bluuebunny/crossref_metadata_2025 huggingface dataset:
Illegitimate recombination induced by benzo[a]pyrene diol epoxide in Escherichia coli.
Authors: Kokontis J M , Vaughan J , Harvey R G , Weiss S B
Published: May 2006

Duplex DNA oligomer constructs (32 base pairs) were prepared that contained a single benzo[a]pyrene (BP) adduct at a specific deoxyadenosine or deoxyguanosine site in either one or both strands. These constructs were inserted into M13 replicative form viral DNA, and the DNA from progeny virus generated by transfection of Escherichia coli was examined by sequence analysis at the site of oligomer insertion. With nonalkylated constructs, and with constructs containing only one BP adduct, no sequence alterations were found in progeny viral DNAs. With constructs containing two BP adducts, one in each strand and closely spaced, some progeny DNAs showed the original oligomer sequence, whereas others exhibited large deletions and illegitimate (nonhomologous) recombination, both of which removed the damaged construct. Increasing the distance between BP adducts in the construct reduced the frequency of recombinant events. These sequence alterations occurred in both recA+ and recA- host cells. We speculate that the closely spaced adducts in opposite construct strands cause a rare distortion in DNA structure, which activates the recombinant machinery, and that mutagenic and carcinogenic agents other than polycyclic aromatic hydrocarbons may cause similar DNA distortions, which induce illegitimate recombination.
A 60-kDa cytoskeletal protein from Trypanosoma brucei brucei can interact with membranes and with microtubules.
Authors: Seebeck T , Küng V , Wyler T , Müller M
Published: May 2006

The cytoskeleton of eukaryotic cells is a major determinant of cellular architecture and of many cellular functions. In addition to or in place of the transcellular cytoskeleton, many eukaryotic cells also contain membrane-associated cytoskeletal structures (membrane skeletons), which are important for cellular structure and function. The membrane skeleton of the parasitic hemoflagellate Trypanosoma brucei consists of a dense array of singlet microtubules (subpellicular microtubules), which are tightly associated to the overlying cell membrane. This study reports the identification of a microtubule-associated protein from Trypanosoma brucei that constitutes a component of the link between this microtubular array and the cell membrane. The protein can bind in vitro both to microtubules and to membrane vesicles or liposomes. Furthermore, it can crosslink microtubules and membrane vesicles, suggesting that it exerts a similar function in the membrane skeleton.
Inoculation of newborn SWR/J females with an ecotropic murine leukemia virus can produce transgenic mice.
Authors: Panthier J J , Condamine H , Jacob F
Published: May 2006

Endogenous ecotropic murine leukemia proviruses that were not present in the parental stock are acquired by the progeny of some SWR/J X RF/J hybrid females. We have made a stock of an ecotropic murine leukemia virus produced by such a hybrid female and inoculated newborn SWR/J females with it. We show that upon crossing of the inoculated females to SWR/J males, some of their progeny acquire ecotropic proviruses. Although most of these proviruses appear to be distributed in somatic tissues in a mosaic way, some are transmitted through the germ line. Thus an exogenous infection is able to mimic the phenomenon observed in SWR/J X RF/J hybrid mice. Available evidence suggests that this infection occurs during oogenesis in the recipient female. Our results document the conversion of an exogenous infectious ecotropic murine leukemia virus to an endogenous provirus without any manipulation of either eggs or embryos.
Glucocorticoids selectively inhibit the transcription of the interleukin 1 beta gene and decrease the stability of interleukin 1 beta mRNA.
Authors: Lee S W , Tsou A P , Chan H , Thomas J , Petrie K , Eugui E M , Allison A C
Published: May 2006

Transcription of the interleukin 1 beta (IL-1 beta) gene was studied by mRNA hybridization with a cDNA probe in the human promonocytic cell line U-937. Phorbol ester and lipopolysaccharide increased the steady-state level of IL-1 beta mRNA. Glucocorticoids markedly decreased IL-1 beta mRNA levels by two mechanisms. Transcription of the IL-1 gene was inhibited, as shown by in vitro transcription assays with nuclei isolated from glucocorticoid-treated cells. Moreover, kinetic analyses and pulse-labeling of mRNAs showed that glucocorticoids selectively decrease the stability of IL-1 beta mRNA, without affecting the stability of beta-actin and FOS mRNAs. Inhibition of the formation and effects IL-1 is a mechanism by which glucocorticoids can exert antiinflammatory and immunosuppressive effects.
Receptor-mediated rapid action of 1 alpha,25-dihydroxycholecalciferol: increase of intracellular cGMP in human skin fibroblasts.
Authors: Barsony J , Marx S J
Published: May 2006

The intracellular cGMP concentration in normal human cultured fibroblasts was increased 2- to 3-fold by 1 alpha,25-dihydroxycholecalciferol [1 alpha,25-(OH)2D3] in a dose-dependent manner between 0.01 nM and 1 microM. The response was detectable within 1 min, reached a maximum (225% +/- 8% of baseline) at 6-8 min, and was no longer detectable at 30 min. The half-maximal effect of 1 alpha,25-(OH)2D3 was at 1.8 nM, and 24,25-dihydroxycholecalciferol showed an estimated EC50 100-fold higher. 1 beta,25-Dihydroxycholecalciferol, 25-hydroxycholecalciferol, and cholecalciferol had no detectable effect. Human skin fibroblasts with three different types of 1 alpha,25-(OH)2D3 receptor defect did not respond to 1 alpha,25-(OH)2D3 exposure with cGMP increase; however, the same cells (like normal cells) responded to testosterone or sodium nitroprusside with a rapid rise of cGMP. We conclude that the rapid rise of cGMP in response to calciferols shows an EC50 for 1 alpha,25-(OH)2D3, a cholecalciferol analog specificity, and a cell line dependency that are all suggestive of mediation through a specific 1 alpha,25-(OH)2D3 receptor.
Gordan—Capelli series in superalgebras
Authors: Brini Andrea , Palareti Aldopaolo , Teolis Antonio G. B.
Published: May 2006

