bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucocorticoids locally disrupt an array of positioned nucleosomes on the rat tyrosine aminotransferase promoter in hepatoma cells.
Authors: Carr K D , Richard-Foy H
Published: May 2006

Transcriptional activation by steroid hormones is often associated with the appearance of a DNase I hypersensitive site resulting from a local alteration of the nucleoprotein structure of the promoter. For the mouse mammary tumor virus long terminal repeat, a viral promoter under glucocorticoid control, a model has been proposed: the appearance of the hormonodependent DNase I hypersensitive site reflects the displacement of a single precisely positioned nucleosome associated with the glucocorticoid responsive elements. To determine if such a mechanism is of general relevance in transcriptional activation by steroid hormones, we have investigated the nucleosomal organization of the rat tyrosine aminotransferase promoter over a 1-kilobase region that contains the glucocorticoid regulatory target. This region displays a hormonodependent DNase I hypersensitive site. In the absence of hormone, micrococcal nuclease digestion of nuclei demonstrates the presence of positioned nucleosomes, with cutting sites centered around positions -3080, -2900, -2700, -2800, -2255, and -2040. Treatment of the cells with dexamethasone induces a disruption of the chromatin structure over a relatively short stretch of DNA (approximately positions -2400 to -2650) that overlaps two nucleosomes. These observations suggest a strong similarity in the role of chromatin structure in glucocorticoid-dependent transcriptional activation of mouse mammary tumor virus and tyrosine aminotransferase promoters.
V(D)J recombination activity in lymphoid cell lines is increased by agents that elevate cAMP.
Authors: Menetski J P , Gellert M
Published: May 2006

V(D)J [variable--(diversity)--joining] recombination is regulated developmentally, being restricted to cells of the early B- and T-lymphocyte lineages. In this report we show that recombination activity can also be regulated in response to chemical effectors. Compounds that increase intracellular cAMP increase V(D)J recombination of extrachromosomal substrates in pre-B-cell lines as much as 10-fold. In contrast, V(D)J recombination is reduced 5- to 8-fold in response to phorbol 12-myristate 13-acetate or to the calcium ionophore A23187. The effect of cAMP agonists on recombination appears to reflect an increase in cellular recombination activity, as indicated by the caffeine-induced rise in the level of mRNA from the recombination-activating genes RAG1 and RAG2. Our data demonstrate that intracellular second messengers modulate recombination activity in lymphoid cell lines, implying that recombination activity can be regulated by these signals in developing B and T lymphocytes.
Inactivation of human alpha-globin gene expression by a de novo deletion located upstream of the alpha-globin gene cluster.
Authors: Liebhaber S A , Griese E U , Weiss I , Cash F E , Ayyub H , Higgs D R , Horst J
Published: May 2006

Synthesis of normal human hemoglobin A, alpha 2 beta 2, is based upon balanced expression of genes in the alpha-globin gene cluster on chromosome 16 and the beta-globin gene cluster on chromosome 11. Full levels of erythroid-specific activation of the beta-globin cluster depend on sequences located at a considerable distance 5' to the beta-globin gene, referred to as the locus-activating or dominant control region. The existence of an analogous element(s) upstream of the alpha-globin cluster has been suggested from observations on naturally occurring deletions and experimental studies. We have identified an individual with alpha-thalassemia in whom structurally normal alpha-globin genes have been inactivated in cis by a discrete de novo 35-kilobase deletion located approximately 30 kilobases 5' from the alpha-globin gene cluster. We conclude that this deletion inactivates expression of the alpha-globin genes by removing one or more of the previously identified upstream regulatory sequences that are critical to expression of the alpha-globin genes.
Thermodynamic characterization of the stability and the melting behavior of a DNA triplex: a spectroscopic and calorimetric study.
Authors: Plum G E , Park Y W , Singleton S F , Dervan P B , Breslauer K J
Published: May 2006

We report a complete thermodynamic characterization of the stability and the melting behavior of an oligomeric DNA triplex. The triplex chosen for study forms by way of major-groove Hoogsteen association of an all-pyrimidine 15-mer single strand (termed y15) with a Watson-Crick 21-mer duplex composed of one purine-rich strand (termed u21) and one pyrimidine-rich strand (termed y21). We find that the near-UV CD spectrum of the triplex can be duplicated by the addition of the B-like CD spectrum of the isolated 21-mer duplex and the CD spectrum of the 15-mer single strand. Spectroscopic and calorimetric measurements show that the triplex (y15.u21.y21) melts by two well-resolved sequential transitions. The first transition (melting temperature, Tm, approximately 30 degrees C) is pH-dependent and involves the thermal expulsion of the 15-mer strand to form the free duplex u21.y21 and the free single strand y15. The second transition (Tm approximately 65 degrees C) is pH-independent between pH 6 and 7 and reflects the thermal disruption of the u21.y21 Watson-Crick duplex to form the component single strands. The thermal stability of the y15.u21.y21 triplex increases with increasing Na+ concentration but is nearly independent of DNA strand concentration. Differential scanning calorimetric measurements at pH 6.5 show the triplex to be enthalpically stabilized by only 2.0 +/- 0.1 kcal/mol of base triplets (1 cal = 4.184 J), whereas the duplex is stabilized by 6.3 +/- 0.3 kcal/mol of base pairs. From the calorimetric data, we calculate that at 25 degrees C the y15.u21.y21 triplex is stabilized by a free energy of only 1.3 +/- 0.1 kcal/mol relative to its component u21.y21 duplex and y15 single strand, whereas the 21-mer duplex is stabilized by a free energy of 17.2 +/- 1.2 kcal/mol relative to its component single strands. The y15 single strand modified by methylation of cytosine at the C-5 position forms a triplex with the u21.y21 duplex, which exhibits enhanced thermal stability. The spectroscopic and calorimetric data reported here provide a quantitative measure of the influence of salt, temperature, pH, strand concentration, and base modification on the stability and the melting behavior of a DNA triplex. Such information should prove useful in designing third-strand oligonucleotides and in defining solution conditions for the effective use of triplex structure formation as a tool for modulating biochemical events.
The Steiner ratio conjecture of Gilbert and Pollak is true.
Authors: Du D Z , Hwang F K
Published: May 2006

