bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of the operator of the lux regulon from the Vibrio fischeri strain ATCC7744.
Authors: Devine J H , Shadel G S , Baldwin T O
Published: May 2006

Escherichia coli that carry a recombinant plasmid bearing the Vibrio fischeri lux regulon express luminescence that mimics the luminescence of V. fischeri. The lux regulon consists of two divergently transcribed operons, the rightward operon (luxICDABE genes) and the leftward operon (luxR gene). The luxR and luxI genes and the control region separating the two operons supply the primary regulatory control over the lux regulon; the regulatory mechanisms result in a dramatic increase in the rate of luciferase synthesis after induction, apparently due to a unique autoregulatory positive feedback mechanism, and in an enormous difference (greater than 10(4] in levels of luminescence in cells before and after induction. The generally accepted model of primary regulation of bioluminescence in V. fischeri involves the interaction of the product of the luxR gene and N-(3-oxohexanoyl)homoserine lactone, the autoinducer produced by the enzyme encoded by luxI, the first gene of the rightward operon, with an operator sequence within the control region to stimulate transcription of the rightward operon in a positive feedback loop. We have used deletion mapping of a transcription reporter vector to determine the approximate location of the operator. By site-directed mutagenesis of the presumed operator, we have demonstrated that the 20-base-pair inverted repeat ACCTGTAGGA/TCGTA CAGGT (where the vertical line is the center of symmetry), which bears striking similarity to the recognition sequence for the pleiotropic repressor protein LexA, is the operator of the lux regulon. We also found that deletion of sequences upstream of the palindrome leads to increased transcription from the rightward promoter (PR), indicative of a cis-acting element that represses transcription in the absence of the LuxR-autoinducer complex. Modifications of the palindrome that eliminate stimulation by LuxR-autoinducer of transcription from PR have no effect on repression by the cis-acting mechanism(s), suggesting that the palindrome is not necessary for repression of the rightward operon. Thus, it appears that the large increase in transcription upon induction of the lux regulon is the result of at least two independent mechanisms, one positive and the other negative.
Implications of a quasispecies genome structure: effect of frequent, naturally occurring amino acid substitutions on the antigenicity of foot-and-mouth disease virus.
Authors: Mateu M G , Martínez M A , Rocha E , Andreu D , Parejo J , Giralt E , Sobrino F , Domingo E
Published: May 2006

We provide evidence that the quasispecies nature (extreme genetic heterogeneity) of foot-and-mouth disease virus is relevant to the virus evading an immune response. A monoclonal antibody neutralizing the viral infectivity (clone SD6) recognizes an epitope located around a highly conserved sequence (amino acid sequence Arg-Gly-Asp-Leu-Ala at positions 141-145) in the capsid protein VP1 of foot-and-mouth disease virus of serotype C1. The amino acid substitutions Ala-138----Thr and Leu-147----Ile (or ----Val) reduced 100-fold the binding titer of monoclonal antibody SD6 to virions or to VP1. The effect of those substitutions was quantitatively reproduced with synthetic peptides representing the relevant sequences. This provides evidence that the two chemically conservative amino acids replacements--and not other substitutions present in the virus quasispecies--are responsible for the modified interaction with neutralizing monoclonal antibody SD6. The three substitutions were fixed in the viral capsid during one occurrence of foot-and-mouth disease and, furthermore, they are of a type found frequently among independent foot-and-mouth disease virus isolates. The results implicate the extreme heterogeneity of foot-and-mouth disease virus as an important element of viral pathogenesis.
Homology of aspartyl- and lysyl-tRNA synthetases.
Authors: Gampel A , Tzagoloff A
Published: May 2006

The yeast nuclear gene MSD1 coding for mitochondrial aspartyl-tRNA synthetase has been cloned and sequenced. The identity of the gene is confirmed by the following evidence. (i) The primary structure of the protein derived from the gene sequence is similar to that of the yeast cytoplasmic aspartyl-tRNA synthetase. (ii) In situ disruption of MSD1 in a respiratory-competent haploid strain of yeast induces a pleiotropic phenotype consistent with a lesion in mitochondrial protein synthesis. (iii) Mitochondria from a mutant with a disrupted chromosomal copy of MSD1 are unable to acylate mitochondrial aspartyl-tRNA. The primary structures of the cytoplasmic and mitochondrial aspartyl-tRNA synthetases are similar to the yeast cytoplasmic lysyl-tRNA synthetase, suggesting that the two types of synthetases may have a common evolutionary origin. Searches of the current protein banks also have revealed a high degree of sequence similarity of the lysyl-tRNA synthetase to the product of the Escherichia coli herC gene and to the partial sequence of a protein encoded by an unidentified reading frame located adjacent to the E. coli frdA gene. Based on the sequence similarities and the map positions of the herC and frdA loci, we propose herC to be the structural gene of the constitutively expressed lysyl-tRNA synthetase of E. coli and the unidentified reading frame to be the structural gene of the heat-inducible lysyl-tRNA synthetase.
Cis-acting elements of the sea urchin histone H2A modulator bind transcriptional factors.
Authors: Palla F , Casano C , Albanese I , Anello L , Gianguzza F , Di Bernardo M G , Bonura C , Spinelli G
Published: May 2006