We derive two Gordan—Capelli series for the supersymmetric algebra of the tensor product of two [unk]
2
-graded [unk]-vector spaces
U
and
V
, being [unk] a field of characteristic zero. These expansions yield complete decompositions of the supersymmetric algebra regarded as a pl(
U
)- and a pl(
V
)- module, where pl(
U
) and pl(
V
) are the general linear Lie superalgebras of
U
and
V
, respectively.
Tn10-encoded tet repressor can regulate an operator-containing plant promoter.
Authors: Gatz C , Quail P H
Published: May 2006

The Tn10-encoded tet repressor-operator system was used to regulate transcription from the cauliflower mosaic virus (CaMV) 35S promoter. Expression was monitored in a transient assay system by using electric field-mediated gene transfer ("electroporation") into tobacco protoplasts. The tet repressor, being expressed in the plant cells under the control of eukaryotic transcription signals, blocks transcription of a CaMV 35S promoter chloramphenicol acetyltransferase (cat) fusion gene when the two tet operators flank the "TATA" box. In the presence of the inducer tetracycline, expression is restored to full activity. Location of the operators 21 base pairs downstream of the transcription start site does not significantly affect transcription in the presence of the repressor. These experiments show that a prokaryotic regulatory protein can function in plant cells. The tet repressor-operator complex may be useful for specifically inducing transferred genes at different stages of plant development.
Phosphorylation of synthetic random polypeptides by protein kinase P and other protein-serine (threonine) kinases and stimulation or inhibition of kinase activities by microbial toxins.
Authors: Abdel-Ghany M , Raden D , Racker E , Katchalski-Katzir E
Published: May 2006

A synthetic random polymer of threonine and glutamate (1:4.4) is readily phosphorylated by protein kinase P but not by five other protein-serine (threonine) kinases. A synthetic random polymer of serine and arginine (1:3) is readily phosphorylated by protein kinase A and protein kinase C but not by protein kinase P. Although the amino acid sequences surrounding the phosphorylated serine (threonine) residue have been demonstrated in studies with small synthetic polypeptides to be decisive factors in the rate at which they are phosphorylated, the findings with the large synthetic polypeptides suggest that in the case of proteins the size, the tertiary structure, and particularly the electrostatic interactions are equally or more important contributing factors. Syringomycin, a toxin from Pseudomonas syringae, and polymyxin B, from Bacillus polymyxa, stimulate protein kinase P, strongly inhibit protein kinase C, and have no effect on protein kinase A. Basic polypeptides with high lysine content are phosphorylated by ATP nonenzymatically.
Detection of activated double-stranded RNA-dependent protein kinase in 3T3-F442A cells.
Authors: Petryshyn R , Chen J J , London I M
Published: May 2006

We have previously reported that cultured mouse 3T3-F442A cells exhibit a transient, double-stranded RNA (dsRNA)-dependent phosphorylation of the dsRNA-dependent eIF-2 alpha kinase (eIF-2 alpha, alpha-subunit of the eukaryotic initiation factor 2) (dsI). When dsI is activated by low levels of dsRNA, it is a potent inhibitor of protein synthesis. The transient expression of dsI is due to an autocrine effect of interferon at specific stages of growth and differentiation, and it may represent a mechanism for regulating cell growth and differentiation in 3T3-F442A cells. In this report, the purification of dsI from 3T3-F442A cell cultures by a two-step procedure is described. A specific immune serum to dsI was prepared by immunizing a rabbit with highly pure preparations. Immune precipitation studies demonstrate that the serum reacts with phosphorylated dsI both in vitro and in vivo and with de novo synthesized dsI after induction with interferon. We find that dsI of 3T3 cells can undergo phosphorylation in vitro without the addition of dsRNA and in vivo in the absence of viral infection. These results are consistent with a physiologic role for dsI in the growth and differentiation of these cells.
A gene activated by growth factors is related to the oncogene v-jun.
Authors: Ryder K , Lau L F , Nathans D
Published: May 2006

We have recently identified by cDNA cloning a set of genes that are rapidly activated in cultured mouse cells by protein growth factors. Here we report that the nucleotide sequence of a cDNA (clone 465) derived from one of these immediate early genes (hereafter called jun-B) encodes a protein homologous to that encoded by the avian sarcoma virus 17 oncogene v-jun. Homology between the jun-B and v-jun proteins is in two regions: one near the N terminus and the other at the C terminus. The latter sequence was shown by Vogt et al. [Vogt, P. K., Bos, T. J. & Doolittle, R. F. (1987) Proc. Natl. Acad. Sci. USA 84, 3316-3319] to have regions of sequence similarity to the DNA-binding domain of the yeast transcriptional regulatory protein GCN4 and to the oncogenic protein fos. Southern blots of human, mouse, and chicken DNA demonstrate that jun-B and c-jun are different genes and that there may be other vertebrate genes related to jun-B and c-jun. These findings suggest that there is a jun family of genes encoding related transcriptional regulatory proteins. The jun-B protein, and perhaps other members of the jun family, may play a role in regulating the genomic response to growth factors.
Targeted mutations induced by a single acetylaminofluorene DNA adduct in mammalian cells and bacteria.
Authors: Moriya M , Takeshita M , Johnson F , Peden K , Will S , Grollman A P
Published: May 2006

