evaluated the cytotoxic activity of immunotoxins composed of monoclonal antibody B3 coupled to native Pseudomonas exotoxin (PE) or two recombinant forms of Pseudomonas exotoxin, PEArg57 or LysPE40, a form of PE with a deletion of the cell binding domain. All three conjugates were cytotoxic to human cell lines expressing the B3 antigen on their surface. The survival of each of the three immunotoxins in the circulation of mice was determined after administering the immunotoxin i.v. The half-life in blood of B3-PE and B3-PEArg57 was 20 hr, whereas the half-life of B3-LysPE40 was 4 hr. The short half-life of B3-LysPE40 may be due to the absence of domain I of PE. To determine the therapeutic effects of the three immunotoxins, they were given intraperitoneally to nude mice bearing subcutaneous A431 tumors. All three immunotoxins caused complete regression of 50-mm3 tumors with no toxic effects to the animals at therapeutic doses. Furthermore, substantial regression was also noted with much larger tumors. Our data indicate that the monoclonal antibody B3, when coupled to PE or recombinant forms of PE, may be useful for the treatment of tumors expressing B3 antigen. The therapeutic window was largest with B3-LysPE40, which can be administered in higher doses because it lacks sequences in domain I of PE that enable PE to bind to nontarget cells.
Characterization of immortal cystic fibrosis tracheobronchial gland epithelial cells.
Authors: Cozens A L , Yezzi M J , Chin L , Simon E M , Finkbeiner W E , Wagner J A , Gruenert D C
Published: June 2006

Tracheobronchial glands were isolated and cultured from a patient with cystic fibrosis (CF). Cultured epithelial cells were transformed with pSVori-. All transformed cell lines express cytokeratin filaments and at early passages express the junctional complex molecule cell CAM 120/80, indicating their epithelial origin. Several gland cell lines express antigens that localize to secretory cells in vivo. Cl- transport measured by 36Cl efflux shows that CF gland epithelial cells, like CF surface airway and nasal polyp epithelial cells, are unable to respond to increases in intracellular cAMP. However, they do produce an increase in intracellular cAMP after treatment with isoproterenol or forskolin. One CF gland cell line shows increased intracellular calcium in response to a number of agents and increased Cl- efflux comparable to that observed in a non-CF airway surface epithelial cell line after addition of calcium ionophore. All cell lines express CF transmembrane conductance regulator mRNA, as measured by PCR amplification of first-strand cDNA. The CF tracheobronchial gland cell lines described here are compound heterozygotes, having a single copy of the delta F508 mutation.
Reversible alterations in myocardial gene expression in a young man with dilated cardiomyopathy and hypothyroidism.
Authors: Ladenson P W , Sherman S I , Baughman K L , Ray P E , Feldman A M
Published: June 2006

Thyroid hormone effects on myocardial gene expression have been well defined in animal models, but their relationship to the pathogenesis of cardiac dysfunction in hypothyroid humans has been uncertain. We evaluated a profoundly hypothyroid young man with dilated cardiomyopathy. Before and during 9 months of thyroxine therapy, serial assessment of myocardial performance documented substantial improvements in the left ventricular ejection fraction (16-37%), left ventricular end-diastolic diameter (7.8-5.9 cm), and cardiac index (1.4-2.7 liters.min-1.m-2). Steady-state levels of mRNAs encoding selected cardiac proteins were measured in biopsy samples obtained before and after thyroxine replacement. In comparison with myocardium from nonfailing control hearts, this patient's pretreatment alpha-myosin heavy-chain mRNA level was substantially lower, the atrial natriuretic factor mRNA level was markedly elevated, and the phospholamban mRNA level was decreased. All of these derangements were reversed 9 months after restoration of euthyroidism. These observations in an unusual patient with profound myxedema and cardiac dilatation permit correlation between the reversible changes in myocardial function and steady-state mRNA levels in a cardiomyopathy. They suggest that alterations in gene expression in the dilated myopathic heart may be correctable when a treatable cause is identified.
Genetic organization of a repeated DNA sequence family in the rice blast fungus.
Authors: Romao J , Hamer J E
Published: June 2006

The fungal rice pathogen Magnaporthe grisea contains repetitive DNA sequences called MGR. We have used a DNA probe, MGR586, derived from these sequences and crosses between rice-pathogenic and non-rice-pathogenic laboratory strains of M. grisea to rapidly map genes in this organism. The rice-pathogenic strain contained 57 EcoRI restriction fragments that hybridize to the MGR586 probe; the other five non-rice-pathogenic parent strains contained a single MGR586 sequence. Genetic analysis of MGR segregation detected eight linkage groups and allowed the mapping of three pigmentation genes (Alb1, Rsy1, and Buf1), the mating type locus (Mat1), the nucleolar organizer (Rdn1), the Smol gene, and two restriction fragment length polymorphisms linked to Smol. Our results indicate that the MGR586 loci are randomly distributed about the M. grisea genome and permit the construction of a well-marked linkage map useful for future studies on genome organization and genetic analysis in M. grisea.
Atomic structures of wild-type and thermostable mutant recombinant human Cu,Zn superoxide dismutase.
Authors: Parge H E , Hallewell R A , Tainer J A
Published: June 2006