Let P be a set of n points on the euclidean plane. Let Ls(P) and Lm(P) denote the lengths of the Steiner minimum tree and the minimum spanning tree on P, respectively. In 1968, Gilbert and Pollak conjectured that for any P, Ls(P) >/= (radical3/2)Lm(P). We provide an abridged proof for their conjecture in this paper.
NF-kappa B: a family of inducible and differentially expressed enhancer-binding proteins in human T cells.
Authors: Molitor J A , Walker W H , Doerre S , Ballard D W , Greene W C
Published: May 2006

A sensitive DNA-protein crosslinking approach has been used to characterize four inducible T-cell proteins (50 kDa, 55 kDa, 75 kDa, and 85 kDa) that specifically bind to kappa B enhancer elements. Partial proteolytic mapping revealed a distinct cleavage pattern for three of these proteins. These polypeptides are sequestered as inactive precursors in the cytosol of unstimulated T cells but can be converted into active forms in vivo by phorbol ester stimulation or in vitro by detergent treatment. The induction of these proteins by phorbol ester results in a strikingly biphasic pattern of nuclear expression with the 55-kDa and 75-kDa species appearing within minutes, whereas the 50-kDa and 85-kDa species appear only several hours after cellular stimulation. These data suggest that NF-kappa B-binding activity may not correspond to a single polypeptide but rather a family of at least four inducible and differentially regulated DNA-binding proteins that are expressed with distinct kinetics in human T lymphocytes.
A fat-specific enhancer is the primary determinant of gene expression for adipocyte P2 in vivo.
Authors: Ross S R , Graves R A , Greenstein A , Platt K A , Shyu H L , Mellovitz B , Spiegelman B M
Published: May 2006

The murine gene for adipocyte P2 encodes an adipocyte-specific member of the family of intracellular lipid binding proteins. The region upstream from the start of transcription of this gene has been found to contain binding sites for the transcription factors c-jun/c-fos and C/EBP (CCAAT/enhancer binding protein) and several short sequence elements found in other adipocyte gene promoters, termed fat-specific elements. To identify DNA sequences that were responsible for the high level of transcription of the gene for adipocyte P2 in vivo, we made a series of transgenic mice containing 168 base pairs (bp), 247 bp, 1.7 kilobases (kb), and 5.4 kb of 5' flanking sequence linked to the bacterial gene chloramphenicol acetyltransferase. Although plasmids containing only 168 bp of 5' sequence including the C/EBP and AP-1 (activation protein 1) binding sites were expressed well in cultured adipocytes, high levels of chloramphenicol acetyltransferase activity in the adipose tissue of transgenic mice were not observed until the 5' flanking region was extended to kb -54. An enhancer mapping between kb -4.9 and kb -5.4 upstream from the start of transcription was identified by transfection of further deletions into cultured adipocytes. This enhancer, when linked to a bp -63 promoter fragment from the gene for adipocyte P2, directed very high level chloramphenicol acetyltransferase expression specifically to adipose tissue in transgenic mice. These results identify a functional adipose-specific enhancer and indicate that it is the major determinant of tissue specificity of the gene for adipocyte P2. These results also demonstrate that the proximal-promoter binding sites for AP-1 and C/EBP are not sufficient or necessary to give adipose-tissue-specific expression in vivo, though they may play an important role in the response of this promoter to glucocorticoids.
Calcium currents recorded from a vertebrate presynaptic nerve terminal are resistant to the dihydropyridine nifedipine.
Authors: Stanley E F , Atrakchi A H
Published: May 2006

The influx of Ca ions into the presynaptic nerve terminal through ion channels is a key link between the action potential and the release of chemical transmitters. It is not clear, however, which types of Ca channel are involved in neurosecretion at vertebrate synapses. In particular, there is disagreement as to whether these channels are sensitive to dihydropyridine blockers, characteristic of L-type Ca channels. We have used the chicken ciliary ganglion calyx synapse to test the effect of the dihydropyridine nifedipine on Ca current recorded directly from a cholinergic presynaptic nerve terminal. We used a control neuron to define the experimental conditions under which L-type Ca channels are blocked by 10 microM nifedipine. We then tested the effect of the dihydropyridine on Ca currents recorded from the presynaptic terminal using the same conditions. Nifedipine did not reduce the calyx Ca current nor did it block chemical transmission through the ganglion. The lack of effect of the dihydropyridine was not due to restricted access since omega-conotoxin GVIA, a peptide toxin that blocks transmission at this synapse, rapidly blocked the calyx Ca current. Thus, the predominant Ca channel in this presynaptic nerve terminal is not dihydropyridine sensitive and, hence, cannot be characterized as L-type.
Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G + C-rich attenuating structure.
Authors: Soreq H , Ben-Aziz R , Prody C A , Seidman S , Gnatt A , Neville L , Lieman-Hurwitz J , Lev-Lehman E , Ginzberg D , Lipidot-Lifson Y
Published: May 2006

