bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific protein-protein interactions between the essential mammalian spliceosome-associated proteins SAP 61 and SAP 114.
Authors: Chiara M D , Champion-Arnaud P , Buvoli M , Nadal-Ginard B , Reed R
Published: May 2006

Spliceosome-associated proteins (SAPs) 61, 62, and 114 can be UV-crosslinked to pre-mRNA in purified spliceosomal complexes and are associated with U2 small nuclear ribonucleoproteins (snRNP). These proteins also compose the essential heterotrimeric splicing factor SF3a, and products of yeast pre-mRNA processing genes PRP9, PRP11, and PRP21 are their likely yeast counterparts. We report the isolation of a cDNA encoding SAP 61 and find that it is 30% identical in amino acid sequence to PRP9. A C-terminal Cys2His2 zinc-finger-like motif, which could be involved in the pre-mRNA binding, is the most highly conserved region of the protein. We also demonstrate specific protein-protein interactions between SAPs 61 and 114 and show that the N terminus of SAP 61 is required for this interaction. Significantly, the corresponding proteins are also known to interact in yeast: PRP9 interacts with PRP21, and the N-terminal portion of PRP9 is required. Previous work showed that direct interactions also occur between SAPs 62 and 114 and between the corresponding PRPs 11 and 21. These observations indicate that the specific protein-protein interactions that occur between the three prespliceosomal factors have been conserved between yeast and mammals.
Rapid morphological radiation and convergence among races of the butterfly Heliconius erato inferred from patterns of mitochondrial DNA evolution.
Authors: Brower A V
Published: May 2006

The neotropical Heliconius butterflies are famous examples of Müllerian mimicry, due to the diverse array of shared, brightly colored wing patterns that advertise the butterflies' unpalatability. The parallel geographical variation in these patterns within several widespread species has been invoked to support the controversial Pleistocene refugium hypothesis of tropical diversification. However, in no Heliconius species have either evolutionary rates or relationships among geographical races been explicitly examined. I present a phylogenetic hypothesis based on mitochondrial DNA sequences for 14 divergent races of Heliconius erato, which reveals that similar wing patterns have evolved rapidly and convergently within the species. There is a basal split between groups of races from east and west of the Andes, reflecting a vicariant separation at the base of the Pleistocene. Within each of these clades, sequence divergence is very low, and some haplotypes are shared between allopatric races with radically different wing patterns. The topology implies a simultaneous radiation of races in these two areas within the last 200,000 years. Ages for the clades are estimated by comparing sequence divergence to a plot of mitochondrial divergence in several arthropod taxa with independently dated divergence times. This plot is linear and suggests that mitochondrial DNA in arthropods evolves in a clocklike manner, at least initially, when sequence divergence is low.
The p75 nerve growth factor receptor mediates survival or death depending on the stage of sensory neuron development.
Authors: Barrett G L , Bartlett P F
Published: May 2006

The function of the low-affinity nerve growth factor (NGF) receptor, p75NGFR, in regulating neuronal survival during development is unclear. The sensory deficit in mice with mutated p75NGFR suggests it is necessary for development of sensory neurons; however, whether it is required, in addition to trkA, for signal transduction or is more involved in localization of NGF is unresolved. In this study we demonstrate, in vitro, that lowering the levels of p75NGFR expression in sensory neurons with antisense oligonucleotides largely prevents the NGF-mediated survival of sensory neurons from embryonic day 12 and 15 mice but increases the survival of embryonic day 19 and postnatal day 2 sensory neurons in the absence of NGF. Thus, the p75NGFR is required for NGF-mediated survival in neurons at the stage of target innervation but can mediate an apoptotic signal at a later stage of cell development. Thus, p75NGFR undergoes a switch in function in the perinatal period: during embryogenesis it is required, probably with trkA, to mediate neuronal survival in the presence of NGF, but in the early postnatal period it acts as a constitutive death signal in the absence of NGF.
Drosophila telomere transposon HeT-A produces a transcript with tightly bound protein.
Authors: Danilevskaya O N , Slot F , Traverse K L , Hogan N C , Pardue M L
Published: May 2006

Telomeres from Drosophila appear to be very different from those of other organisms. A transposable element, HeT-A, plays a major role in forming telomeres and may be the sole structural element, since telomerase-generated repeats are not found. The structure of the HeT-A element, deduced from cloned fragments of DNA, suggests that transposition of the element is mediated by a polyadenylylated RNA intermediate. We now report analyses of HeT-A transcripts. The major RNA is of the appropriate size and strandedness to serve as a transposition intermediate. This RNA is found in cultured cells and in intact flies and is unusual in that it is associated with protein after treatments that apparently remove all protein from other RNAs.
Enzymatic synthesis of anandamide, an endogenous ligand for the cannabinoid receptor, by brain membranes.
Authors: Devane W A , Axelrod J
Published: May 2006