Mutagenic specificity of 2-acetylaminofluorene (AAF) has been established in mammalian cells and several strains of bacteria by using a shuttle plasmid vector containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene (C8-dG-AAF) adduct. The nucleotide sequence of the gene conferring tetracycline resistance was modified by conservative codon replacement so as to accommodate the sequence d(CCTTCGCTAC) flanked by two restriction sites, Bsm I and Xho I. The corresponding synthetic oligodeoxynucleotide underwent reaction with 2-(N-acetoxy-N-acetylamino)-fluorene (AAAF), forming a single dG-AAF adduct. This modified oligodeoxynucleotide was hybridized to its complementary strand and ligated between the Bsm I and Xho I sites of the vector. Plasmids containing the C8-dG-AAF adduct were used to transfect simian virus 40-transformed simian kidney (COS-1) cells and to transform several AB strains of Escherichia coli. Colonies containing mutant plasmids were detected by hybridization to 32P-labeled oligodeoxynucleotides. Presence of the single DNA adduct increased the mutation frequency by 8-fold in both COS cells and E. coli. Over 80% of mutations detected in both systems were targeted and represented G.C----C.G or G.C----T.A transversions or single nucleotide deletions. We conclude that modification of a deoxyguanosine residue with AAF preferentially induces mutations targeted at this site when a plasmid containing a single C8-dG-AAF adduct is introduced into mammalian cells or bacteria.
Retroviral transfer of a murine cDNA for multidrug resistance confers pleiotropic drug resistance to cells without prior drug selection.
Authors: Guild B C , Mulligan R C , Gros P , Housman D E
Published: May 2006

We have constructed a retrovirus expression vector that carries the murine mdr cDNA transcribed under the control of the human H4 histone promoter to examine the feasibility of efficiently transferring a multidrug resistance phenotype to cells without requiring drug selection. This approach will facilitate the transfer of mdr cDNA to hematopoietic progenitor cells for the study of multidrug resistance in vivo. The retrovirus vector pHmdr has been used for transmission and expression of the mdr cDNA in initially drug-sensitive NIH 3T3 fibroblasts. Selection of pHmdr infectants in the cytotoxic agents colchicine or doxorubicin gave rise to highly multidrug-resistant colonies containing a single gene copy of the vector. Moreover, in the analysis of 12 cloned unselected NIH 3T3 cell infectants, a multidrug resistance phenotype was conferred by as few as two copies of the pHmdr vector. Overexpression of the mdr cDNA in drug-selected and unselected pHmdr infectants was directly related to cell survival in three cytotoxic agents tested. These results hold significant implications for the study of multidrug resistance in vivo.
Effects of glycyl-L-glutamine in vitro on the molecular forms of acetylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.
Authors: Koelle G B , Massoulié J , Eugène D , Melone M A
Published: May 2006

Normal and preganglionically denervated cat superior cervical ganglia were sectioned and cultured for 24 or 48 hr, with or without preliminary inactivation of acetylcholinesterase, and in the presence or absence of 10(-5) M glycyl-L-glutamine. They were then homogenized, and the molecular forms of acetylcholinesterase were analyzed by sucrose gradient sedimentation. We observed an increased proportion of the globular monomeric G1 form, and to a lesser extent of the dimeric G2 and tetrameric membranous G4 forms, of acetylcholinesterase in the glycyl-L-glutamine-treated compared with the control cultures. There was only a small increase in the total acetylcholinesterase activity and no significant variation in the activity of the metabolic enzyme lactate dehydrogenase. It therefore seems likely that glycyl-L-glutamine, or the endogenous neurotrophic factor, maintains acetylcholinesterase in the preganglionically denervated ganglia in vivo by specifically increasing the biosynthesis of the monomeric G1 form, but not that of other proteins; these trophic factors do not seem to promote the polymerization of G1 into the more complex G2 and G4 forms.
Repair of O-alkylpyrimidines in mammalian cells: a present consensus.
Authors: Brent T P , Dolan M E , Fraenkel-Conrat H , Hall J , Karran P , Laval L , Margison G P , Montesano R , Pegg A E , Potter P M
Published: May 2006

Enzymatic repair of the O-alkylpyrimidines (O2- and O4-alkylthymine, O2-alkylcytosine) and alkyl phosphotriesters has been studied in Escherichia coli, and the two proteins involved, a glycosylase (DNA-3-methyladenine glycosylase) and a methyltransferase (DNA-O6-methylguanine:protein-L-cysteine S-methyltransferase, EC 2.1.1.63), have been well characterized. In mammals or mammalian cells treated with carcinogenic alkylating agents, loss of these derivatives has been demonstrated repeatedly. Nevertheless, mammalian repair proteins that are analogous to those from E. coli do not detectably act on these alkyl derivatives. A variety of techniques has been used by many investigators in the United States and Europe, who conclude here that the mode of O-alkylpyrimidine and alkyl phosphotriester repair in mammalian cells differs from that in E. coli. New approaches and methods are needed to characterize these processes at the biochemical and molecular level.
Binding sites of the 19-kDa and 68/72-kDa signal recognition particle (SRP) proteins on SRP RNA as determined in protein-RNA "footprinting".
Authors: Siegel V , Walter P
Published: May 2006