To study the primary structure of human acetylcholinesterase (AcChoEase; EC 3.1.1.7) and its gene expression and amplification, cDNA libraries from human tissues expressing oocyte-translatable AcChoEase mRNA were constructed and screened with labeled oligodeoxynucleotide probes. Several cDNA clones were isolated that encoded a polypeptide with greater than or equal to 50% identically aligned amino acids to Torpedo AcChoEase and human butyrylcholinesterase (BtChoEase; EC 3.1.1.8). However, these cDNA clones were all truncated within a 300-nucleotide-long G + C-rich region with a predicted pattern of secondary structure having a high Gibbs free energy (-117 kcal/mol) downstream from the expected 5' end of the coding region. Screening of a genomic DNA library revealed the missing 5' domain. When ligated to the cDNA and constructed into a transcription vector, this sequence encoded a synthetic mRNA translated in microinjected oocytes into catalytically active AcChoEase with marked preference for acetylthiocholine over butyrylthiocholine as a substrate, susceptibility to inhibition by the AcChoEase inhibitor BW284C51, and resistance to the BtChoEase inhibitor tetraisopropylpyrophosphoramide. Blot hybridization of genomic DNA from different individuals carrying amplified AcChoEase genes revealed variable intensities and restriction patterns with probes from the regions upstream and downstream from the predicted G + C-rich structure. Thus, the human AcChoEase gene includes a putative G + C-rich attenuator domain and is subject to structural alterations in cases of AcChoEase gene amplification.
Studies on the initiation and elongation reactions in the simian virus 40 DNA replication system.
Authors: Matsumoto T , Eki T , Hurwitz J
Published: May 2006

The synthesis of oligoribonucleotides by DNA primase in the presence of duplex DNA containing the simian virus 40 (SV40) origin of replication was examined. Small RNA chains (10-15 nucleotides) were synthesized in the presence of the four common ribonucleoside triphosphates, SV40 large tumor antigen (T antigen), the human DNA polymerase alpha (pol alpha)-DNA primase complex, the human single-stranded DNA-binding protein (HSSB), and topoisomerase I isolated from HeLa cells. The DNA primase-catalyzed reaction showed an absolute requirement for T antigen, HSSB, and pol alpha. The requirement for HSSB was not satisfied by other SSBs that can support the T-antigen-catalyzed unwinding of DNA containing the SV40 origin of replication. Oligoribonucleotide synthesis occurred with a lag that paralleled the lag observed in DNA synthesis. These results indicate that the specificity for the HSSB in the SV40 replication reaction is due to the pol alpha-primase-mediated synthesis of the Okazaki fragments. In contrast to this specificity, the elongation of Okazaki fragments can be catalyzed by a variety of different DNA polymerases, including high levels of pol alpha, the polymerase delta holoenzyme, T4 polymerase holoenzyme, the Escherichia coli polymerase III holoenzyme, and other polymerases. These observations suggest that leading-strand synthesis in the in vitro SV40 replication system can be nonspecific.
Boron neutron capture therapy of intracerebral rat gliosarcomas.
Authors: Joel D D , Fairchild R G , Laissue J A , Saraf S K , Kalef-Ezra J A , Slatkin D N
Published: May 2006

The efficacy of boron neutron capture therapy (BNCT) for the treatment of intracerebrally implanted rat gliosarcomas was tested. Preferential accumulation of 10B in tumors was achieved by continuous infusion of the sulfhydryl borane dimer, Na4(10)B24H22S2, at a rate of 45-50 micrograms of 10B per g of body weight per day from day 11 to day 14 after tumor initiation (day 0). This infusion schedule resulted in average blood 10B concentrations of 35 micrograms/ml in a group of 12 gliosarcoma-bearing rats and 45 micrograms/ml in a group of 10 similar gliosarcoma-bearing rats treated by BNCT. Estimated tumor 10B levels in these two groups were 26 and 34 micrograms/g, respectively. On day 14, boron-treated and non-boron-treated rats were exposed to 5.0 or 7.5 MW.min of radiation from the Brookhaven Medical Research Reactor that yielded thermal neutron fluences of approximately 2.0 x 10(12) or approximately 3.0 x 10(12) n/cm2, respectively, in the tumors. Untreated rats had a median postinitiation survival time of 21 days. Reactor radiation alone increased median postinitiation survival time to 26 (5.0 MW.min) or 28 (7.5 MW.min) days. The 12 rats that received 5 MW.min of BNCT had a median postinitiation survival time of 60 days. Two of these animals survived greater than 15 months. In the 7.5 MW.min group, the median survival time is not calculable since 6 of the 10 animals remain alive greater than 10 months after BNCT. The estimated radiation doses to tumors in the two BNCT groups were 14.2 and 25.6 Gy equivalents, respectively. Similar gliosarcoma-bearing rats treated with 15.0 or 22.5 Gy of 250-kilovolt peak x-rays had median survival times of only 26 or 31 days, respectively, after tumor initiation.
Adenovirus 12S E1A gene represses differentiation of F9 teratocarcinoma cells.
Authors: Weigel R J , Devoto S H , Nevins J R
Published: May 2006