Anandamide, an endogenous eicosanoid derivative (arachidonoylethanolamide), binds to the cannabinoid receptor, a member of the G protein-coupled superfamily. It also inhibits both adenylate cyclase and N-type calcium channel opening. The enzymatic synthesis of anandamide in bovine brain tissue was examined by incubating brain membranes with [14C]ethanolamine and arachidonic acid. Following incubation and extraction into toluene, a radioactive product was identified which had the same Rf value as authentic anandamide in several thin-layer chromatographic systems. When structurally similar fatty acid substrates were compared, arachidonic acid exhibited the lowest EC50 and the highest activity for enzymatic formation of the corresponding ethanolamides. The concentration-response curve of arachidonic acid exhibited a steep slope, and at higher concentrations arachidonate inhibited enzymatic activity. When brain homogenates were separated into subcellular fractions by sucrose density gradient centrifugation, anandamide synthase activity was highest in fractions enriched in synaptic vesicles, myelin, and microsomal and synaptosomal membranes. When several areas of brain were examined, anandamide synthase activity was found to be highest in the hippocampus, followed by the thalamus, cortex, and striatum, and lowest in the cerebellum, pons, and medulla. The ability of brain tissue to enzymatically synthesize anandamide and the existence of specific receptors for this eicosanoid suggest the presence of anandamide-containing (anandaergic) neurons.
The superoxide dismutase molecular clock revisited.
Authors: Fitch W M , Ayala F J
Published: May 2006

The Cu,Zn superoxide dismutase (SOD) was examined earlier and found to behave in a very unclock-like manner despite (accepted point mutation, or PAM) corrections for multiple replacements per site. Depending upon the time span involved, rates could differ 5-fold. We have sought to determine whether the data might be clock-like if a covarion model were used. We first determined that the number of concomitantly variable codons (covarions) in SOD is 28. With that value fixed we found that the observations for SOD could fit reasonably well a molecular clock if, given 28 covarions, (i) there are approximately six replacements every 10 million years, (ii) the total number of codons is 162, (iii) the number of codons that are permanently invariable across the range of taxa from fungi to mammals is 44, and (iv) the persistence of variability is quite low (0.01). Thus, the inconsistent number of amino acid differences between various pairs of descendent sequences could well be the result of a fairly accurate molecular clock. The general conclusion has two sides: (i) the inference that a given gene is a bad clock may sometimes arise through a failure to take all the relevant biology into account and (ii) one should examine the possibility that different subsets of amino acids are evolving at different rates, because otherwise the assumption of a clock may yield erroneous estimates of divergence times on the basis of the observed number of amino acid differences.
Genome structure and evolution in Drosophila: applications of the framework P1 map.
Authors: Hartl D L , Nurminsky D I , Jones R W , Lozovskaya E R
Published: May 2006

Physical maps showing the relative locations of cloned DNA fragments in the genome are important resources for research in molecular genetics, genome analysis, and evolutionary biology. In addition to affording a common frame of reference for organizing diverse types of genetic data, physical maps also provide ready access to clones containing DNA sequences from any defined region of the genome. In this paper, we present a physical map of the genome of Drosophila melanogaster based on in situ hybridization with 2461 DNA fragments, averaging approximately 80 kilobase pairs each, cloned in bacteriophage P1. The map is a framework map in the sense that most putative overlaps between clones have not yet been demonstrated at the molecular level. Nevertheless, the framework map includes approximately 85% of all genes in the euchromatic genome. A continuous physical map composed of sets of overlapping P1 clones (contigs), which together span most of the euchromatic genome, is currently being assembled by screening a library of 9216 P1 clones with single-copy genetic markers as well as with the ends of the P1 clones already assigned positions in the framework map. Because most P1 clones from D. melanogaster hybridize in situ with chromosomes from related species, the framework map also makes it possible to determine the genome maps of D. pseudoobscura and other species in the subgenus Sophophora. Likewise, a P1 framework map of D. virilis affords potential access to genome organization and evolution in the subgenus Drosophila.
Molecular phylogeny of the Anopheles gambiae complex suggests genetic introgression between principal malaria vectors.
Authors: Besansky N J , Powell J R , Caccone A , Hamm D M , Scott J A , Collins F H
Published: May 2006

The six Afrotropical species of mosquitoes comprising the Anopheles gambiae complex include the most efficient vectors of malaria in the world as well as a nonvector species. The accepted interpretation of evolutionary relationships among these species is based on chromosomal inversions and suggests that the two principal vectors, A. gambiae and Anopheles arabiensis, are on distant branches of the phylogenetic tree. However, DNA sequence data indicate that these two species are sister taxa and suggest gene flow between them. These results have important implications for malaria control strategies involving the replacement of vector with nonvector populations.
JIP60, a methyl jasmonate-induced ribosome-inactivating protein involved in plant stress reactions.
Authors: Reinbothe S , Reinbothe C , Lehmann J , Becker W , Apel K , Parthier B
Published: May 2006

Plant tissues treated with the naturally occurring cyclopentanone compound methyl jasmonate or exposed to stress causing in planta jasmonate accumulation express distinctive proteins and, concomitantly, reduce the synthesis of most preexisting proteins. One of the recently identified jasmonate-induced proteins, designated JIP60, in barley is a ribosome-inactivating protein that cleaves polysomes of both animal and plant origin into their ribosomal subunits. By attacking foreign and self ribosomes, respectively, JIP60 appears to be both a defense protein and a potent regulator of protein synthesis in stressed plant tissues.
High-affinity urokinase receptor antagonists identified with bacteriophage peptide display.
Authors: Goodson R J , Doyle M V , Kaufman S E , Rosenberg S
Published: May 2006