Functional tests, performed by microinjection into Xenopus laevis oocytes, show that a DNA fragment containing the modulator of the early histone H2A gene of Paracentrotus lividus enhances transcription of a reporter gene when located, in the physiological orientation, upstream of the tk basal promoter. Gel retardation and DNase I footprinting assays further reveal that the H2A modulator contains at least two binding sites [upstream sequence elements 1 and 2 (USE 1 and USE 2)] for nuclear factors extracted from sea urchin embryos, which actively transcribe the early histone gene set. Interestingly, USE 1 is highly homologous to a cis-acting element previously identified in the H2A modulator of Psammechinus miliaris [Grosschedl, R., Mächler, M., Rohrer, U. & Birnstiel, M. L. (1983) Nucleic Acids Res. 11, 8123-8136]. Finally, a cloned oligonucleotide containing the USE 1 sequence competes efficiently in Xenopus oocytes with the H2A modulator to prevent enhancement of transcription of the reporter gene. From these results, we conclude that USE 1 and perhaps USE 2 in the H2A modulator are upstream transcriptional elements that are recognized by trans-acting factors common to Xenopus and sea urchin.
Resolution and partial characterization of factors required for in vitro transcription by mammalian RNA polymerase II.
Authors: Kitajima S , Kawaguchi T , Yasukochi Y , Weissman S M
Published: May 2006

Multiple protein factors from HeLa cells are necessary for the accurate initiation of transcription on minimal promoters in vitro. We have partially purified these factors by chromatographic methods. In addition to RNA polymerase II, six factors A-F (FA, FB, FC, FD, FE, and FF) necessary for initiation at the beta-globin promoter start site in vitro have been identified. Certain of these (FA, FC, and FE) have been purified to near homogeneity. The present purification scheme yields sufficient amounts of purified material for the more detailed characterization and cloning of the genes for these activities. Among these factors, FD and FF were required with template DNA at an early step of formation of the initiation complex, whereas FB, FA together with FC, and FE were effective when added at successively later stages in the process of complex formation.
Oncogene activation in human benign tumors of the skin (keratoacanthomas): is HRAS involved in differentiation as well as proliferation?
Authors: Corominas M , Kamino H , Leon J , Pellicer A
Published: May 2006

In vitro DNA amplification followed by oligonucleotide mismatch hybridization was used to study the frequency of HRAS mutations in the benign self-regressing skin tumors keratoacanthomas and in squamous cell carcinomas. We used freshly obtained keratoacanthomas as well as Formalin-fixed paraffin-embedded tissues from both types of tumors. DNA from 50 samples of each tumor type was analyzed for activating mutations involving codons 12 and 61. A relatively high percentage (30%) of HRAS mutations was found in the keratoacanthomas compared with 13% in the squamous cell carcinomas. The most frequent mutation identified is the A-T-to-T.A transversion in the second position of codon 61. The present findings demonstrate the involvement of the HRAS oncogene in human benign tumors. Moreover, they indicate that an activated HRAS oncogene is not sufficient to maintain a neoplastic phenotype and argue against a role of HRAS in the progression of skin tumorigenesis.
Ascorbate is an outstanding antioxidant in human blood plasma.
Authors: Frei B , England L , Ames B N
Published: May 2006

We have shown recently that the temporal order of antioxidant consumption in human blood plasma exposed to a constant flux of aqueous peroxyl radicals is ascorbate = protein thiols greater than bilirubin greater than urate greater than alpha-tocopherol and that detectable lipid peroxidation starts only after ascorbate has been consumed completely. In this paper, we show that it is indeed ascorbate that completely protects plasma lipids against detectable peroxidative damage induced by aqueous peroxyl radicals and that ascorbate is the only plasma antioxidant that can do so. Plasma devoid of ascorbate, but no other endogenous antioxidant, is extremely vulnerable to oxidant stress and susceptible to peroxidative damage to lipids. The plasma proteins' thiols, although they become oxidized immediately upon exposure to aqueous peroxyl radicals, are inefficient radical scavengers and appear to be consumed mainly by autoxidation. Our data demonstrate that ascorbate is the most effective aqueous-phase antioxidant in human blood plasma and suggest that in humans ascorbate is a physiological antioxidant of major importance for protection against diseases and degenerative processes caused by oxidant stress.
Coordinated regulation of intracellular K+ in the proximal tubule: Ba2+ blockade down-regulates the Na+,K+-ATPase and up-regulates two K+ permeability pathways.
Authors: Kone B C , Brady H R , Gullans S R
Published: May 2006