The F9 teratocarcinoma cell line differentiates in vitro after treatment with retinoic acid and cAMP and has been a widely used model system for the study of the molecular events that are responsible for cellular commitment and differentiation during early development. Previous experiments have suggested intriguing parallels between the control of gene expression during F9 cell differentiation and the regulation of gene expression by adenovirus E1A. Transfection of a 12S E1A-expressing plasmid into terminally differentiated, nonproliferating F9 cells generates, at high frequency, colonies of dividing cells, each of which expresses E1A. Cell lines established from these colonies proliferate in the presence of retinoic acid and have lost the fully differentiated phenotype as characterized by the absence of expression of a series of differentiation-specific genes. We conclude that expression of the viral 12S E1A gene product interferes with retinoic acid-induced F9 cell differentiation. Moreover, the results suggest that the differentiation process, as defined by markers of terminal differentiation, may not be a permanent event but can be reversed by E1A expression.
Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster.
Authors: Eissenberg J C , James T C , Foster-Hartnett D M , Hartnett T , Ngan V , Elgin S C
Published: May 2006

We report here that a point mutation in the gene which encodes the heterochromatin-specific nonhistone chromosomal protein HP-1 in Drosophila melanogaster is associated with dominant suppression of position-effect variegation. The mutation, a G-to-A transition at the first nucleotide of the last intron, causes missplicing of the HP-1 mRNA. This suggests that heterochromatin-specific proteins play a central role in the gene suppression associated with heterochromatic position effects.
Lesion-induced synthesis and secretion of proteins by nonneuronal cells resident in frog peripheral nerve.
Authors: Rotshenker S , Reichert F , Shooter E M
Published: May 2006

Transection of a peripheral nerve results in Wallerian degeneration of the nerve segment distal to the lesion site and the initiation of axonal regeneration just proximal to it (neuroma site). Nonneuronal cells resident in peripheral nerve are suggested to play an important role in neural repair mechanisms through diffusable molecules that they synthesize and secrete. We examined the array of proteins synthesized and secreted by nonneuronal cells resident in the frog peripheral nerve, which is known for its high regenerative capacity. Nerve segments were incubated in medium containing [35S]methionine, and the secreted radioactively labeled proteins were analyzed by gel electrophoresis. Nerve injury resulted in the complete down-regulation of a group of proteins synthesized and secreted by nonneuronal cells in intact nerve. At the same time, the synthesis and secretion of several proteins were up-regulated in the neuroma and degenerating nerve segments, proximal and distal to the axotomy site, respectively. Proteins secreted by the proximal segment were of apparent kDa/pI (mass/isoelectric point) values of 215/5.6, 76/6.7, 73/7.0, 44/5.2, 36.5/5.6, 35.5/6.0, and 32/6.0. Similar proteins were secreted by the degenerating distal segment but with the exception of variable reductions in the 44- and 32-kDa proteins and increases in proteins of apparent kDa/pI values of 39/5.2 and 29/7.3-7.4. Step gradient ultracentrifugation suggested that the latter two are apolipoproteins. Comparison with plasma apolipoproteins further indicated that nerve and plasma apolipoproteins differ. The up-regulation of the synthesis and secretion of these proteins concurrently with nerve degeneration and regeneration strongly imply that these molecules are involved in neuronal repair mechanisms.
Infection of cultured central nervous system neurons with a defective herpes simplex virus 1 vector results in stable expression of Escherichia coli beta-galactosidase.
Authors: Geller A I , Freese A
Published: May 2006

We have developed a defective herpes simplex virus (HSV) vector system that permits the introduction of virtually any gene into mammalian central nervous system neurons. The prototype vector, pHSVlac, contains a transcription unit that places the Escherichia coli lacZ gene under the control of the HSV-1 immediate early 4/5 promoter. pHSVlac was propagated using the HSV-1 temperature-sensitive mutant ts K as helper virus. Infection of rat neurons in primary culture derived from various regions throughout the central nervous system, including spinal cord, cerebellum, thalamus, basal ganglia, hippocampus, occipital cortex, temporal cortex, and frontal cortex, resulted in stable expression of high levels of beta-galactosidase for at least 2 weeks, without cell damage. Since other genes can be expressed from pHSVlac, HSV-1 vectors may prove useful for delivery of genes into central nervous system neurons for studies on nervous system physiology or to perform gene therapy for neurological conditions.
Serum and depolarizing agents cause acute neurotoxicity in cultured cerebellar granule cells: role of the glutamate receptor responsive to N-methyl-D-aspartate.
Authors: Schramm M , Eimerl S , Costa E
Published: May 2006