Affinity selection of a 15-mer random peptide library displayed on bacteriophage M13 has been used to identify potent ligands for the human urokinase receptor, a key mediator of tumor cell invasion. A family of receptor binding bacteriophage ligands was obtained by sequentially and alternately selecting the peptide library on COS-7 monkey kidney cells and baculovirus-infected Sf9 insect cells overexpressing the human urokinase receptor. Nineteen peptides encoded by the random DNA regions of the selected bacteriophage were synthesized and tested in a urokinase receptor binding assay, where they competed with the labeled N-terminal fragment of urokinase with IC50 values ranging from 10 nM to 10 microM. All of the isolated peptides were linear and showed two relatively short conserved subsequences: LWXXAr (Ar = Y, W, F, or H) and XFXXYLW, neither of which is found in urokinase or its receptor. Competition experiments demonstrated that the most potent peptide, clone 20, prevented binding of bacteriophage displaying the urokinase receptor binding sequence (urokinase residues 13-32). In addition, this peptide blocked other apparently unrelated receptor binding bacteriophage, suggesting overlapping receptor interaction sites for all of these sequences. These results provide a demonstration of bacteriophage display identifying peptide ligands for a receptor expressed on cells and yield leads for the development of urokinase receptor antagonists.
Antiproliferative gastrin/cholecystokinin receptor antagonists target the 78-kDa gastrin-binding protein.
Authors: Baldwin G S
Published: May 2006

Inhibition of colon carcinoma cell growth by the nonselective gastrin/cholecystokinin (CCK) receptor antagonists proglumide and benzotript provided evidence that gastrin functions as an autocrine growth factor. However, the molecular properties of the receptor mediating the antagonist effects have not been identified. A 78-kDa gastrin-binding protein (GBP), the sequence of which is related to the family of enzymes possessing enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities, has been previously purified from porcine gastric mucosal membranes. I now report that covalent cross-linking of 125I-labeled [Nle15]gastrin2,17 to the 78-kDa GBP is inhibited by crotonyl-CoA and by acetoacetyl-CoA. Gastrin, CCK, and their analogues also inhibit cross-linking, and the spectrum of analogue affinities correlates better with the values previously reported for binding to the gastrin/CCK-C receptor than with the values reported for binding to either the CCK-A or the gastrin/CCK-B receptor. Cross-linking is also inhibited by proglumide and benzotript, but no inhibition is seen with either the CCK-A receptor-selective antagonist L364,718 or the gastrin/CCK-B receptor-selective antagonist L365,260. The affinities of antagonists for the GBP correlate well with their affinities for the gastrin/CCK-C receptor and with their potencies for inhibition of colon carcinoma cell growth. I conclude that the 78-kDa gastrin-binding protein is (i) a member of the hydratase/dehydrogenase family of fatty acid oxidation enzymes, (ii) the gastrin/CCK-C receptor, and (iii) the target for the antiproliferative action of two gastrin/CCK receptor antagonists.
A critical test of the role of population density in producing transformation.
Authors: Yao A , Rubin H
Published: May 2006

Cells of the NIH 3T3 line gain the capacity to produce neoplastically transformed foci when they are maintained at high density for more than 1 week and transferred in a standard assay for focus formation. This change in cell behavior has been variously attributed to an adaptive response to the constraint of the high population density or to a spontaneous genetic change that increases in probability for a culture with the increase in the total number of cell divisions. To distinguish between these alternatives, 200 cells of the 28H subline were seeded in many culture dishes of two size classes differing 6-fold in surface area and allowed to multiply for 1, 2, and 3 weeks. At each weekly interval, 18 dishes of each class were assayed for focus formation, and two of the original dishes were stained for focus formation. The cells in the small (S) and large (L) dishes multiplied to the same extent at 1 week and produced only a few small light foci in some of the assay dishes. At 2 weeks, cells in the S dishes had become confluent and had only one-third the number of cells as those in the nonconfluent L dishes. Upon assay, 14 of the 18 S cultures produced some foci whereas only 9 of the L cultures did so. In addition, 4 of the S cultures produced large dense foci while none of the L cultures did. By 3 weeks, the L cultures were confluent and had four times as many cells as the S cultures. When assayed at this time, both sets produced dense foci in many of the cultures and light foci in the remaining ones, indicating a narrowing of the differences between the S and L cultures between 2 and 3 weeks of incubation. There were differences in the morphology of the foci produced in parallel assays from different cultures. The results showed that transformation is a diverse graded response to the growth constraint of high population density and not a spontaneous event dependent on the number of cell divisions in a cell culture. Transformation thus is basically an epigenetic process since it represents a response to physiological restraint, but the final form of response may be modulated by genetic alterations.
The genome of the THE I human transposable repetitive elements is composed of a basic motif homologous to an ancestral immunoglobulin gene sequence.
Authors: Hakim I , Amariglio N , Grossman Z , Simoni-Brok F , Ohno S , Rechavi G
Published: May 2006

Amplification of rearranged human immunoglobulin heavy-chain genes using the polymerase chain reaction resulted unexpectedly in the amplification of human transposable repetitive element genomes. These were identified as members of the THE I (transposon-like human element I) transposable element family. Analysis of the THE I sequences revealed the presence of several copies of the ancestral building block described > 10 years ago by Ohno and coworkers as the primordial immunoglobulin sequence. The frequency and degree of homology of the repeats of the basic unit were similar for the two genes, as well as for two murine intracisternal A particles. These findings suggest that both the transposable genetic elements and the immunoglobulin genes originated from a common ancestral building block.
A parathyroid hormone antagonist stimulates epidermal proliferation and hair growth in mice.
Authors: Holick M F , Ray S , Chen T C , Tian X , Persons K S
Published: May 2006