To avoid large changes in cell K+ content and volume during variations in Na+,K+-ATPase activity, Na+-transporting epithelia must adjust the rate of K+ exit through passive permeability pathways. Recent studies have shown that a variety of passive K+ transport mechanisms may coexist within a cell and may be functionally linked to the activity of the Na+,K+-ATPase. In this study, we have identified three distinct pathways for passive K+ transport that act in concert with the Na+,K+-ATPase to maintain intracellular K+ homeostasis in the proximal tubule. Under control conditions, the total K+ leak of the tubules consisted of discrete Ba2+-sensitive (approximately 65%), quinine-sensitive (approximately 20%), and furosemide-sensitive (approximately 10%) pathways. Following inhibition of the principal K+ leak pathway with Ba2+, the tubules adaptively restored cell K+ content to normal levels. This recovery of cell K+ content was inhibited, in an additive manner, by quinine and furosemide. Following adaptation to Ba2+, the tubules exhibited a 30% reduction in Na+-K+ pump rate coupled with an increase in K+ leak by means of the quinine-sensitive (approximately 70%) and furosemide-sensitive (approximately 280%) pathways. Thus, the proximal tubule maintains intracellular K+ homeostasis by the coordinated modulation of multiple K+ transport pathways. Furthermore, these results suggest that, like Ba2+, other inhibitors of K+ conductance will cause compensatory changes in both the Na+-K+ pump and alternative pathways for passive K+ transport.
Genetic switching in the flagellar gene hierarchy of Caulobacter requires negative as well as positive regulation of transcription.
Authors: Newton A , Ohta N , Ramakrishnan G , Mullin D , Raymond G
Published: May 2006

Caulobacter crescentus flagellar (fla, flb, or flg) genes are periodically expressed in the cell cycle and they are organized in a regulatory hierarchy. We have analyzed the genetic interactions required for fla gene expression by determining the effect of mutations in 30 known fla genes on transcription from four operons in the hook gene cluster. These results show that the flaO (transcription unit III) and flbF (transcription unit IV) operons are located at or near the top of the hierarchy. They also reveal an extensive network of negative transcriptional controls that are superimposed on the positive regulatory cascade described previously. The strong negative autoregulation observed for the flaN (transcription unit I), flbG (transcription unit II), and flaO (transcription unit III) promoters provides one possible mechanism for turning off fla gene expression at the end of the respective synthetic periods. We suggest that these positive and negative transcriptional interactions are components of genetic switches that determine the sequence in which fla genes are turned on and off in the C. crescentus cell cycle.
Exceptional chromosomal mutations in a rodent population are not strongly underdominant.
Authors: Nachman M W , Myers P
Published: May 2006

The observation of karyotypic uniformity in most species has led to the widespread belief that selection limits chromosomal change. We report an unprecedented amount of chromosomal variation in a natural population of the South American marsh rat Holochilus brasiliensis. This variation consists of four distinct classes of chromosomal rearrangements: whole-arm translocations, pericentric inversions, variation in the amount of euchromatin, and variation in number and kind of supernumerary (B) chromosomes. Twenty-six karyotypes are present among 42 animals. Observations of the natural population over a 7-year period and breeding experiments with captive animals indicate that heterozygous individuals suffer no detectable reduction in fitness. This is at odds with a central assumption in current models of chromosomal speciation and provides a firm rejection of the view that selection necessarily restricts chromosomal change.
Marked increases in hepatic NAD(P)H:oxidoreductase gene transcription and mRNA levels correlated with a mouse chromosome 7 deletion.
Authors: Petersen D D , Gonzalez F J , Rapic V , Kozak C A , Lee J Y , Jones J E , Nebert D W
Published: May 2006

The NAD(P)H:menadione oxidoreductase gene (Nmo-1) codes for a quinone reductase (also called DT diaphorase; EC 1.6.99.2) believed to play a central role in protection against oxidative stress. We have studied mice with a radiation-induced chromosomal deletion involving the albino locus (c) on chromosome 7 and found that Nmo-1 mRNA levels and the rate of Nmo-1 gene transcription are markedly increased (greater than 100-fold and greater than 12-fold, respectively) in the untreated c14CoS/c14CoS deletion homozygote, compared with the untreated Cch/Cch wild-type and the Cch/C14CoS heterozygote. These data suggest that a gene located on chromosome 7 encodes a trans-acting regulatory factor that might be a negative effector of the Nmo-1 gene, which we show here is located on chromosome 8 approximately 1.4 centimorgans (about 1000 kilobase pairs) from the Es-2 gene. Conversely, there are no detectable basal levels of cytochrome P1450 (Cyp1a1 gene) or cytochrome P3450 (Cyp1a2 gene) mRNA, indicating that the regulation of basal expression of the Cyp1a1 and Cyp1a2 genes is distinct from that of the Nmo-1 gene. Moreover, the Cyp1a1 and Cyp1a2 genes and the Nmo-1 gene are induced by tetrachlorodibenzo-p-dioxin in the cch/cch, cch/c14CoS, and c14CoS/c14CoS mice. The mechanism of tetrachlorodibenzo-p-dioxin inducibility of the Cyp1a1, Cyp1a2, and Nmo-1 genes is, therefore, independent of the mechanism of Nmo-1 gene activation in untreated c14CoS/c14CoS mice.
Trans-inactivation of the Drosophila brown gene: evidence for transcriptional repression and somatic pairing dependence.
Authors: Henikoff S , Dreesen T D
Published: May 2006

Position-effect variegation in Drosophila is the variable inactivation of a gene that occurs when it is juxtaposed to heterochromatic regions of chromosomes. The brown gene, required for pteridine pigment in the eye, is unusual in that expression of the unrearranged homolog also is affected. This dominant effect can be very strong, as inactivation is detectable when as many as three trans copies of the gene are present. We show that pteridine reductions coincide with similar reductions in the accumulation of brown mRNA. The dominant effect is suppressed by certain altered structural configurations of the brown region, suggesting that somatic pairing is involved in the phenomenon. We propose that direct transmission of the altered chromatin structure characteristic of position-effect variegation (heterochromatinization) occurs between paired homologs in the region of the brown locus.
A repertoire of monoclonal antibodies with human heavy chains from transgenic mice.
Authors: Brüggemann M , Caskey H M , Teale C , Waldmann H , Williams G T , Surani M A , Neuberger M S
Published: May 2006