The life span of neonatal rat cerebellar granule cells, grown in basal minimal Eagle's medium containing 10% (vol/vol) fetal calf serum, was extended to 21-30 days by weekly supplementation with glucose. Addition of 1% fetal calf serum to the culture at 14 days killed 85% of the cells within 1 hr. This lethal effect could be prevented by the N-methyl-D-aspartate (NMDA) receptor antagonists dibenzocyclohepteneimine (MK-801) and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP). These findings suggested that the glutamate in the serum caused the dramatic neuronal death through action on the NMDA receptor. Indeed, a 5-min incubation in a Locke physiological salt solution containing 20 microM glutamate and 5 microM glycine killed 55-90% of the cells. This acute toxicity could be prevented by a lyso-GM1 ganglioside with N-acetylated sphingosine. The relatively low glutamate content of the sera analyzed suggests that factors in addition to glycine potentiate serum neurotoxicity. The above noted antagonists of the NMDA receptor also greatly reduced the lethal effect of depolarization by 90 mM KCl or 10 microM veratridine. Therefore, it is likely that the toxicity of the depolarizing agents is mediated by glutamate released from the cells. It is concluded that survival of cerebellar neurons in primary culture may be strongly affected by unsuspected neurotoxic phenomena elicited by brief action of a rather low glutamate concentration.
G-DNA: a twice-folded DNA structure adopted by single-stranded oligo(dG) and its implications for telomeres.
Authors: Panyutin I G , Kovalsky O I , Budowsky E I , Dickerson R E , Rikhirev M E , Lipanov A A
Published: May 2006

Our dimethyl sulfate modification experiments suggest that (dG)n stretches within single-stranded DNA fragments, which represent the simplest model for telomeric sequences, adopt a complex intrastrand structure other than a simple hairpin. We present a molecular model for the DNA structure that conforms to dimethyl sulfate methylation data. The principal element of this G-DNA structure is a quadruple helix formed by pairwise antiparallel segments of the twice-folded (dG)n stretch. This quadruple core has two wide and two narrow grooves connected by three loop-shaped segments. The strong stacking interactions of the neighboring guanine tetrads and the large number of hydrogen bonds formed can be the primary reasons that such structures are favored over a common hairpin for long (dG)n stretches. Such compact structures may be formed from (dG)n stretches of telomeric sequences.
Design of crystalline helices of short oligopeptides as a possible model for nucleation of alpha-helix: role of water molecules in stabilizing helices.
Authors: Parthasarathy R , Chaturvedi S , Go K
Published: May 2006

We have designed, synthesized, crystallized, and performed x-ray analysis of several hydrophobic tripeptides that show an extended near alpha-helical structure in the crystalline state. All of the tripeptides that show this remarkably stable helix crystallize with two or three water molecules; they all have glycine at the N terminus and have increasing hydrophobicity as one moves from the N to C terminus. Even though three residues in the oligomer are not sufficient to complete a turn, one of the water molecules acts as an added residue and links up adjacent tripeptide segments along the helix axis so that in the crystal, the helix appears effectively as one long continuous helix. Two of these tripeptides are stabilized by two water molecules that enable the peptides to complete a turn of the helix and extend the helical structure throughout the crystal by linking translationally related peptides by hydrogen bonds. In two other peptides, these roles are played by three rather than two water molecules. Though these tripeptides have different crystal symmetry, they all show the basic pattern of hydrated helix and packing, indicating the strong conformational preference for a stable structure even for these tripeptides. Such conformationally stable hydrated structures for short specific related sequences illustrate their possible importance in nucleating protein folding and in the role water molecules play in such events.
A pair of tandemly repeated genes code for gp24, a putative adhesion protein of Dictyostelium discoideum.
Authors: Loomis W F , Fuller D L
Published: May 2006

The glycoprotein gp24 has been implicated in cell-cell adhesion of Dictyostelium discoideum. We have used a cDNA clone that codes for gp24 to screen cloned genomic fragments. Two closely linked genes (GP24A and GP24B) were recognized that generate mRNAs of about 650 base pairs after excision of a small intron and addition of poly(A). They appear to have arisen by tandem duplication of about 800 base pairs, followed by divergence. These genes are expressed within a few hours of the initiation of development; their mRNAs accumulate to a peak at 12 hr and persist until culmination. Both genes have short guanine-rich sequences (G boxes) upstream that have been shown to be involved in transcriptional regulation of other genes expressed during development of Dictyostelium. Their mRNAs code for proteins that are 85% identical. GP24A and GP24B mRNAs code for proteins with a hydrophobic domain followed by a highly charged carboxyl-terminal domain.
The 5HT2 receptor defines a family of structurally distinct but functionally conserved serotonin receptors.
Authors: Julius D , Huang K N , Livelli T J , Axel R , Jessell T M
Published: May 2006

Serotonin exerts its diverse physiological effects by interacting with multiple distinct receptor subtypes. We have isolated a rat brain 5HT2 serotonin receptor cDNA by virtue of its homology with the 5HT1c receptor. The 5HT2 receptor is a member of the family of receptors that are linked to guanine nucleotide-binding proteins and are predicted to span the lipid bilayer seven times. Overall sequence identity between the 5HT2 and 5HT1c receptors is 49%, but identity within the transmembrane domains is 80%. Expression of both the 5HT2 and 5HT1c receptors in transfected mouse fibroblasts activates phospholipase C signaling pathways and promotes cellular transformation. However, RNA blotting shows that these two receptor subtypes are differentially expressed in the central nervous system. In this manner, structurally and functionally homologous receptor subtypes may elicit distinct physiologic actions.
Release of peptide cotransmitters from a cholinergic motor neuron under physiological conditions.
Authors: Cropper E C , Price D , Tenenbaum R , Kupfermann I , Weiss K R
Published: May 2006