The biologic action of parathyroid hormone (PTH)-related peptide (PTHrP) in normal skin was investigated in cultured human keratinocytes and in SKH-1 hairless mice. The results indicate that the PTHrP agonists human PTHrP-(1-34) and PTH(1-34) are potent inhibitors of epidermal cell proliferation. [Nle8,18,Tyr34]bovine PTH-(7-34)-amide, an antagonist of the PTH/PTHrP receptor, blocked the inhibitory effect of PTH-(1-34) in cultured keratinocytes. In the SKH-1 mice, PTH-(7-34) caused a 244% increase of [3H]thymidine incorporation into isolated epidermal DNA and 246% and 180% increases in the number and length of hair shafts, respectively. Thus, PTH and PTHrP may play an important role in the normal physiology of skin, and their agonists and antagonists have potentially wide therapeutic applications in the treatment of hyperproliferative skin disorders and aging skin and could also be effective in stimulating and maintaining hair growth.
Cyclin D1 induction in breast cancer cells shortens G1 and is sufficient for cells arrested in G1 to complete the cell cycle.
Authors: Musgrove E A , Lee C S , Buckley M F , Sutherland R L
Published: May 2006

The sequential transcriptional activation of cyclins, the regulatory subunits of cell-cycle-specific kinases, is thought to regulate progress through the cell cycle. Cyclins are therefore potential oncogenes, and cyclin D1 overexpression and/or amplification at its genomic locus, 11q13, are common features of several human cancers. Induction of cyclin D1 is an early response to mitogenic stimulation in several cell types, but the consequences of altered expression of this gene in human cells of epithelial origin remain undefined. We assessed the effects of alterations of cyclin D1 expression in human breast cancer cells by generating T-47D cells expressing human cyclin D1 under the control of a zinc-responsive metallothionein promoter. In cycling cells induction of cyclin D1 after zinc treatment resulted in an increase in the number of cells progressing through G1 and in the rate of transition from G1 to S phase, indicating that cyclin D1 is rate-limiting for progress through G1 phase. In cells arrested in early G1 phase after growth factor deprivation, zinc induction of cyclin D1 was sufficient for completion of the cell cycle, a process requiring growth factor stimulation in control cells. These data demonstrate a critical role for cyclin D1 in human breast cancer cell-cycle control and suggest that deregulated expression of cyclin D1 is likely to reduce dependence on normal physiological growth stimuli, thereby providing a growth advantage to tumor cells and a potential mechanism of resistance to endocrine therapy.
Nonpeptide ligands for human gamma delta T cells.
Authors: Tanaka Y , Sano S , Nieves E , De Libero G , Rosa D , Modlin R L , Brenner M B , Bloom B R , Morita C T
Published: May 2006

gamma delta T cells respond to a variety of microbial pathogens and transformed cells. Their limited receptor repertoire and activation by mycobacterial antigens resistant to proteases suggest that they may recognize nonpeptide antigens. We have tested a variety of nonpeptide molecules for stimulation of human gamma delta T cells. Synthetic alkyl phosphates, particularly monoethyl phosphate (MEP), selectively activated gamma delta T cells and stimulated their proliferation in vitro. All gamma delta T cells stimulated by MEP expressed V gamma 2/V delta 2 receptors. The purified natural ligand of mycobacteria is chemically similar to, though distinct from, MEP and contains a phosphate residue that is critical for biological activity. Recognition and expansion of a specific T-cell receptor-bearing population to non-peptide ligands is unprecedented among T cells. We suggest that MEP mimics small natural ligands capable of expanding one subset of gamma delta T cells and that this recognition of nonpeptide antigens may play an important role in human immunity to pathogens.
Alcohol action on a neuronal membrane receptor: evidence for a direct interaction with the receptor protein.
Authors: Li C , Peoples R W , Weight F F
Published: May 2006

For almost a century, alcohols have been thought to produce their effects by actions on the membrane lipids of central nervous system neurons--the well known "lipid theory" of alcohol action. The rationale for this theory is the correlation of potency with oil/water or membrane/buffer partition coefficient. Although a number of recent studies have shown that alcohols can affect the function of certain neuronal neurotransmitter receptors, there is no evidence that the alcohols interact directly with these membrane proteins. In the present study, we report that inhibition of a neuronal neurotransmitter receptor, an ATP-gated ion channel, by a series of alcohols exhibits a distinct cutoff effect. For alcohols with a molecular volume of < or = 42.2 ml/mol, potency for inhibiting ATP-activated current was correlated with lipid solubility (order of potency: 1-propanol = trifluoroethanol > monochloroethanol > ethanol > methanol). However, despite increased lipid solubility, alcohols with a molecular volume of > or = 46.1 ml/mol (1-butanol, 1-pentanol, trichloroethanol, and dichloroethanol) were without effect on the ATP-activated current. The results suggest that alcohols inhibit the function of this neurotransmitter receptor by interacting with a small hydrophobic pocket on the receptor protein.
Differential expression of mRNAs for JC virus large and small tumor antigens in brain tissues from progressive multifocal leukoencephalopathy patients with and without AIDS.
Authors: Ishaq M , Stoner G L
Published: May 2006