We have used the nuclease alpha-sarcin to map the binding sites of the 19-kDa and the 68/72-kDa proteins of signal recognition particle (SRP) on SRP RNA. We found that the regions of protection to nuclease afforded by the two proteins were distinct. p19 protected primarily the two tips in the RNA secondary structure. p68/72 protected a large region extending across the center of the particle and altered the nuclease pattern in the regions that p19 would bind, suggesting that these two proteins may be in close proximity in the particle. The protection afforded by the two proteins in combination was equal to the sum of the individual protections. We have not observed cooperativity in the binding of these two proteins as assessed by the protection assay; nor do we have any evidence that the structure becomes more compact as it assembles. The map derived from this "footprint" analysis places the signal recognition domain (p54 bound to the RNA via the 19-kDa protein) and the elongation arrest domain (associated with the Alu end of the particle) on opposite ends of the particle. Thus, it is possible that SRP recognizes signals by the direct interaction of p54 with the signal sequence at the nascent chain exit site and simultaneously blocks elongation by the entrance of p9/14 into the aminoacyl tRNA site 16 nm away.
Leukocyte chemoattraction by 1,2-diacylglycerol.
Authors: Wright T M , Hoffman R D , Nishijima J , Jakoi L , Snyderman R , Shin H S
Published: May 2006

Previous reports have demonstrated the hydrolysis of inositol phospholipids in polymorphonuclear leukocytes (PMN) in response to chemoattractants and in lymphocytes in response to the mitogen phytohemagglutinin. We investigated the role of 1,2-diacylglycerol, one of the products of receptor-linked phosphatidylinositol hydrolysis, in mediating the migratory response of leukocytes. In an under-agarose migration system, we found 1,2-dioctanoylglycerol to be a strong chemoattractant for human PMN, 6C3HED (a mouse thymic lymphoma), and Jurkat (a human T-cell leukemia). By using a modified Boyden chamber assay, the migratory response of PMN to 1,2-dioctanoylglycerol was determined to be primarily chemotactic. Analysis of structural analogs indicated that both the position and number of acyl chains are important in determining chemoattractant activity. These studies demonstrate that exogenous 1,2-diacylglycerol can stimulate the directed migration of leukocytes. They further suggest that the formation of 1,2-diacylglycerol following receptor-mediated stimulation may represent a common step in the migratory responses of myeloid and lymphoid cells.
Cytotoxic activity of an interleukin 2-Pseudomonas exotoxin chimeric protein produced in Escherichia coli.
Authors: Lorberboum-Galski H , FitzGerald D , Chaudhary V , Adhya S , Pastan I
Published: May 2006

A cDNA clone for human interleukin 2 (IL-2) has been fused to the 5' end of a modified Pseudomonas exotoxin (PE) gene that lacks the sequences encoding the cell recognition domain. The chimeric protein IL-2-PE40 was produced in Escherichia coli. It was extremely toxic to IL-2 receptor-positive cells but had no measurable effect on cells lacking the IL-2 receptor. IL-2-PE40 might be a useful cytotoxic agent in the treatment of diseases involving IL-2 receptor-positive cells and in the treatment of allograft rejection.
Isolation of an insulin-like growth factor II cDNA with a unique 5' untranslated region from human placenta.
Authors: Shen S J , Daimon M , Wang C Y , Jansen M , Ilan J
Published: May 2006

Human insulin-like growth factor II (IGF-II) cDNA from a placental library was isolated and sequenced. The 5' untranslated region (5'-UTR) sequence of this cDNA differs completely from that of adult human liver and has considerable base sequence identity to the same region of an IGF-II cDNA of a rat liver cell line, BRL-3A. Human placental poly(A)+ RNA was probed with either the 5'-UTR of the isolated human placental IGF-II cDNA or the 5'-UTR of the IGF-II cDNA obtained from adult human liver. No transcripts were detected by using the 5'-UTR of the adult liver IGF-II as the probe. In contrast, three transcripts of 6.0, 3.2, and 2.2 kilobases were detected by using the 5'-UTR of the placental IGF-II cDNA as the probe or the probe from the coding sequence. A fourth IGF-II transcript of 4.9 kilobases presumably containing a 5'-UTR consisting of a base sequence dissimilar to that of either IGF-II 5'-UTR was apparent. Therefore, IGF-II transcripts detected may be products of alternative splicing as their 5'-UTR sequence is contained within the human IGF-II gene or they may be a consequence of alternative promoter utilization in placenta.
Comparison of ion channels in multidrug-resistant and -sensitive human leukemic cells.
Authors: Lee S C , Deutsch C , Beck W T
Published: May 2006