The introduction of human immunoglobulin gene segments in their unrearranged configuration into the germ line of mice might allow the production of a repertoire of human antibodies. Such transgenic mice could be used for the production of human monoclonal antibodies against human antigens. To test the feasibility of this approach, mice were created that carry a human heavy-chain minilocus comprising unrearranged immunoglobulin variable, diversity, and joining elements linked to a human mu-chain gene. The gene segments of this minilocus are rearranged in a large proportion of cells in thymus and spleen but not in nonlymphoid tissue. Some 4% of the B lymphocytes synthesize human mu chains resulting in a serum titer of about 50 micrograms of transgenic IgM antibody per ml. Hybridomas were established from the transgenic mice that stably secreted several micrograms of antibodies containing human mu heavy chains per milliliter.
Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: determination by reconstitution and affinity-labeling.
Authors: Baxter R C , Martin J L
Published: May 2006

To determine the structure of the high molecular weight, growth hormone-dependent complex between the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins in human serum, we have reconstituted the complex from its purified component proteins and analyzed it by gel electrophoresis and autoradiography after covalent cross-linking. The proteins tested in reconstitution mixtures were an acid-labile Mr 84,000-86,000 glycoprotein doublet (alpha subunit), an acid-stable Mr 47,000-53,000 glycoprotein doublet with IGF-binding activity (BP-53 or beta subunit), and IGF-I or IGF-II (gamma subunit). In incubations containing any one of the three subunits 125I-labeled and the other two unlabeled, identical 125I-labeled alpha-beta-gamma complexes of Mr 140,000 were formed. Minor bands of Mr 120,000 and 90,000 were also seen, thought to represent a partially deglycosylated form of the alpha-beta-gamma complex, and an alpha-gamma complex arising as a cross-linking artifact. When serum samples from subjects of various growth hormone status were affinity-labeled with IGF-II tracer, a growth hormone-dependent Mr 140,000 band was seen, corresponding to the reconstituted alpha-beta-gamma complex. Other growth hormone-dependent labeled bands, of Mr 90,000 (corresponding to alpha-gamma), Mr 55,000-60,000 (corresponding to labeled beta-subunit doublet), and smaller bands of Mr 38,000, 28,000, and 23,000-25,000 (corresponding to labeled beta-subunit degradation products), were also seen in the affinity-labeled serum samples and in the complex reconstituted from pure proteins. All were immunoprecipitable with an anti-BP-53 antiserum. We conclude that the growth hormone-dependent Mr 140,000 IGF-binding protein complex in human serum has three components: the alpha (acid-labile) subunit, the beta (binding) subunit, and the gamma (growth factor) subunit.
Topography of glycosylation in yeast: characterization of GDPmannose transport and lumenal guanosine diphosphatase activities in Golgi-like vesicles.
Authors: Abeijon C , Orlean P , Robbins P W , Hirschberg C B
Published: May 2006

"Outer-chain" addition of mannose residues to yeast glycoproteins occurs in the Golgi compartment of the cell. Essential steps in this process are thought to include transport of GDPmannose from the cytoplasm into the lumen of Golgi vesicles, transfer of mannose to glycoprotein acceptors, hydrolysis of the resulting GDP to GMP, and return of GMP and inorganic phosphate to the cytoplasm. We report detection and characterization of a GDPmannose transport activity and a GDPase by yeast vesicles. The active transport of GDPmannose as well as the GDPase and another presumed Golgi enzyme, alpha 1,2-mannosyltransferase, are concentrated in a subcellular fraction that can be partially separated, by velocity sucrose gradient centrifugation, from a fraction enriched in an endoplasmic reticulum marker enzyme.
Long-range intramolecular electron transfer in azurins.
Authors: Farver O , Pecht I
Published: May 2006

The Cu(II) sites of azurins, the blue single copper proteins, isolated from Pseudomonas aeruginosa and Alcaligenes spp. (Iwasaki) are reduced by CO2- radicals, produced by pulse radiolysis, in two distinct reaction steps: (i) a fast bimolecular phase, at the rates (5.0 +/- 0.8) x 10(8) M-1.s-1 (P. aeruginosa) and (6.0 +/- 1.0) x 10(8) M-1.s-1 (Alcaligenes); (ii) a slow unimolecular phase with specific rates of 44 +/- 7 s-1 in the former and 8.5 +/- 1.5 s-1 for the latter (all at 298 K, 0.1 M ionic strength). Concomitant with the fast reduction of Cu(II), the single disulfide bridge linking cysteine-3 to -26 in these proteins is reduced to the RSSR- radical ion as evidenced by its characteristic absorption band centered at 410 nm. This radical ion decays in a unimolecular process with a rate identical to that of the slow Cu(II) reduction phase in the respective protein, thus clearly suggesting that a long-range intramolecular electron transfer occurs between the RSSR- radicals and the Cu(II) site. The temperature dependence of the internal electron transfer process in both proteins was measured over the 4 degrees C to 42 degrees C range. The activation parameters derived are delta H* = 47.5 +/- 4.0 and 16.7 +/- 1.5 kJ.mol-1; and delta S not equal to = -56.5 +/- 7.0 and -171 +/- 18 J.K-1.mol-1, respectively. Using the Marcus theory, we found that the intramolecular electron transfer rates and their activation parameters observed for the two azurins correlate well with the distances between the reactive sites, their redox potential, and the nature of the separating medium. Thus, azurins with distinct structural and reactivity characteristics isolated from different bacteria or modified by site-directed mutagenesis can be used in comparing long-range electron transfer process between their conserved disulfide bridge and the Cu(II) sites.
Evidence for two pathways of meiotic intrachromosomal recombination in yeast.
Authors: Gottlieb S , Wagstaff J , Esposito R E
Published: May 2006