In previous studies, we demonstrated that B15, one of the two cholinergic motor neurons of the accessory radula closer muscle of Aplysia, synthesizes two peptides, small cardioactive peptides A and B (SCPA and SCPB), that, when exogenously applied, increase the size and relaxation rate of muscle contractions elicited by motor neuron stimulation. In the present experiments, we obtained evidence that the SCPs are released under physiological conditions. Specifically, we characterized firing patterns of motor neuron B15 during normal behavior, simulated them in vitro, and demonstrated that this type of neuronal activity produces decreases in SCP levels in neuronal processes and terminals. We also obtained evidence that suggests that enough SCP is released under physiological conditions to modulate neuromuscular activity in the accessory radula closer. We demonstrated that physiological activity of neuron B15 produces significant increases in muscle cAMP levels. Furthermore, increases in the size and relaxation rate of muscle contractions can be produced by changes in stimulation parameters that are also likely to maximize effects of released endogenous SCPA and SCPB.
Molecular cloning of human protein 4.2: a major component of the erythrocyte membrane.
Authors: Sung L A , Chien S , Chang L S , Lambert K , Bliss S A , Bouhassira E E , Nagel R L , Schwartz R S , Rybicki A C
Published: May 2006

Protein 4.2 (P4.2) comprises approximately 5% of the protein mass of human erythrocyte (RBC) membranes. Anemia occurs in patients with RBCs deficient in P4.2, suggesting a role for this protein in maintaining RBC stability and integrity. We now report the molecular cloning and characterization of human RBC P4.2 cDNAs. By immunoscreening a human reticulocyte cDNA library and by using the polymerase chain reaction, two cDNA sequences of 2.4 and 2.5 kilobases (kb) were obtained. These cDNAs differ only by a 90-base-pair insert in the longer isoform located three codons downstream from the putative initiation site. The 2.4- and 2.5-kb cDNAs predict proteins of approximately 77 and approximately 80 kDa, respectively, and the authenticity was confirmed by sequence identity with 46 amino acids of three cyanogen bromide-cleaved peptides of P4.2. Northern blot analysis detected a major 2.4-kb RNA species in reticulocytes. Isolation of two P4.2 cDNAs implies existence of specific regulation of P4.2 expression in human RBCs. Human RBC P4.2 has significant homology with human factor XIII subunit a and guinea pig liver transglutaminase. Sequence alignment of P4.2 with these two transglutaminases, however, revealed that P4.2 lacks the critical cysteine residue required for the enzymatic crosslinking of substrates.
Genotypic variation within asexual lineages of Taraxacum officinale.
Authors: King L M , Schaal B A
Published: May 2006

Restriction site variation in DNA that encodes rRNA (rDNA) was surveyed among 714 offspring within 31 lineages (26 genotypes) of obligate asexually reproducing Taraxacum officinale (dandelions). Although clonal offspring are expected, plants with nonparental rDNA were produced from two parents that were themselves siblings (same genotype). The variation is best characterized by the loss of an EcoRI restriction site that maps to the spacer region in the parental rDNA and is most likely involved in amplification of rare or unique rDNA repeats. In one family, 41 surveyed offspring lacked the EcoRI site. In the other family, only 1 of 26 offspring lost the EcoRI site. Other classes of DNA surveyed, chloroplast DNA and the alcohol dehydrogenase 2 gene (Adh2), showed no variation. However, offspring with nonparental rDNA also had nonparental alcohol dehydrogenase 1 (Adh1) restriction fragments. Because somatic mutations in plants can be incorporated into reproductive tissue, we propose that somatic events affecting at least both multicopy rDNA and DNA homologous to the maize Adh1 gene occurred at different developmental times in the two families. An event early in development would result in all variant offspring; an event late in development would result in a single variant offspring. These results support the view that mutation (in the broad sense) influences the level of genotypic variation in asexual organisms, which may facilitate adaptive evolution of asexual species.
Antibodies against membrane interleukin 1 alpha activate accessory cells to stimulate proliferation of T lymphocytes.
Authors: Eugui E M , Almquist S J
Published: May 2006

Some monoclonal antibodies (mAbs) against interleukin (IL) 1 alpha have been found to activate antigen-presenting cells (APC, human peripheral blood monocytes and B lymphocytes), so that unstimulated T lymphocytes cultured with them are induced to proliferate and secrete IL-2. Control mAbs of the same isotypes and mAbs against IL-1 beta do not activate APC. In the absence of APC, mAbs against IL-1 alpha do not induce proliferation of T lymphocytes. Mitomycin C-treated activated APC still induce T-cell proliferation. Proliferation of T lymphocytes cannot be induced by culture supernatants and requires contact with APC activated by mAbs against IL-1 alpha. The observations imply that surface membrane IL-1 alpha can function as a triggering molecule on APC, which could play an important role in the initiation of immune responses by T lymphocytes.
Purification of a glycoprotein vascular endothelial cell mitogen from a rat glioma-derived cell line.
Authors: Conn G , Soderman D D , Schaeffer M T , Wile M , Hatcher V B , Thomas K A
Published: May 2006

A growth factor that is mitogenic for vascular endothelial cells, with an ED50 of approximately 1 ng/ml, has been purified 170,000-fold to apparent homogeneity from tissue culture medium conditioned by a rat glioma-derived cell line. The pure protein is a 46-kDa dimer composed of two subunits of equivalent mass as established by comparison of migration in SDS/polyacrylamide gels with and without prior reduction. This glioma-derived growth factor is a glycoprotein and is not mitogenic for BALB/c 3T3 fibroblasts, properties that further distinguish it from other well-characterized vascular endothelial cell mitogens. In contrast to acidic and basic fibroblast growth factors and to platelet-derived endothelial cell growth factor, which have no secretory leader sequences and might only be released by leakage from damaged cells, the glycoprotein nature of this mitogen implies that it is processed through the glycosylating secretory pathway. This secretable growth factor could, therefore, be readily available in the extracellular space under normal physiological conditions in vivo to promote vascular endothelial cell proliferation associated with blood-vessel growth and maintenance.
Expression of gap junction channels in communication-incompetent cells after stable transfection with cDNA encoding connexin 32.
Authors: Eghbali B , Kessler J A , Spray D C
Published: May 2006