Tumor cell lines selected to grow in the presence of one "natural product" antineoplastic drug often develop cross-resistance to others. This multidrug resistance (MDR) is believed to be a major problem in cancer therapy. Organic Ca2+-channel blockers, such as verapamil, can reverse this resistance and render MDR cells in culture nearly as sensitive to the antineoplastic drugs as the drug-sensitive cells from which they were derived. It has therefore been suggested that Ca2+ channels may play a role in MDR. To determine directly whether there are electrophysiological correlates of MDR, we used whole-cell and single-channel patch-clamp techniques to survey the ion channels in a drug-sensitive human T-cell leukemia line, CCRF-CEM, and a MDR variant, CEM/VLB100. We found no evidence for a voltage-gated Ca2+ channel. However, we did identify three other current/channel types: a voltage-gated tetrodotoxin-sensitive inward current carried by Na+, a voltage-gated labile outward current carried by K+, and a nonselective cation channel reversing at 0 mV. Drug-sensitive and -resistant cells were the same with respect to the level of expression of these channels.
Microcloning reveals a high frequency of repetitive sequences characteristic of chromosome 4 and the beta-heterochromatin of Drosophila melanogaster.
Authors: Miklos G L , Yamamoto M T , Davies J , Pirrotta V
Published: May 2006

Microdissection and microcloning of the euchromatin-heterochromatin transition region of the Drosophila melanogaster polytene X chromosome and part of the euchromatin of chromosome 4 reveals that they share certain features characteristic of beta-heterochromatin, which is morphologically defined as the loosely textured material at the bases of some polytene chromosome arms. Both are mosaics of many different middle-repetitive DNA sequences interspersed with single-copy DNA sequences. Sixty percent of cloned inserts derived from division 20 and about 40 percent from subdivisions 19EF of the X chromosome harbor at least one repetitive DNA sequence in an average insert of 4.5 kilobases. No repeats have significant cross-hybridization to any of the eleven satellite DNAs, or to the clustered-scrambled sequences present in pDm1. The repetitive elements are, in general, confined to the beta-heterochromatic regions of polytene chromosomes, but some are adjacent to nomadic elements. Chromosome 4, however, has some repeats spread throughout its entire euchromatin. These data have implications for the structure of transition zones between euchromatin and heterochromatin of mitotic chromosomes and also provide a molecular basis for reexamining some of the unusual classical properties of chromosome 4.
Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro.
Authors: Bae Y S , Kawasaki I , Ikeda H , Liu L F
Published: May 2006

We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase. The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits. Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes.
High-sensitivity neutron diffraction of membranes: Location of the Schiff base end of the chromophore of bacteriorhodopsin
Authors: Heyn M. P. , Westerhausen J. , Wallat I. , Seiff F.
Published: May 2006

Three important events in the functional cycle of bacteriorhodopsin occur at the chromophore: the primary absorption of light, the isomerization from the alltrans to the 13-cis form, and the deprotonation and reprotonation of its Schiff base. The protonated Schiff base linkage of the chromophore with lysine-216 plays an essential role in the color regulation of the pigment and is most likely directly involved in the charge translocation of this light-driven proton pump. Although much is known about the structure of the protein, the position of this key functional group has not yet been determined. We have synthesized a retinal in which the five protons closest to the Schiff base are replaced by deuterons. The labeled retinal was spontaneously incorporated into bacteriorhodopsin by using a mutant of
Halobacterium halobium
that is deficient in the synthesis of retinal. The position of the labeled Schiff base end of the chromophore was determined in the two-dimensional projected density of dark-adapted bacteriorhodopsin by neutron diffraction. The result fits very well with our previous work using retinals that were selectively deuterated in the middle of the polyene chain or in the cyclohexene ring. A coherent structure emerges with the three labeled positions on one line, separated by distances that are in good agreement with the tilt angle of the polyene chain (about 20°). The chromophore is located in the interior of the protein with the nitrogen of the Schiff base between helices 2 and 6 and with its ring in the vicinity of helix 4. Our results show that it is possible to locate a small group containing as few as five deuterons in a membrane protein of molecular weight 27,000.
Cell-cycle regulation as a mechanism for targeting proteins to specific DNA sequences in Tetrahymena thermophila.
Authors: Wu M , Allis C D , Gorovsky M A
Published: May 2006

Transcriptionally active macronuclei and transcriptionally inert micronuclei of the ciliated protozoan Tetrahymena thermophila contain similar DNA sequences but have very different histones associated with the linker regions of chromatin. In situ hybridization showed that a gene coding for micronuclear linker histone is expressed only in association with micronuclear DNA replication, whereas the gene for macronuclear H1 histone is expressed during macronuclear (but not during micronuclear) S phase. These results indicate that cell-cycle regulation plays an important role in directing proteins to the appropriate nucleus in Tetrahymena and that the replication-expression model [Gottesfeld, J. & Bloomer, L. S. (1982) Cell 28, 781-791; Wormington, W. M., Schlissel, M. & Brown, D. D. (1983) Cold Spring Harbor Symp. Quant. Biol. 47, 879-884] for establishing appropriate transcriptionally active or repressed chromatin complexes during DNA replication is generally applicable.
Evidence that associative interactions between synapses during the induction of long-term potentiation occur within local dendritic domains.
Authors: White G , Levy W B , Steward O
Published: May 2006