JC virus (JCV) causes progressive multifocal leukoencephalopathy (PML), the fatal demyelinating infection of oligodendrocytes, in up to 5% of AIDS patients. An intron-differential RNA PCR was developed to study the expression of alternately spliced JCV early mRNAs in brain tissues from PML patients with and without AIDS and in JCV-induced hamster brain tumors. The method utilizes primers that span the large tumor (T) and small tumor (t) antigen introns allowing amplification of specific cDNAs in the presence of contaminating viral genomic DNA. Hybridization with specific junctional probes and DNA sequence analysis confirmed the identity of the PCR products. Sequencing showed that JCV early mRNA is alternatively spliced as previously predicted by analogy to simian virus 40. Large T antigen mRNA was detected in all the brain tissues from PML patients with and without AIDS. The expression of small t antigen mRNA varied depending upon the association of PML with AIDS and upon other unknown factors. Of the 12 PML/AIDS brain tissue samples, 11 (92%) expressed small t antigen mRNA, whereas only 8 of 13 (62%) brain samples from patients with PML alone showed detectable levels of small t antigen mRNA. Human immunodeficiency virus 1 proviral DNA was detected in 10 of 12 PML/AIDS brain samples. The results indicate that alternative splicing of JCV early mRNA is regulated in the human brain and that the production of small t antigen may not be essential for the pathogenesis of PML.
Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activity.
Authors: Borchelt D R , Lee M K , Slunt H S , Guarnieri M , Xu Z S , Wong P C , Brown R H , Price D L , Sisodia S S , Cleveland D W
Published: May 2006

Familial amyotrophic lateral sclerosis (FALS) has been linked to mutations in the homodimeric enzyme Cu/Zn superoxide dismutase 1 (SOD1). Assay by transient expression in primate cells of six FALS mutant enzymes revealed a continuum of enzymatic activity bounded by the enzyme carrying the mutation Gly-85-->Arg, which was inactive, and mutant enzyme G37R carrying the Gly-37-->Arg change, which retained full specific activity but displayed a 2-fold reduction in polypeptide stability. The G37R mutant displayed similar properties in transformed lymphocytes from an individual heterozygous for the G37R and wild-type SOD1 genes; heterodimeric enzymes composed of mutant and wild-type subunits were detected, but there was no measurable diminution in the stability and activity of the wild-type subunits. Thus, for mutants such as G37R, either surprisingly modest losses in activity (involving only the mutant subunit) can yield motor neuron death, or alternatively, mutant SOD1 may acquire properties that injure motor neurons by one or more mechanisms unrelated to the metabolism of oxygen radicals.
Transthyretin sequesters amyloid beta protein and prevents amyloid formation.
Authors: Schwarzman A L , Gregori L , Vitek M P , Lyubski S , Strittmatter W J , Enghilde J J , Bhasin R , Silverman J , Weisgraber K H , Coyle P K
Published: May 2006

The cardinal pathological features of Alzheimer disease are depositions of aggregated amyloid beta protein (A beta) in the brain and cerebrovasculature. However, the A beta is found in a soluble form in cerebrospinal fluid in healthy individuals and patients with Alzheimer disease. We postulate that sequestration of A beta precludes amyloid formation. Failure to sequester A beta in Alzheimer disease may result in amyloidosis. When we added A beta to cerebrospinal fluid of patients and controls it was rapidly sequestered into stable complexes with transthyretin. Complexes with apolipoprotein E, which has been shown to bind A beta in vitro, were not observed in cerebrospinal fluid. Additional in vitro studies showed that both purified transthyretin and apolipoprotein E prevent amyloid formation.
Nerve growth factor-induced ocular dominance plasticity in adult cat visual cortex.
Authors: Gu Q , Liu Y , Cynader M S
Published: May 2006

Activity-dependent modifiability of cortical ocular dominance occurs only during early postnatal life, within the so-called "critical period," but not thereafter in adult visual cortex. To examine the role of neurotrophins in the activity- and age-dependent stimulation-induced modifiability of visual cortex, we tested whether intracortical infusion of nerve growth factor could induce ocular dominance plasticity in adult visual cortex. Nerve growth factor was continuously infused, by means of osmotic minipumps, into striate cortex of adult cats for 2 weeks. At the time of minipump implantation, one eyelid of the experimental animals was sutured closed. After 3 weeks of monocular deprivation, the ocular dominance distribution of neurons in the striate cortex was assessed using single unit recording. We found that monocular deprivation imposed on adult animals in conjunction with nerve growth factor infusion causes an ocular dominance shift toward the deprived eye. Although the underlying mechanisms remain uncertain, the results indicate that nerve growth factor can enhance activity-dependent synaptic modification and remodeling in adult visual cortex.
Two distinctly regulated events, priming and triggering, during retinoid-induced maturation and resistance of NB4 promyelocytic leukemia cell line.
Authors: Ruchaud S , Duprez E , Gendron M C , Houge G , Genieser H G , Jastorff B , Doskeland S O , Lanotte M
Published: May 2006