Superoxide dismutase enzymes protect aerobic organisms from oxygen-mediated free-radical damage. Crystallographic structures of recombinant human Cu,Zn superoxide dismutase have been determined, refined, and analyzed at 2.5 A resolution for wild-type and a designed thermostable double-mutant enzyme (Cys-6----Ala, Cys-111----Ser). The 10 subunits (five dimers) in the crystallographic asymmetric unit form an unusual stable open lattice with 80-A-diameter channels. The 10 independently fit and refined subunits provide high accuracy, error analysis, and insights on loop conformations. There is a helix dipole interaction with the Zn site, and 14 residues form two or more structurally conserved side-chain to main-chain hydrogen bonds that appear critical to active-site architecture, loop conformation, and the increased stability resulting from the Cys-111----Ser mutation.
Primary structure, expression, and signal-dependent tyrosine phosphorylation of a Drosophila homolog of extracellular signal-regulated kinase.
Authors: Biggs W H , Zipursky S L
Published: June 2006

The extracellular signal-regulated kinases (ERKs) comprise a class of protein-serine/threonine kinases that are activated in response to a wide variety of extracellular signals transduced via receptor tyrosine kinases. Activation of the ERKs requires both threonine and tyrosine phosphorylation suggestive of a key role in mediating intracellular events in response to extracellular cues. To critically assess the role of ERKs in intracellular signaling, a genetically tractable receptor tyrosine kinase system would be invaluable. In this paper we report the identification of a Drosophila homolog of ERK1 and -2, designated DmERK-A. DmERK-A is 80% identical to rat ERK1 and -2 and is rapidly phosphorylated on tyrosine in response to an extracellular signal activating a receptor tyrosine kinase. Biochemical and histological studies reveal its expression in the eye imaginal disc. These studies provide a first step in a genetic analysis of ERK function.
Note on the form of the metric for an isolated vortex in general relativity.
Authors: Pekeris C L , Frankowski K
Published: June 2006

Calling a metric semidiagonal if it has a single off-diagonal element g t, we show that the stationary interior solution for a cylindrically symmetrical perfect fluid possessing an angular momentum cannot have a semidiagonal metric, unless the motion of the fluid particles is purely rotational around the axis of symmetry. A discussion is given of the relativistic spherical vortex in flat space, with a view of seeking a solution for such an isolated vortex in which gravitation is not neglected.
Delineation of putative mechanisms involved in antibody-mediated clearance of rabies virus from the central nervous system.
Authors: Dietzschold B , Kao M , Zheng Y M , Chen Z Y , Maul G , Fu Z F , Rupprecht C E , Koprowski H
Published: June 2006

The in vitro biological activities of several rabies virus-neutralizing monoclonal antibodies (mAbs) were compared with their ability to prevent a lethal rabies virus encephalomyelitis. The protective activity of a particular mAb in vivo did not correlate with its virus-neutralizing activity in vitro; rather it was related to the mAb's ability to inhibit virus spread from cell to cell and to restrict rabies virus RNA transcription. Since treatment of rabies virus-infected cells with virus-neutralizing mAbs results in an endocytosis of the antibody, we hypothesize that an antibody may exert its inhibitory activity even after uptake by the cell. Post-exposure treatment of rats with a mAb that inhibited both virus spread and virus RNA transcription in vitro resulted in viral clearance from the central nervous system and protected the animals against a lethal rabies virus infection.
Classification and evolution of alpha-amylase genes in plants.
Authors: Huang N , Stebbins G L , Rodriguez R L
Published: June 2006

The DNA sequences for 17 plant genes for alpha-amylase (EC 3.2.1.1) were analyzed to determine their phylogenetic relationship. A phylogeny for these genes was obtained using two separate approaches, one based on molecular clock assumptions and the other based on a comparison of sequence polymorphisms (i.e., small and localized insertions) in the alpha-amylase genes. These polymorphisms are called "alpha-amylase signatures" because they are diagnostic of the gene subfamily to which a particular alpha-amylase gene belongs. Results indicate that the cereal alpha-amylase genes fall into two major classes: AmyA and AmyB. The AmyA class is subdivided into the Amy1 and Amy2 subfamilies previously used to classify alpha-amylase genes in barley and wheat. The AmyB class includes the Amy3 subfamily to which most of the alpha-amylase genes of rice belong. Using polymerase chain reaction and oligonucleotide primers that flank one of the two signature regions, we show that the AmyA and AmyB gene classes are present in approximately equal amounts in all grass species examined except barley. The AmyB (Amy3 subfamily) genes in the latter case are comparatively underrepresented. Additional evidence suggests that the AmyA genes appeared recently and may be confined to the grass family.
Torsional stress generated by RecA protein during DNA strand exchange separates strands of a heterologous insert.
Authors: Jwang B , Radding C M
Published: June 2006

Previous studies have shown that the helical RecA nucleoprotein filament formed on a circular single strand of DNA causes the progressive, directional transfer of a complementary strand from naked linear duplex DNA to the nucleoprotein filament, even when the duplex contains a sizable heterologous insertion. Since RecA protein lacks demonstrable helicase activity, the mechanism by which it pushes strand exchange through long heterologous inserts has been a quandary. In the present study, a linear duplex substrate with an insertion of 110 base pairs in its middle yielded the expected products, whereas much less of the heteroduplex product was seen when the insertion was located at either end of the duplex substrate or 160 base pairs from the far end of the duplex substrate. In an ongoing reaction of the substrate with an insertion in its middle, P1 nuclease cleaved intermediates from the point of the insertion to various distal sites. Acting on a duplex substrate that contained a single nick located in the complementary strand just beyond the insertion, RecA protein formed joint molecules but failed to complete strand exchange. These data show that negative torsional stress is generated by distant homologous interactions that occur beyond the heterologous insertion and that such stress is essential for unwinding a heterologous insertion that otherwise halts strand exchange.