The present study evaluates whether the associative interactions between synapses that lead to long-term potentiation and depression (LTP and LTD) can occur between spatially segregated synapses of the medial and lateral temporodentate pathway of the rat. Coconditioning of crossed and ipsilateral pathways resulted in LTP of the crossed system only when the current sinks of the two conditioned pathways overlapped sufficiently. Likewise, conditioning of an ipsilateral pathway alone resulted in LTD of the crossed pathway only when those current sinks overlapped sufficiently. These observations support the idea that associative events that lead to LTP or LTD can be restricted to a local dendritic domain. The postsynaptic cell can therefore serve as more than one unit of integration for synaptic modification.
Bidirectional control of the chicken beta- and epsilon-globin genes by a shared enhancer.
Authors: Nickol J M , Felsenfeld G
Published: May 2006

An enhancer specific to erythroid cells was identified previously in the 3' flanking sequence of the chicken adult beta-globin gene and shown to act on the beta-globin promoter. This enhancer lies between the adult beta-globin gene and the embryonic epsilon-globin gene, about equidistant from the two promoters. To determine whether this enhancer acts also on the epsilon-globin promoter, we constructed plasmids containing the enhancer and either the beta- or the epsilon-globin promoter fused to the bacterial chloramphenicol acetyltransferase gene. Primary chicken erythrocytes of both primitive and definitive lineages were transfected with these plasmids. We show that the enhancer is able to stimulate expression from the epsilon-globin promoter as well as the beta-globin promoter. Levels of expression change with the developmental stage of the cell in a way that is partially consistent with the observed developmental regulation of the beta- and epsilon-globin genes in vivo. There appear to be no other enhancer elements either 5' of the epsilon-globin gene or within 6 kilobase pairs of its 3' end. Thus, the enhancer between the beta- and epsilon-globin genes apparently serves to regulate both genes.
Water-stress-induced changes in the abscisic acid content of guard cells and other cells of
Vicia faba
L. leaves as determined by enzyme-amplified immunoassay
Authors: Harris Michael J. , Outlaw William H. , Mertens Rüdiger , Weiler Elmar W.
Published: May 2006

A highly sensitive, solid-phase, enzyme-amplified immunoassay for the plant growth regulator (+)-abscisic acid (ABA) was developed. The assay sensitivity (0.2-10 fmol) was sufficient for analyzing free ABA in homogeneous tissue samples dissected from
Vicia faba
L. leaves. Eight hours after detached leaves had been desiccated to 10% loss of fresh weight, the bulk leaf ABA content increased from ≤0.2 to 6.2 ng·(mg dry weight)
-1
. Epidermal tissue, spongy parenchyma cells, and palisade parenchyma cells from this water-stressed leaf had the following ABA contents, respectively: 4.8, 9.4, and 9.0 ng·(mg dry weight)
-1
. Guard cells, which respond to exogenous ABA by losing solutes and volume, were also assayed. When they were dissected from control (fully hydrated) leaves, their ABA content was ≈0.7 fg·(cell pair)
-1
[[unk]0.2 ng·(mg dry weight)
-1
]. In contrast, the ABA content of guard cells of water-stressed leaves was ≈17.7 fg·(cell pair)
-1
. These results indicate that ABA accumulation in a highly stressed
V. faba
leaflet is generalized; guard cells contain only 0.15% of bulk leaf ABA. The time course for loss of ABA from guard cells of a floating epidermal peel was studied. There was little loss within 30 min, but after 4 hr, the ABA content was only 17% of the original value. These results indicate that the bulk of guard cell ABA is not readily diffusible (i.e., probably not apoplastic). The results also indicate that common laboratory procedures results in lowered guard cell ABA content.
Sequence analyses of herpesviral enzymes suggest an ancient origin for human sexual behavior.
Authors: Gentry G A , Lowe M , Alford G , Nevins R
Published: May 2006

Comparison of the amino acid sequences of the deoxythymidine kinases of herpes simplex (HSV) and of marmoset herpes viruses (MHV) suggests a divergence time of 8 to 10 million years ago for HSV-1 and -2. Like MHV, HSV-1 and -2 cause local infections in their natural hosts, and direct contact between two individuals during the brief period of infectivity is needed for transmission. Because B virus, a nearer relative of HSV, depends on both oral and genital routes of transmission, we postulate that ancestral HSV (aHSV) was similar, and that for HSV-1 and -2 to diverge, genital and oral sites had to become microbiologically somewhat isolated from each other, while oral--oral and genital--genital contact had to be facilitated to maintain both aHSV strains. We propose that acquisition of continual sexual attractiveness by the ancestral human female and the adoption of close face-to-face mating, two hallmarks of human sexual behavior, provided the conditions for the divergence.
Saturation mutagenesis of a yeast his3 "TATA element": genetic evidence for a specific TATA-binding protein.
Authors: Chen W , Struhl K
Published: May 2006