The gene family encoding gap junction proteins (connexins) consists of several known members, and multiple connexins are frequently coexpressed by coupled cells. To characterize the channel properties of the major rat liver gap junction protein (connexin 32) in isolation from other gap junction proteins, we have introduced the cDNA encoding it into a human hepatoma cell line (SKHep1) in which we have identified a gap junction deficiency. In this cell line, dye coupling was absent and junctional conductance was near zero. Connexins and connexin 32 mRNA were not detectable by immunocytochemistry and Northern blot analysis. After transfection and selection, cells were strongly coupled with regard to dye and electrical current, and connexin 32 mRNA and punctate connexin 32-immunoreactive membrane contacts were abundant. Functional gap junction channels were still expressed after 19 passages of the cells, indicating stable transfection. When junctional conductance was rendered reversibly low by exposing the cells to agents that uncouple other cell types, currents through single gap junction channels could be observed. The unitary conductance of these expressed channels was about 120-150 pS, a value that is distinctly larger than in heart cells, which express a different gap junction protein.
Protective immunogenicity of two synthetic peptides selected from the amino acid sequence of Bordetella pertussis toxin subunit S1.
Authors: Askelöf P , Rodmalm K , Wrangsell G , Larsson U , Svenson S B , Cowell J L , Undén A , Bartfai T
Published: May 2006

Two peptides, corresponding to amino acids 1-17 and 169-186 of the amino acid sequence of pertussis toxin (PT) subunit S1, were synthesized and coupled to the diphtheria toxin cross-reactive mutant protein CRM 197 and evaluated for immunogenicity and protective capacity against PT challenge in vivo. The peptide-CRM conjugates induced high antibody titers against native toxin in mice (BALB/c, C57/Black, and outbred NMRI) as measured by ELISA. Upon PT challenge (0.5 microgram of toxin) of the NMRI mice, the CRM conjugates of peptides 1-17 and 169-186 fully protected the mice from PT-induced leukocytosis. Immunization with the corresponding bovine serum albumin conjugates of these two peptides also fully protected mice. Rabbit antiserum to the peptide 1-17-CRM conjugate was highly efficient in inhibiting the ADP-ribosylating activity of PT but did not neutralize the clustering effect of PT on Chinese hamster ovary cells. In contrast, the rabbit antiserum raised against the peptide 169-186-CRM conjugate neutralized the clustering effect of PT on Chinese hamster ovary cells but did not inhibit the enzymatic activity of PT. Peptide 169-186-CRM conjugates mimic the immunoglobulin binding properties of PT and also cause clustering of Chinese hamster ovary cells. The CRM conjugates of these two peptides constitute a synthetic pertussis vaccine candidate with the ability to provide a chemically well-defined, safe, and efficient pertussis vaccine.
Induction of acetylcholine receptor alpha-subunit gene expression in chicken myotubes by blocking electrical activity requires ongoing protein synthesis.
Authors: Duclert A , Piette J , Changeux J P
Published: May 2006

The level of acetylcholine receptor alpha-subunit mRNA in primary cultures of chicken myotubes increases when the spontaneous electrical activity of the myotube is suppressed by the sodium channel blocker tetrodotoxin. This increase was prevented by two translational inhibitors: cycloheximide and anisomycin. Neither the basal level of alpha-subunit mRNA nor that of muscle-specific creatine phosphokinase mRNA was affected by these inhibitors. In contrast, cycloheximide potentiated the limited increase of alpha-subunit mRNA levels evoked by the neuropeptide calcitonin gene-related peptide. The high level of alpha-subunit mRNA elicited by tetrodotoxin treatment did not persist after subsequent addition of cycloheximide in the presence of tetrodotoxin, indicating that the continuous synthesis of protein factor(s) is necessary for this regulation. Moreover, cycloheximide decreased the high level of alpha-subunit mRNA present at early stages of in vitro maturation of muscle primary cultures without blocking the further increase of the muscle-specific creatine phosphokinase mRNA. Implications of the requirement for constant synthesis of protein factors on the induction of muscle-specific gene expression by blocking myotube electrical activity are discussed.
High-efficiency oligonucleotide-directed plasmid mutagenesis.
Authors: Olsen D B , Eckstein F
Published: May 2006

A number of single- and double-base substitutions have been introduced into either the polylinker region or the lacZ gene in the plasmid vector pUC19. The efficiencies of these changes upon transfection of TG-1 bacterial cells were generally 70-80%. A strategy has been devised by which the wild-type DNA can be selectively destroyed. It is primarily based on the resistance of phosphorothioate internucleotide linkages to some restriction enzymes. A mismatch oligonucleotide is introduced into a gapped region and the gap is filled using three deoxynucleoside 5'-triphosphates and one deoxynucleoside 5'-[alpha-thio]triphosphate. Reaction with a restriction enzyme that is unable to hydrolyze phosphorothioates ensures that the DNA containing the mismatch oligonucleotide is only nicked. Concomitantly, the DNA that does not contain the desired mutation is linearized. Subsequent reactions with an exonuclease and DNA polymerase I yield mutant homoduplex DNA for transfection.
A time-dependent bacterial bioluminescence emission spectrum in an in vitro single turnover system: energy transfer alone cannot account for the yellow emission of Vibrio fischeri Y-1.
Authors: Eckstein J W , Cho K W , Colepicolo P , Ghisla S , Hastings J W , Wilson T
Published: May 2006