This study shows that RAD50, a yeast DNA repair gene required for meiotic interchromosomal exchange between homologs, also is required for meiotic intrachromosomal recombination. However, only intrachromosomal events in nonribosomal DNA are dependent on RAD50; those in ribosomal DNA (rRNA-encoding DNA) occur in the absence of this gene. Furthermore, nonribosomal DNA sequences retain their RAD50-dependence even when inserted into the ribosomal DNA array. We argue that these data provide evidence for at least two pathways of meiotic intrachromosomal recombination whose activity depends on the specific sequences involved or their structural context in the chromosome. In contrast to its role in meiosis, RAD50 is not required for either inter- or intrachromosomal spontaneous mitotic recombination.
Two proteins targeted to the same lytic granule compartment undergo very different posttranslational processing.
Authors: Burkhardt J K , Hester S , Argon Y
Published: May 2006

The granules of natural killer (NK) cells contain cytolysin and serine proteases, proteins that are expressed specifically in cytolytic cells and are released in response to target binding. We have used immunofluorescence and immunoelectron microscopy to localize these proteins with respect to the various morphological compartments of granules in a rat NK cell line. Both cytolysin and the proteases are limited to the core regions of the dense core granules. While these proteins are targeted to the same compartment, they differ markedly in their posttranslational processing. Cytolysin bears N-linked oligosaccharides that are converted to the complex type, while the major trypsin-like protease, granzyme A, bears only high-mannose-type oligosaccharides. The glycans of granzyme A, but not those of cytolysin, are modified with phosphomannose moieties. These results suggest that one possible mechanism for packaging proteins into NK granules is the mannose 6-phosphate-dependent lysosomal targeting system. However, the absence of the mannose 6-phosphate modification from cytolysin suggests the existence of yet another targeting system.
Regulation of human monocyte adherence by granulocyte-macrophage colony-stimulating factor.
Authors: Gamble J R , Elliott M J , Jaipargas E , Lopez A F , Vadas M A
Published: May 2006

Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to increase the adherence of purified peripheral blood monocytes to plastic surfaces and to monolayers of human umbilical vein endothelial cells. With plastic surfaces as a model 9-hr culture with GM-CSF was necessary for enhancement, and maximum levels were obtained after 24-hr stimulation. GM-CSF-stimulated adherence must require new RNA and protein synthesis because actinomycin D and cycloheximide abolished existing adherence and prevented further monocyte attachment. Interestingly, shorter incubations (1-2 hr) with cycloheximide increased adherence, suggesting a labile inhibitor. Formaldehyde fixation of monocytes but not of human vein endothelial cells abolished adherence, indicating the need for actively metabolizing monocytes. Thus, a hemopoietic growth factor, responsible for the proliferation and differentiation of monocytes, can also alter their adhesive characteristics. These observations may have important implications in pathological situations and in the in vivo use of GM-CSF.
cGMP-induced differentiation of the promyelocytic cell line HL-60.
Authors: Boss G R
Published: May 2006

cGMP is a second messenger that mediates numerous metabolic events; in the present work a role in myeloid cell differentiation was demonstrated. Nitroprusside and NaNO2, which activate cytosolic guanylate cyclase and increase the intracellular cGMP concentration, induced granulocytic differentiation of the human promyelocytic cell line HL-60; differentiation was measured by acquisition of the OKM1 antigen, morphological changes, and nitroblue tetrazolium reduction. When theophylline, a phosphodiesterase inhibitor, which by itself induced modest differentiation, was added to nitroprusside or NaNO2, differentiation increased in an additive fashion. The degree of differentiation correlated with the increase in the intracellular cGMP concentration. 8-Bromoguanosine 3',5'-cyclic monophosphate, a membrane-permeable cGMP analogue, also induced differentiation of HL-60 cells but was much more effective in the presence of theophylline, with the two agents interacting synergistically. The effect of theophylline in these studies could not be attributed to increasing the intracellular cAMP concentration. Dimethyl sulfoxide, and established inducer of differentiation of HL-60 cells, markedly enhanced the differentiation induced by nitroprusside and NaNO2.
High levels of a parathyroid hormone-like protein in milk.
Authors: Budayr A A , Halloran B P , King J C , Diep D , Nissenson R A , Strewler G J
Published: May 2006