The yeast his3 promoter region contains two distinct classes of "TATA elements," constitutive (TC) and regulatory (TR), that are defined by their interactions with upstream promoter elements, selectivity of initiation sites, and chromatin structure. TC is localized between positions -83 and -53, and TR is localized between positions -55 and -35, regions in which there are several TATA-like sequences. In this study, we used saturation mutagenesis to examine the structural requirements of the his3 TR element necessary for transcriptional induction. To avoid the complications of redundant elements, the phenotypic analysis was carried out by using a gal-his3 hybrid promoter whose function depends on a short oligonucleotide containing the prospective his3 TR element. In this context, an oligonucleotide containing the sequence TATAAA is sufficient for TR function. However, 17 out of the 18 possible single-base substitutions and 9 out of 10 double mutations of this sequence abolish TR function. This strict sequence requirement for TR function strongly suggests that the TR element is a target site for a sequence-specific DNA-binding protein. Further, as the region encoding TC and promoters of certain other yeast genes do not contain a sequence that is compatible with TR function, we suggest that yeast cells contain multiple proteins with distinct sequence specificities that carry out a related "TATA function" and that yeast promoters can be divided into classes based on their downstream promoter elements.
Characterization of histamine H1-receptor binding peptides in guinea pig brain using [125I]iodoazidophenpyramine, an irreversible specific photoaffinity probe.
Authors: Ruat M , Körner M , Garbarg M , Gros C , Schwartz J C , Tertiuk W , Ganellin C R
Published: May 2006

Aminophenpyramine--i.e., N-(5-[2-(4-aminophenyl)ethanamidopentyl])-N'-(4-methoxybenzyl)-N-m ethyl-N'- (2-pyridinyl)-1,2-ethanediamine, a derivative of mepyramine (pyrilamine), a typical antagonist of histamine at its H1 receptor--was synthesized and converted into [125I)iodoazidophenpyramine, a potential photoaffinity probe for the H1 receptor. In the dark, reversible binding of this probe to cerebellar membranes occurred with a Kd of 1.2 x 10(-11) M and a Bmax of 240 fmol/mg of protein and was inhibited by various H1-receptor antagonists with the expected potencies. These features establish the compound as one of the most potent H1-receptor antagonists known so far. Upon UV irradiation, 5% of the bound radioactivity was covalently incorporated into cerebellar membrane polypeptides as shown by standard NaDodSO4/PAGE. Two bands of 47 and 56 kDa were consistently labeled, labeling being prevented by various H1-receptor antagonists with the expected potencies and stereoselectivity. In the presence of protease inhibitors, labeling of the 56-kDa peptide increased at the expense of the 47-kDa peptide, suggesting that the latter was produced by hydrolysis of the former under the action of membrane proteases. In the absence of 2-mercaptoethanol, a band of 350-400 kDa appeared, apparently at the expense of the lighter bands, suggesting that the latter might be linked by one or more disulfide bridges to a higher molecular mass complex. We propose that at least part of the ligand binding domain of the histamine H1 receptor resides within a subunit of apparent molecular mass 56,000.
Immunohistochemical evidence for the derivation of a peptide ligand from the amyloid beta-protein precursor of Alzheimer disease.
Authors: Allsop D , Wong C W , Ikeda S , Landon M , Kidd M , Glenner G G
Published: May 2006

A monoclonal antibody to a synthetic peptide consisting of residues 8-17 of the amyloid beta protein of Alzheimer disease was used in immunohistochemical studies to reveal binding sites for this peptide in vesicular elements in the islets of Langerhans of the pancreas and the zona reticularis of the adrenal gland. These binding sites may represent a specific membrane receptor. These results, together with similarities in structural features between the precursors for epidermal growth factor and beta protein, suggest that the beta-protein precursor may be processed to release an active peptide ligand rather than acting as a membrane receptor. In Alzheimer disease, abnormal processing of this active peptide precursor may result in the deposition of beta-protein amyloid fibrils in the brain.
myc protooncogene linked to retroviral promoter, but not to enhancer, transforms embryo cells.
Authors: Zhou R P , Duesberg P H
Published: May 2006

To define conditions under which the chicken protooncogene p-myc is converted to a viral and possibly to a cellular transforming gene, we assayed transforming function of hybrid genes put together from cloned retroviral and p-myc elements and of p-myc genes isolated from spontaneous viral lymphomas. Transforming function was measured in quail embryo cells transfected with cloned myc genes. We found that only myc genes with a promoter of a retroviral long terminal repeat (LTR) located between the native p-myc promoter and the second p-myc exon have transforming function. Transforming efficiencies decreased with increasing lengths of unspliced sequences between the LTR and p-myc exon 2. p-myc DNAs with LTRs downstream of the coding region or upstream but in the opposite transcriptional orientation failed to transform embryo cells. Likewise, only those retroviral-p-myc combinations from chicken B-cell lymphomas with a LTR positioned as promoter upstream of p-myc exon 2 had transforming function. We conclude that substitution of a retroviral LTR for the promoter and for as yet poorly defined, untranscribed regulatory elements of p-myc is sufficient to convert chicken p-myc to a transforming gene. However, retroviral LTRs can only convert p-myc genes to embryo-cell-transforming genes from a limited number of positions, and not as position-independent enhancers. Further, we deduce that there are two classes of viral chicken B-cell lymphomas, those with and those without embryo-cell-transforming p-myc genes.
In situ
localization of light-induced chalcone synthase mRNA, chalcone synthase, and flavonoid end products in epidermal cells of parsley leaves
Authors: Schmelzer Elmon , Jahnen Willi , Hahlbrock Klaus
Published: May 2006

Methods involving
in situ
RNA hybridization, immunohistochemistry, and microspectrophotometry of individual cells were used to localize the mRNA encoding chalcone synthase (the key enzyme of flavonoid biosynthesis), the enzyme protein, and the biosynthetic end products in cross sections of parsley leaves (
Petroselinum crispum
). The light-dependent, sequential occurrence of all three components was restricted to epidermal cells. The results are in agreement with the putative function of flavonoids (transcriptionally inducible, UV-protective pigments) and suggest that all biosynthetic steps occur in those cells in which the products accumulate.
Common regulatory elements control symbiotic and microaerobic induction of nifA in Rhizobium meliloti.
Authors: Virts E L , Stanfield S W , Helinski D R , Ditta G S
Published: May 2006