In t(15;17) acute promyelocytic leukemia, all-trans retinoic acid (RA) induces leukemic cell maturation in vitro and remission in acute promyelocytic leukemia patients, but in vivo treatments invariably lead to relapse with resistance to RA. NB4, a maturation-inducible cell line, and NB4-RAr sublines (R1 and R2) displaying no maturation in the presence of RA have been isolated from a patient in relapse. We show that resistance to maturation is not a mere unresponsiveness to RA: rather, R1 "resistant" cells do respond to RA (1 microM) by sustained growth, become competent to undergo terminal maturation, and up-regulate CD11c/CD18 integrins. Interestingly, maturation of "resistant" cells, rendered competent by RA, can be achieved by cAMP-elevating agents (prostaglandin E, isoproterenol, cholera toxin, or phosphodiesterase inhibitor) or stable agonistic cAMP analogs such as (SP)-8-chloroadenosine cyclic 3',5'-phosphorothioate. This shows that activation of cAMP-dependent protein kinase (cA kinase) can override the RA resistance and suggests interdependent RA and cAMP signaling pathways in acute promyelocytic leukemia maturation. No such cooperation was observed in the R2 resistant cells, though their cA-kinase was functional. (RP)-8-Chloroadenosine cyclic 3',5'-phosphorothioate, which by displacing endogenous cAMP inhibits the basal cA-kinase activity, decreased the response of sensitive cells to RA. This raises the possibility that cA-kinase plays a key role in the maturation also of RA-sensitive cells. Our results define two discrete steps in the maturation process: an RA-dependent priming step that maintains proliferation while cells become competent to undergo maturation in response to retinoids and a cAMP-dependent step that triggers RA-primed cells to undergo terminal maturation. Uncoupling RA and cAMP action might cause the so-called "resistance."
Amygdala modulation of hippocampal-dependent and caudate nucleus-dependent memory processes.
Authors: Packard M G , Cahill L , McGaugh J L
Published: May 2006

These experiments investigated the effects, on memory, of injections of d-amphetamine (10 micrograms/0.5 microliter) administered into the amygdala, hippocampus, or caudate nucleus immediately after training in cued or spatial water-maze tasks. In experiment 1, rats received an eight-trial training session on one of the two tasks followed by injections of d-amphetamine or saline. Retention was tested 24 hr later. On the spatial task, intrahippocampal, but not intracaudate, injections of d-amphetamine facilitated retention. In contrast, on the cued task intracaudate, but not intrahippocampal, injections of d-amphetamine facilitated retention. Posttraining intraamygdala injections of d-amphetamine enhanced retention of both tasks. In experiment 2, lidocaine (2% solution; 1.0 microliter) injected intraamygdally prior to the retention test did not block the memory enhancement induced by posttraining intraamygdala injections of d-amphetamine. The findings (i) provide further evidence of a dissociation between the roles of the hippocampus and caudate nucleus in different forms of memory, (ii) indicate that the modulatory role of the amygdala is not limited to either of the two different forms of memory represented in spatial and cued discriminations in a water maze, and (iii) are consistent with previous findings indicating that amygdala influences on memory storage are not mediated by lasting neural changes located within the amygdala.
Evolution of the Glx-tRNA synthetase family: the glutaminyl enzyme as a case of horizontal gene transfer.
Authors: Lamour V , Quevillon S , Diriong S , N'Guyen V C , Lipinski M , Mirande M
Published: May 2006

An important step ensuring the fidelity in protein biosynthesis is the aminoacylation of tRNAs by aminoacyl-tRNA synthetases. The accuracy of this process rests on a family of 20 enzymes, one for each amino acid. One exception is the formation of Gln-tRNA(Gln) that can be accomplished by two different pathways: aminoacylation of tRNA(Gln) with Gln by glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) or transamidation of Glu from Glu-tRNA(Gln) mischarged by glutamyl-tRNA synthetase (GluRS; EC 6.1.1.17). The latter pathway is widespread among bacteria and organelles that, accordingly, lack GlnRS. However, some bacterial species, such as Escherichia coli, do possess a GlnRS activity, which is responsible for Gln-tRNA(Gln) formation. In the cytoplasm of eukaryotic cells, both GluRS and GlnRS activities can be detected. To gain more insight into the evolutionary relationship between GluRS and GlnRS enzyme species, we have now isolated and characterized a human cDNA encoding GlnRS. The deduced amino acid sequence shows a strong similarity with other known GlnRSs and with eukaryotic GluRSs. A molecular phylogenetic analysis was conducted on the 14 GlxRS (GluRS or GlnRS) sequences available to date. Our data suggest that bacterial GlnRS has a eukaryotic origin and was acquired by a mechanism of horizontal gene transfer.
Using bulked extremes and recessive class to map genes for photoperiod-sensitive genic male sterility in rice.
Authors: Zhang Q , Shen B Z , Dai X K , Mei M H , Saghai Maroof M A , Li Z B
Published: May 2006