Expression of a parathyroid hormone-like protein (PLP), which is associated with hypercalcemia in malignancy, has recently been localized to normal lactating mammary tissue. We examined the possibility of an extramammary role of PLP by measuring its levels in serum and milk of lactating women. The levels of PLP by radioimmunoassay in serum of lactating and nonlactating women were indistinguishable [4.2 +/- 1.8 and 3.6 +/- 1.0 pg equivalents (eq) of PLP-(1-34) amide per ml, respectively]. As PLP was undetectable in some serum samples, this result does not exclude the possibility that lactation results in a small increase in serum levels of PLP. In contrast, high concentrations of immunoreactive PLP [40,000-75,000 pg eq of PLP-(1-34) amide per ml] and correspondingly high concentrations of bioactive PLP were found in human, rat, and bovine milk. A variety of processed bovine milk products had a PLP content similar to that of fresh bovine milk, whereas infant formulas had lower concentrations, ranging down to undetectable. Although the physiological role of PLP in lactation is unknown, the data establish the presence of PLP in milk and suggest the possibility that PLP may be important in neonatal calcium homeostasis.
Extensive diversity of branched-RNA-linked multicopy single-stranded DNAs in clinical strains of Escherichia coli.
Authors: Sun J , Herzer P J , Weinstein M P , Lampson B C , Inouye M , Inouye S
Published: May 2006

Recently it was shown that a clinical strain of Escherichia coli contains a reverse transcriptase that is essential for the synthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA). We now have examined 113 independent clinical isolates of E. coli for the existence of msDNA and found that 7 strains contained msDNA. Four of them were further analyzed by hybridization analysis, which indicated that three of the msDNAs were different, having little sequence homology. When the reverse transcriptase gene associated with one of these msDNAs was used as a probe, it did not hybridize with chromosomal DNA from the other strains containing msDNA. These results indicate that some clinical E. coli strains carry their own unique msDNA-synthesizing systems; msDNAs produced by these systems have little, if any, sequence homology in their RNA and DNA molecules and the reverse transcriptases required for the production of msDNA also have little sequence similarity. Such extensive diversity of the msDNA-synthesizing systems supports the notion that they were acquired by the E. coli genome late during the evolution of E. coli.
Single-channel recording in myelinated nerve fibers reveals one type of Na channel but different K channels.
Authors: Jonas P , Bräu M E , Hermsteiner M , Vogel W
Published: May 2006

Amphibian myelinated nerve fibers were treated with collagenase and protease. Axons with retraction of the myelin sheath were patch-clamped in the nodal and paranodal region. One type of Na channel was found. It has a single-channel conductance of 11 pS (15 degrees C) and is blocked by tetrodotoxin. Averaged events show the typical activation and inactivation kinetics of macroscopic Na current. Three potential-dependent K channels were identified (I, F, and S channel). The I channel, being the most frequent type, has a single-channel conductance of 23 pS (inward current, 105 mM K on both sides of the membrane), activates between -60 and -30 mV, deactivates with intermediate kinetics, and is sensitive to dendrotoxin. The F channel has a conductance of 30 pS, activates between -40 and 60 mV, and deactivates with fast kinetics. The former inactivates within tens of seconds; the latter inactivates within seconds. The third type, the S channel, has a conductance of 7 pS and deactivates slowly. All three channels can be blocked by external tetraethylammonium chloride. We suggest that these distinct K channel types form the basis for the different components of macroscopic K current described previously.
Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the alpha subunit.
Authors: Neumann D , Barchan D , Horowitz M , Kochva E , Fuchs S
Published: May 2006

The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind alpha-bungarotoxin. Numerous studies indicate that the ligand-binding site of the AcChoR includes cysteine residues at positions 192 and 193 of the alpha subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR alpha subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion of the snake AcChoR alpha subunit. First, a mouse AcChoR alpha-subunit cDNA probe was used to screen a size-selected snake (Natrix tessellata) genomic library. A genomic clone was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR alpha subunit. The domain of the alpha subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake alpha subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR alpha subunits. Sequence comparison revealed that the cloned region of the snake alpha subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind alpha-bungarotoxin. In the presumed ligand-binding site, in the vicinity of Cys-192 and Cys-193, four major substitutions occur in the snake sequence--at positions 184 (Trp----Phe), 185 (Lys----Trp), 187 (Trp----Ser), and 194 (Pro----Leu). In addition, Asn-189 is a putative N-glycosylation site, present only in the snake. These changes, or part of them, may explain the lack of alpha-bungarotoxin-binding to snake AcChoR.
A 13-amino acid peptide in three yeast glycosyltransferases may be involved in dolichol recognition.
Authors: Albright C F , Orlean P , Robbins P W
Published: May 2006