Yellow fluorescent protein (YFP), which has a bound FMN, was isolated from the marine bacterium Vibrio fischeri strain Y-1b. Its presence in a luciferase [alkanal monooxygenase (FMN-linked); alkanal, reduced-FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] reaction mixture causes a striking color change, and an increase in bioluminescence intensity, as well as a faster rate of intensity decay, so that the quantum yield is not changed. The emission spectrum shows two distinct color bands, one at 490 nm attributed to the unaltered emission of the luciferase system, the other peaking in the yellow around 540 nm due to YFP emission. The kinetics of the two color bands differ, so the spectrum changes with time. The yellow emission reaches its initial maximum intensity later than the blue, and then both blue and yellow emissions decay exponentially with nearly the same pseudo-first-order rate constants, linearly dependent on [YFP] (from 0.01 sec-1 with no YFP to a maximum of approximately 0.1 sec-1 at 4 degrees C) but exhibiting a saturation behavior. The data can be interpreted by assuming the interaction of YFP with the peroxyhemiacetal intermediate in the luciferase reaction to form an unstable new complex whose breakdown gives the yellow emitter in its excited state. This simple model fits well the data at [YFP] less than 15 microM. The results indicate that a single primary excited state cannot be responsible for the blue and the yellow emissions.
Neuronal potentialities of cells in the optic nerve of the chicken embryo are revealed in culture.
Authors: Giess M C , Cochard P , Duprat A M
Published: May 2006

Neuronal potentialities in neuroepithelial cells of the chicken embryonic optic nerve were studied in culture by using neurofilament antibodies as neuronal markers. Embryonic day-4 and -5 (E4 and E5) optic stalks were explanted in vitro. Within the first few days of culture, numerous morphologically identifiable neurons extending long neurites developed. These neurons and their processes were specifically labeled with neurofilament antibodies. Similar results were obtained by explanting only the medial portion of E7 optic stalks away from possibly contaminating cerebral or retinal tissue. To determine whether neuronal potentialities persisted at later embryonic stages, cultures of dissociated optic stalks were established at E11, E15, and E18. Neurons labeled with the various neurofilament antibodies appeared in all cultures of E11 and E15 optic stalks. However, typical neurons could not be recognized in cultures of E18 optic nerves. These results indicate that cells with neuronal potentialities are present in the embryonic optic nerve from early stages of development and persist until at least E15. Since the adult optic nerve is devoid of nerve cell bodies, our observations are consistent with the hypothesis that axons of retinal ganglion cells, which course through the optic stalk, repress neuronal potentialities within a subpopulation of precursor cells during normal development.
Endothelial leukocyte adhesion molecule 1: direct expression cloning and functional interactions.
Authors: Hession C , Osborn L , Goff D , Chi-Rosso G , Vassallo C , Pasek M , Pittack C , Tizard R , Goelz S , McCarthy K
Published: May 2006

A cDNA for endothelial leukocyte adhesion molecule 1 (ELAM-1) was isolated by transient expression in COS-7 cells of a subtracted cDNA library from cytokine-treated human umbilical vein endothelial cells (HUVECs), with selection of ELAM-1-expressing clones by adhesion of transfected cells to the human promyelocytic cell line HL-60. This cloning method requires neither antibody nor purified ligand. ELAM-1-expressing COS cells bind the promyelocytic cell line HL-60 by a Ca2(+)-dependent but temperature-independent mechanism. Although ELAM-1 is homologous to mammalian lectins, its interaction with HL-60 cells is not inhibited by simple carbohydrate structures. ELAM-1-expressing COS cells also bind human neutrophils and the human colon carcinoma cell line HT-29, but not the B-cell line Ramos. However, Ramos cells adhere to cytokine-treated HUVECs but not control HUVECs, confirming the existence of other inducible adhesion molecules. In addition, the binding of HL-60 cells or neutrophils to ELAM-1-expressing COS cells is not inhibited by a monoclonal antibody (60.3) directed to an inhibitory epitope on CD18, indicating that the ELAM-1 ligand, although uncharacterized, is not a member of the CD11/CD18 family.
"Site-selected" transposon mutagenesis of Drosophila.
Authors: Kaiser K , Goodwin S F
Published: May 2006

Despite the wide range of techniques that can be brought to bear on the study of basic processes in Drosophila, there are still deficiencies in our armory. One of these is an ability to select mutants in cases where the gene is known and has been cloned, but where we are ignorant of the associated phenotype. We describe here a solution to this problem as applied to a model system, the singed (sn) locus. Our method is a combination of classical genetics and molecular biology: sib selection plus the polymerase chain reaction. We have used the method to isolate rare individuals with P-element-induced alleles of sn merely by recognition of the DNA structures induced at the locus by transposon insertion. Phenotypic criteria were used only retrospectively to verify our diagnoses. There are obvious implications of this technique for the mutagenesis of other organisms.