Photoperiod-sensitive genic male sterile (PS-GMS) rice has a number of desirable characteristics for hybrid rice production. In this study we made use of a published rice genetic linkage map to determine the locations of PSGMS genes and we have characterized the effects of these genes on sterility by using molecular markers. A two-step approach was designed for mapping the genes: (i) identifying possible PSGMS gene-containing chromosome regions with bulked DNA from extreme fertile and extreme sterile plants of a very large F2 population and (ii) determining the map locations of the genes in extreme sterile individuals. We show that this mapping method is much more cost effective and statistically efficient than using a random sample of an F2 population. We identified two chromosomal regions each containing a PSGMS locus, one designated pms1 on chromosome 7 and one designated pms2 on chromosome 3. The existence of these two loci was confirmed by a large sample assay and with data on rationing progenies of the F2 plants. A marker-based analysis shows that the effect of pms1 is 2-3 times larger than that of pms2 and that dominance is almost complete at both loci. Implications in the breeding of PSGMS rice lines are discussed.
Atomic-level accuracy in simulations of large protein crystals.
Authors: York D M , Wlodawer A , Pedersen L G , Darden T A
Published: May 2006

Proper treatment of long-range Coulombic forces presents a major obstacle to providing realistic molecular dynamics simulations of macromolecules. Traditional approximations made to lessen computational cost ultimately lead to unrealistic behavior. The particle mesh Ewald method accommodates long-range Coulombic forces accurately and efficiently by use of fast Fourier transform techniques. We report a 1-ns simulation of bovine pancreatic trypsin inhibitor in a crystal unit cell using the particle mesh Ewald methodology. We find an rms backbone deviation from the x-ray structure (0.33 A) that is lower than that observed between bovine pancreatic trypsin inhibitor in different crystal forms and much lower than those of previous simulations. These results bridge the gap between structures obtained from molecular simulation and those from experiment.
CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program.
Authors: Lin F T , Lane M D
Published: May 2006

Previous studies showed that CCAAT/enhancer binding protein alpha (C/EBP alpha) is required for differentiation of 3T3-L1 preadipocytes induced by exogenous hormonal agents. It was not possible to ascertain, however, whether C/EBP alpha alone is sufficient to induce differentiation because its antimitogenic activity precluded propagating 3T3-L1 cell lines that constitutively express C/EBP alpha at high levels. This problem was circumvented by using 3T3-L1 preadipocytes stably transfected with an isopropyl beta-D-thiogalactoside (IPTG)-inducible p42 C/EBP alpha expression vector system. IPTG-induced expression of the 42-kDa isoform of C/EBP alpha in preadipocytes caused expression of several endogenous adipocyte-specific genes (genes encoding the 422 adipose P2 protein, glucose transporter 4, and C/EBP alpha) and the accumulation of cytoplasmic triglyceride. Thus, C/EBP alpha is not only necessary but also is sufficient to trigger differentiation of growth-arrested 3T3-L1 preadipocytes without use of exogenous hormonal agents.
Integrin alpha v beta 3 rescues melanoma cells from apoptosis in three-dimensional dermal collagen.
Authors: Montgomery A M , Reisfeld R A , Cheresh D A
Published: May 2006

Human melanoma cells required ligation of the integrin alpha v beta 3 to sustain viability and growth in three-dimensional dermal collagen. Variant melanoma cells, lacking the alpha v subunit, progressed rapidly to apoptosis within this matrix, whereas transfection of these cells with an alpha v cDNA restored alpha v beta 3 expression and prevented apoptosis. Furthermore, inhibition of alpha v beta 3 ligation with a monoclonal antibody promoted cell death. Apoptosis of alpha v(-) cells within this matrix could be overcome by the addition of insulin or serum. However, alpha v(+) melanoma cells had a significant growth advantage in the presence of these growth factors. Initial adhesion of the melanoma cells to type I collagen depended on ligation of alpha 2 beta 1, but these cells can degrade this collagen to expose cryptic alpha v beta 3 binding sites. These findings provide evidence that the survival and growth of transformed cells may be regulated by collagen degradation and integrin-dependent anchorage to this proteolysed matrix.
Heteromeric olfactory cyclic nucleotide-gated channels: a subunit that confers increased sensitivity to cAMP.
Authors: Bradley J , Li J , Davidson N , Lester H A , Zinn K
Published: May 2006

Olfactory receptor neurons respond to odorant stimulation with a rapid increase in intracellular cAMP that opens cyclic nucleotide-gated (cng) cation channels. cng channels in rat olfactory neurons are activated by cAMP in the low micromolar range and are outwardly rectifying. The cloned rat olfactory cng channel (rOCNC1), however, is much less sensitive to cAMP and exhibits very weak rectification. Here we describe the cloning and characterization of a second rat cng channel subunit, denoted rOCNC2. rOCNC2 does not form functional channels when expressed alone. When rOCNC1 and rOCNC2 are coexpressed, however, an outwardly rectifying cation conductance with cAMP sensitivity near that of the native channel is observed. In situ hybridization with probes specific for the two subunits shows that they are coexpressed in olfactory receptor neurons. These data indicate that the native olfactory cng channel is likely to be a heterooligomer of the rOCNC1 and rOCNC2 subunits.
BCL2 translocation frequency rises with age in humans.
Authors: Liu Y , Hernandez A M , Shibata D , Cortopassi G A
Published: May 2006