A 13-amino acid peptide was identified in three glycosyltransferases of the yeast endoplasmic reticulum. These enzymes, the products of the ALG1, ALG7, and DPM1 genes, catalyze the transfer of sugars from nucleotide sugars to dolichol phosphate derivatives. The consensus sequence for the conserved peptide was Leu-Phe-Val-Xaa-Phe-Xaa-Xaa-Ile-Pro-Phe-Xaa-Phe-Tyr. A sequence resembling the conserved peptide was also found in the predicted SEC59 protein, which is suspected to participate in assembly of the lipid-linked precursor oligosaccharide, although its specific function is unknown. All of the identified sequences contain an isoleucine at position 8 and phenylalanine or tyrosine at positions 2, 5, and 12. We believe this peptide may be involved in dolichol recognition for the following reasons. (i) The conserved sequence occurs in potential membrane-spanning regions. (ii) The ALG7 and DPM1 proteins are known to recognize the isoprenoid region of dolichol phosphate specifically; this recognition presumably occurs in the membrane since dolichol is very hydrophobic. (iii) The consensus sequence is similar to a region of two halobacterial proteins implicated in binding of the isoprenoid region of retinal. (iv) If the consensus sequence is represented as an alpha-helix, the conserved residues lie on one face of the helix. An alpha-helical structure is likely since the conserved regions are in potential membrane-spanning domains.
Production of platelet-derived endothelial cell growth factor by normal and transformed human cells in culture.
Authors: Usuki K , Heldin N E , Miyazono K , Ishikawa F , Takaku F , Westermark B , Heldin C H
Published: May 2006

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa endothelial cell mitogen which has angiogenic properties in vivo. We report here that human foreskin fibroblasts, a human squamous cell carcinoma cell line, and 2 out of the 3 human thyroid carcinoma cell lines investigated produce PD-ECGF, whereas 21 other cell lines examined do not. The positive cell lines contained a 1.8-kilobase PD-ECGF mRNA, and a 45-kDa protein could be demonstrated in lysates of the cell lines by immunoblotting and immunoprecipitation using a specific antiserum against PD-ECGF. Furthermore, the cell lysates contained mitogenic activity for endothelial cells that was neutralized by the PD-ECGF antiserum. PD-ECGF was found to be secreted only slowly from the producer cells, consistent with the previous finding that the primary translation product lacks a signal sequence. The restricted expression and intracellular sequestration of PD-ECGF imply a strictly controlled function in endothelial cell proliferation and angiogenesis. Aberrant production of PD-ECGF may play a role in tumor angiogenesis.
Persistent poliovirus infection of human neuroblastoma cells.
Authors: Colbère-Garapin F , Christodoulou C , Crainic R , Pelletier I
Published: May 2006

Two human neuroblastoma cell lines were persistently infected with poliovirus strains of all three serotypes. In persistently infected IMR-32 cells, which were studied in greatest detail, viral antigens were present in most cells, and over a 9-month period virions were found in the medium at high titers. Persistently infected cells were resistant to superinfection by Sabin 1, 2, and 3 poliovirus but sensitive to coxsackievirus B3. The viruses recovered from persistently infected cells were studied for conservation of epitopes, host cell specificity, and temperature resistance phenotype. The antigenic site 1 carried by the major capsid protein VP1 was modified on the persistent viruses of all three serotypes. This was confirmed for one virus by sequencing the corresponding genomic region in which two mutations were detected. The titers of persistent viruses were 1-3 log10 units higher on IMR-32 cells than on nonneuronal HEp-2 cells, while parental viruses had similar titers on both lines. When thermosensitive viruses were used to initiate the infection, the persistent viruses were found to be thermoresistant at 39 degrees C. Together the results indicate that the persistent infection correlated with the selection of highly mutated viral strains. Poliovirus-infected neuroblastoma cell lines thus constitute an in vitro model of chronic viral infections, which are increasingly implicated in human neural diseases.
Classical conditioning of the Aplysia siphon-withdrawal reflex exhibits response specificity.
Authors: Hawkins R D , Lalevic N , Clark G A , Kandel E R
Published: May 2006

The gill- and siphon-withdrawal reflex of Aplysia undergoes classical conditioning of its amplitude and duration when siphon stimulation (the conditioned stimulus, CS) is paired with tail or mantle shock (the unconditioned stimulus, US). This conditioning of a preexisting response exhibits both temporal and stimulus specificities, which can be accounted for by activity-dependent enhancement of presynaptic facilitation of the siphon sensory neurons. To test whether conditioning of the reflex also exhibits response specificity (development of a new type of response to the CS that often resembles the response to the US), we measured the direction of siphon withdrawal in response to siphon stimulation (the CS) with tail or mantle shock as the US. The unlearned response to siphon stimulation is straight contraction, the response to tail shock is backward bending, and the response to mantle shock is forward bending. In the first experiment, we trained different animals with the tail or mantle US paired or unpaired with the CS; in a second experiment, we trained each animal with two CSs, one of which was paired with the US; in a third experiment, we varied US intensity; and in a fourth experiment, we trained each animal with two USs, one of which was paired with the CS. There was a significant, pairing-specific tendency for the direction of the response to the CS to resemble the response to the US after training in each experiment, demonstrating response specificity in conditioning of the withdrawal reflex. This feature of conditioning could in principle be accounted for by an elaboration of activity-dependent facilitation.
Determination of the primary charge separation rate in isolated photosystem II reaction centers with 500-fs time resolution
Authors: Wasielewski Michael R. , Johnson Douglas G. , Seibert Michael , Govindjee
Published: May 2006