We have previously demonstrated that the nifA promoter (nifAp) of Rhizobium meliloti is inducible under microaerobic conditions in the absence of alfalfa. Here we show that microaerobic activation of nifAp involves both cis- and trans-acting regulatory controls identical to those used symbiotically. The start site for nifA mRNA synthesis was found to be the same during symbiosis and microaerobiosis, and a deletion analysis of nifAp demonstrated that DNA between positions -62 and -45 is essential for induction. Mutants isolated as being unable to induce nifA microaerobically also were found to be defective in symbiotic nitrogen fixation with alfalfa. Such mutants form nodules that are equivalent cytologically to those induced by nifA::Tn5 mutants. Genetic and structural studies have localized the mutations to a cluster of fix genes 200 kilobases distant from the nod-nif region on the pSym megaplasmid [Renalier, M.-H., Batut, J., Ghai, J., Terzaghi, B., Gherardi, M., David, M., Garnerone, A.-M., Vasse, J., Truchet, G., Huguet, T. & Boistard, P. (1987) J. Bacteriol. 169, 2231-2238].
Tissue localization and fate in mice of injected multipotential colony-stimulating factor.
Authors: Metcalf D , Nicola N A
Published: May 2006

The hemopoietic regulator multipotential colony-stimulating factor [Multi-CSF (interleukin 3)] has proliferative effects on a wide range of hemopoietic cells in vitro and in vivo. Native or recombinant Multi-CSF injected intravenously into adult mice had an initial half-life of 3-5 min and a second phase of 50 min. Clear labeling of hemopoietic cells was observed in the bone marrow and spleen of mice injected intravenously with recombinant 125I-labeled Multi-CSF showing that injected Multi-CSF can obtain access to such cells in situ. A high proportion of injected 125I-labeled Multi-CSF of both types became localized in the liver and in the kidney (in cells of the Bowman's capsule and proximal renal tubules). The kidney appeared to be an active site of degradation of Multi-CSF with the early appearance of low molecular weight labeled material in the urine.
Pattern of expression of glutamic acid decarboxylase mRNA in the developing rat brain.
Authors: Bond R W , Jansen K R , Gottlieb D I
Published: May 2006

The time and pattern of appearance of glutamic acid decarboxylase (glutamate decarboxylase; EC 4.1.1.15) (GAD) mRNA during the development of the rat brain were analyzed. RNA transfer blot analysis of poly(A)+ RNA from whole brain shows that a 3.7-kilobase transcript is the most abundant form of the message from embryonic day 15 (E15) through adulthood. By E15 this form is present at about 50% of its adult abundance relative to other poly(A)+ mRNA species. At birth the abundance is approximately the same as in the adult. In contrast, the enzyme activity level is only 8% of the adult level at birth and takes 3 weeks to reach adult levels. There are qualitative changes in GAD mRNA during development. Several large (7-9 kilobases) transcripts with strong homology to GAD are enriched in early developmental stages but are barely detectable in the adult. A nuclease protection assay shows a developmentally regulated heterogeneity in a coding portion of the mRNA.
Bradykinin as a pain mediator: receptors are localized to sensory neurons, and antagonists have analgesic actions.
Authors: Steranka L R , Manning D C , DeHaas C J , Ferkany J W , Borosky S A , Connor J R , Vavrek R J , Stewart J M , Snyder S H
Published: May 2006

Autoradiographic studies localize [3H]bradykinin receptor binding sites to the substantia gelatinosa, dorsal root, and a subset of small cells in both the dorsal root and trigeminal ganglia of the guinea pig. [3H]Bradykinin labeling is also observed over myocardial/coronary visceral afferent fibers. The localization of [3H]bradykinin receptors to nociceptive pathways supports a role for bradykinin in pain mediation. Several bradykinin antagonists block bradykinin-induced acute vascular pain in the rat. The bradykinin antagonists also relieve bradykinin- and urate-induced hyperalgesia in the rat paw. These results indicate that bradykinin is a physiologic mediator of pain and that bradykinin antagonists have analgesic activity in both acute and chronic pain models.
Transplantation tolerance correlates with high levels of T- and B-lymphocyte activity.
Authors: Bandeira A , Coutinho A , Carnaud C , Jacquemart F , Forni L
Published: May 2006

Mice tolerized (treated to make them tolerant) at birth to transplantation antigens by injection of semiallogeneic cells contain very high numbers of activated T and B lymphocytes in their spleen. Lymphoid hyperactivity correlates with the tolerant state: it is present only in animals accepting skin allografts. Tolerized mice that reject the allogeneic skin graft have approximately the same numbers of total and activated lymphocytes as normal mice. The high level of lymphocyte activation in tolerant mice persists for up to 1 year of age, although it declines with age, and is markedly increased by a secondary allograft. The magnitudes of both primary and secondary tolerant responses are significantly higher than the immunological response of a normal mouse rejecting the same type of allograft. These observations contradict concepts of clonal deletion or anergy as the basis of neonatally induced transplantation tolerance and may contribute additional approaches to experimentation and control of transplantation reactions.