The background frequency of t(14;18) (q32;q21) chromosomal translocations at the locus associated with B-cell leukemia/lymphoma-2 (BCL2) was determined from a survey of the peripheral blood lymphocytes (PBLs) of 53 living individuals and from tissues of 31 autopsies by using a nested PCR assay. The translocation was detected in 55% of PBLs and 35% of autopsied spleens with a frequency of between less than 1 to 853 translocations per million cells. Translocations copurified with B lymphocytes. The frequency of translocations significantly increased with age in PBLs and spleens, as does human risk for lymphoma. Average translocation frequency was more than 40 times greater in the spleen and 13 times greater in the peripheral blood in the oldest individuals (61 yr and older) compared with the youngest individuals (20 yr or younger). Particular t(14;18)-bearing clones persisted over a period of 5 months in two individuals. These findings demonstrate that clones harboring the oncogenic t(14;18) chromosomal translocation are commonly present in normal humans, that such clones are long-lived, and that they rise in frequency with age. A multihit model of lymphomagenesis involving t(14;18) translocation followed by antigen stimulation is proposed.
Isolation of phytoalexin-deficient mutants of Arabidopsis thaliana and characterization of their interactions with bacterial pathogens.
Authors: Glazebrook J , Ausubel F M
Published: May 2006

A genetic approach was used to assess the extent to which a particular plant defense response, phytoalexin biosynthesis, contributes to Arabidopsis thaliana resistance to Pseudomonas syringae pathogens. The A. thaliana phytoalexin, camalexin, accumulated in response to infection by various P. syringae strains. No correlation between pathogen avirulence and camalexin accumulation was observed. A biochemical screen was used to isolate three mutants of A. thaliana ecotype Columbia that were phytoalexin deficient (pad mutants). The mutations pad1, pad2, and pad3 were found to be recessive alleles of three different genes. pad1 and pad2 were mapped to chromosome IV and pad3 was mapped to chromosome III. Infection of pad mutant plants with strains carrying cloned avirulence genes revealed that the pad mutations did not affect the plants' ability to restrict the growth of these strains. This result strongly suggests that in A. thaliana, phytoalexin biosynthesis is not required for resistance to avirulent P. syringae pathogens. Two of the pad mutants displayed enhanced sensitivity to isogenic virulent P. syringae pathogens, suggesting that camalexin may serve to limit the growth of virulent bacteria.
An in vitro polysome display system for identifying ligands from very large peptide libraries.
Authors: Mattheakis L C , Bhatt R R , Dower W J
Published: May 2006

We have used an in vitro protein synthesis system to construct a very large library of peptides displayed on polysomes. A pool of DNA sequences encoding 10(12) random decapeptides was incubated in an Escherichia coli S30 coupled transcription/translation system. Polysomes were isolated and screened by affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin B. The mRNA from the enriched pool of polysomes was recovered, copied into cDNA, and amplified by the polymerase chain reaction (PCR) to produce template for the next round of in vitro synthesis and selection. A portion of the amplified template from each round was cloned into a filamentous phagemid vector to determine the specificity of peptide binding by phage ELISA and to sequence the DNA. After four rounds of affinity selection, the majority of clones encoded peptides that bound specifically to the antibody and contained a consensus sequence that is similar to the known epitope for the antibody. Synthetic peptides corresponding to several of these sequences have binding affinities ranging from 7 to 140 nM. The in vitro system described here has the potential to screen peptide libraries that are three to six orders of magnitude larger than current biological peptide display systems.
A cytolysin encoded by Salmonella is required for survival within macrophages.
Authors: Libby S J , Goebel W , Ludwig A , Buchmeier N , Bowe F , Fang F C , Guiney D G , Songer J G , Heffron F
Published: May 2006

A Salmonella gene encoding a cytolysin has been identified by screening for hemolysis on blood agar. DNA sequence analyses together with genetic mapping in Salmonella suggest that it is unrelated to other toxins or hemolysins. The gene (slyA) is present in every strain of Salmonella examined, in Shigella, and in enteroinvasive Escherichia coli but not in other Enterobacteriaceae. SlyA (salmolysin) purified from a derivative of the original clone has hemolytic and cytolytic activity and has a molecular weight predicted by the DNA sequence. The median lethal dose and infection kinetics in mice suggest that the toxin is required for virulence and facilitates Salmonella survival within mouse peritoneal macrophages.
The parasitophorous vacuole membrane surrounding intracellular Toxoplasma gondii functions as a molecular sieve.
Authors: Schwab J C , Beckers C J , Joiner K A
Published: May 2006

The obligate intracellular protozoan parasite Toxoplasma gondii creates and enters into a unique membrane-bounded cytoplasmic compartment, the parasitophorous vacuole, when invading mammalian host cells. By microinjecting polar fluorescent molecules into individual T. gondii-infected fibroblasts, we show here that the parasitophorous vacuole membrane (PVM) surrounding the parasite functions as a molecular sieve. Lucifer yellow (457 Da) displayed free bidirectional flux across the PVM and distinctly outlined the parasites, which did not take up the dye, within the vacuole. This dye movement was not appreciably delayed by pretreatment of cells with 5 mM probenecid or chilling the monolayer to 5 degrees C, suggesting that dye movement was due to passive permeation through a membrane pore rather than active transport. Calcein, fluo-3, and a series of fluorescein isothiocyanate-labeled peptides up to 1291 Da crossed the PVM in a size-restricted fashion. A labeled peptide of 1926 Da and labeled dextrans and proteins (> or = 3000 Da) failed to transit the PVM. This putative channel in the PVM therefore allows exchange of molecules up to 1300-1900 Da between the host cell cytoplasm and the parasitophorous vacuolar space.