We have measured directly the rate of formation of the oxidized chlorophyll a electron donor (P680
+
) and the reduced electron acceptor pheophytin a
-
(Pheo
a
-
) following excitation of isolated spinach photosystem II reaction centers at 4°C. The reaction-center complex consists of D
1
, D
2
, and cytochrome
b
-559 proteins and was prepared by a procedure that stabilizes the protein complex. Transient absorption difference spectra were measured from 440 to 850 nm as a function of time with 500-fs resolution following 610-nm laser excitation. The formation of P680
+
-Pheo
a
-
is indicated by the appearance of a band due to P680
+
at 820 nm and corresponding absorbance changes at 505 and 540 nm due to formation of Pheo
a
-
. The appearance of the 820-nm band is monoexponential with τ = 3.0 ± 0.6 ps. The time constant for decay of
1*
P680, the lowest excited singlet state of P680, monitored at 650 nm, is τ = 2.6 ± 0.6 ps and agrees with that of the appearance of P680
+
within experimental error. Treatment of the photosystem II reaction centers with sodium dithionite and methyl viologen followed by exposure to laser excitation, conditions known to result in accumulation of Pheo
a
-
, results in formation of a transient absorption spectrum due to
1*
P680. We find no evidence for an electron acceptor that precedes the formation of Pheo
a
-
.
Structure of the rat argininosuccinate lyase gene: close similarity to chicken delta-crystallin genes.
Authors: Matsubasa T , Takiguchi M , Amaya Y , Matsuda I , Mori M
Published: May 2006

Argininosuccinate lyase (EC 4.3.2.1) is an enzyme of arginine biosynthesis and is also involved in the urea cycle in the liver of ureotelic animals. A comparison of cDNA-derived amino acid sequences revealed that argininosuccinate lyase is highly homologous with chicken delta-crystallin, a major structural protein of the eye lens. The gene for the rat argininosuccinate lyase was cloned and its structure was determined. This gene is a single-copy gene about 14 kilobases long and is split into 16 exons. A comparison with chicken delta-crystallin genes revealed that all introns interrupt the protein-coding regions at homologous positions. This close similarity in structural organization provides strong evidence for the view of Piatigorsky et al. [Piatigorsky, J., O'Brien, W. E., Norman, B. L., Kalmuck, K., Wistow, G., Borras, T., Nickerson, J. M. & Wawrousek, E. F. (1988) Proc. Natl. Acad. Sci. USA 85, 3479-3483] that chicken delta 1- and delta 2-crystallin genes evolved by recruitment and duplication of the preexisting argininosuccinate lyase gene and that delta 2-crystallin is probably the direct homologue argininosuccinate lyase.
Three-dimensional structure of Fab R19.9, a monoclonal murine antibody specific for the p-azobenzenearsonate group.
Authors: Lascombe M B , Alzari P M , Boulot G , Saludjian P , Tougard P , Berek C , Haba S , Rosen E M , Nisonoff A , Poljak R J
Published: May 2006

The crystal structure of Fab R19.9, derived from an anti-p-azobenzenearsonate monoclonal antibody, has been determined and refined to 2.8-A resolution by x-ray crystallographic techniques. Monoclonal antibody R19.9 (IgG2b kappa) shares some idiotopes with a major idiotype (CRIA) associated with A/J anti-p-azobenzenearsonate antibodies. The amino acid sequences of the variable (V) parts of the heavy (VH) and light (VL) polypeptide chains of monoclonal antibody R19.9 were determined through nucleotide sequencing of their mRNAs. The VL region is very similar to that of CRIA-positive anti-p-azobenzenearsonate antibodies as is VH, except for its third complementarity-determining region, which is three amino acids longer; it makes a loop, unique to R19.9, that protrudes into the solvent. A large number of tyrosine residues in the complementarity-determining region of VH and VL, with their side chains pointing towards the solvent, may have an important function in antigen binding.
Monoclonal antibodies specific for murine CD2 reveal its presence on B as well as T cells.
Authors: Yagita H , Nakamura T , Karasuyama H , Okumura K
Published: May 2006

Monoclonal antibodies specific for the murine CD2 antigen were identified by an efficient screening method utilizing murine CD2 cDNA transfectants. An unexpected expression of CD2 on murine B cells was revealed by immunofluorescence and immunoprecipitation studies with these monoclonal antibodies and by RNA blot analysis for the murine CD2 transcript.
Interaction of [3H]MK-801 with multiple states of the N-methyl-D-aspartate receptor complex of rat brain.
Authors: Javitt D C , Zukin S R
Published: May 2006

N-Methyl-D-aspartate (N-Me-D-Asp) and phencyclidine receptors interactively mediate central nervous system processes including psychotomimetic effects of drugs as well as neurodegenerative, cognitive, and developmental events. To elucidate the mechanism of this interaction, effects of N-Me-D-Asp agonists and antagonists and of glycine-like agents upon binding of the radiolabeled phencyclidine receptor ligand [3H]MK-801 were determined in rat brain. Scatchard analysis revealed two discrete components of [3H]MK-801 binding after 4 hr of incubation. Incubation in the presence of L-glutamate led to an increase in apparent densities but not in affinities of both components of [3H]MK-801 binding as well as conversion of sites from apparent low to high affinity. Incubation in the presence of combined D-serine and L-glutamate led to an increase in the apparent density of high-affinity [3H]MK-801 binding compared with incubation in the presence of either L-glutamate or D-serine alone. These data support a model in which phencyclidine receptor ligands bind differentially to closed as well as open conformations of the N-Me-D-Asp receptor complex and in which glycine-like agents permit or factilitate agonist-induced conversion of N-Me-D-Asp receptors from closed to open conformations.