bluuebunny/crossref_metadata_2025 huggingface dataset:
A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen.
Authors: van Loon A P , Pesold-Hurt B , Schatz G
Published: May 2006

The nuclear gene for manganese-containing superoxide dismutase (MnSOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) of yeast mitochondria was mapped on chromosome VIII and inactivated by gene disruption. The resulting mutant lacked any protein cross-reacting with anti-MnSOD antibodies, and its mitochondria exhibited less than 1% of the cyanide-insensitive superoxide dismutase activity found in mitochondria of the wild-type parent strain. In the absence of oxygen, the mutant grew as rapidly as the wild-type parent. However, increasing concentrations of oxygen led to a progressive inhibition of growth. The properties of this mutant provide direct evidence that MnSOD contributes to the natural protection of cells against oxygen toxicity.
Actively transcribed genes in the raf oncogene group, located on the X chromosome in mouse and human.
Authors: Huebner K , ar-Rushdi A , Griffin C A , Isobe M , Kozak C , Emanuel B S , Nagarajan L , Cleveland J L , Bonner T I , Goldsborough M D
Published: May 2006

Murine and human cDNAs, related to but distinct from c-raf-1, have been isolated and designated mA-raf and hA-raf, respectively. The mA-raf and hA-raf cDNAs detect the same murine and human fragments in Southern blots of restriction enzyme-cleaved murine and human cellular DNA. The murine restriction enzyme fragments homologous to mA-raf cDNA cosegregate with mouse chromosome X in a panel of Chinese hamster-mouse hybrid cells, thus localizing the mA-raf locus to mouse chromosome X. Two independently segregating loci, detected by the hA-raf cDNA (or mA-raf cDNA), hA-raf-1 and hA-raf-2, are located on human chromosomes X and 7, respectively. The mA-raf locus and the hA-raf-1 locus are actively transcribed in several mouse and human cell lines.
Antibodies of predetermined specificity to the major charged species of human interleukin 1.
Authors: Limjuco G , Galuska S , Chin J , Cameron P , Boger J , Schmidt J A
Published: May 2006

The development of highly specific antisera to human interleukin 1 (IL-1) has been an elusive goal hampered mainly by the availability of only limited amounts of pure immunogen. To surmount this difficulty, three peptides of the major charged species of IL-1 (pI 6.8) were synthesized and covalently coupled to keyhole limpet hemocyanin (KLH). All three peptide-KLH conjugates raised rabbit heterologous antisera that bound intact pure IL-1 in a dose-dependent and domain-specific manner. Immunoblot analysis of crude concentrated culture supernatants with these antisera showed each of them to be highly specific for mature 18-kDa IL-1. Immunoblot analysis of monocyte lysates revealed a single 33-kDa band consistent with the size of the IL-1 precursor molecule deduced from cloned cDNA. These reagents should prove to be valuable tools in the localization and measurement of IL-1 in cells and fluids and may permit the separate study of individual IL-1 species as well as discrete domains of intact IL-1 molecules.
Monoclonal antibodies to a particulate superoxide-forming system stimulate a respiratory burst in intact guinea pig neutrophils.
Authors: Berton G , Rosen H , Ezekowitz R A , Bellavite P , Serra M C , Rossi F , Gordon S
Published: May 2006

Monoclonal rat antibodies were produced against a subcellular preparation of phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig neutrophils that retains NADPH-oxidase activity. Two antibodies, 1A10.4 and IG4, were isolated that bind to a surface antigen restricted to guinea pig neutrophils from bone marrow and peritoneal exudate and to macrophages and that trigger a respiratory burst in neutrophils in the presence of cytochalasin B. Intact antibody 1A10.4, subclass IgG2c, can trigger superoxide anion release directly; F(ab')2 fragments of 1A10.4 and intact IG4 require further cross-linking by F(ab')2 fragments of anti-rat immunoglobulin antibody. Both antibodies recognize the same antigen, a proteolipid of apparent molecular mass 10 kDa. Immunoprecipitation of solubilized oxidase activity with 1A10.4 brings down this activity as part of a macromolecular complex. Surface expression of the antigen is increased on treatment of cells with both PMA and cytochalasin B. 1A10.4 also triggers release of the granule enzyme beta-glucuronidase. Triggering of a respiratory burst by the antibodies appears distinct from the PMA and fMet-Leu-Phe signalling systems. These studies indicate that the antigen defined by antibodies 1A10.4 and IG4 becomes associated with the superoxide anion-generating system of neutrophils but may play a more general role in signal transduction in phagocytic cells.
Electrical coupling between bipolar cells in carp retina
Authors: Kujiraoka Toru , Saito Takehiko
Published: May 2006

Intracellular recordings were made simultaneously from pairs of neighboring bipolar cells by advancing two independent microelectrodes into retinas of carp (
Cyprinus carpio
). Bipolar cells were identified by their response properties and in several samples were verified by intracellular injection of Lucifer yellow. Current of either polarity injected into one member of the bipolar cell pair elicited a signconserving, sustained potential change in the other bipolar cell without any significant delay. This electrical coupling was reciprocal, and it was observed between cell types similar in function and in morphology. Our results strongly suggest that there is a spatial summation of signals at the level of bipolar cells, which makes central receptive field areas much larger than their dendritic fields.
Antibody to a molecular marker of cell position inhibits synapse formation in retina.
Authors: Trisler D , Bekenstein J , Daniels M P
Published: May 2006

A topographic gradient of TOP molecules in retina can be used to identify neuron position. Antibody to TOP from hybridoma cells that were injected into in vivo embryo eyes diffused into the retina and bound in a topographic gradient of [antibody.TOP] ([Ab.TOP]) complexes. Synapse formation in retina was inhibited in the presence of anti-TOP antibody. This suggests that TOP is involved in synapse formation and that recognition of position by neurons is necessary for normal synapse formation.
Primary structure of bovine vitamin K-dependent protein S.
Authors: Dahlbäck B , Lundwall A , Stenflo J
Published: May 2006

Protein S is a vitamin K-dependent plasma protein that functions as a cofactor to activated protein C in the inactivation of coagulation factors Va and VIIIa. The nucleotide sequence of a full-length cDNA clone, obtained from a bovine liver library, was determined and the amino acid sequence was deduced. In addition, 95% of the structure was determined by protein sequencing. Protein S consists of 634 amino acids in a single polypeptide chain and has one asparagine-linked carbohydrate side chain. The cDNA sequence showed that the protein has a leader sequence, 41 amino acid residues long. The amino-terminal part of the molecule containing gamma-carboxyglutamic acid is followed by a region, residues 42-75, with two peptide bonds that are very sensitive to cleavage by thrombin. Residues 76-244 have four cysteinerich repeat sequences, each about 40 residues long, that are homologous to the precursor of mouse epidermal growth factor. In contrast to the other vitamin K-dependent plasma proteins, the carboxyl-terminal part of protein S is not homologous to the serine proteases.
Antigenic crossreactivity of the alpha subunit of complement component C8 with the cysteine-rich domain shared by complement component C9 and low density lipoprotein receptor.
Authors: Tschopp J , Mollnes T E
Published: May 2006

Complement component C9 contains two distinct cysteine-rich domains exhibiting high sequence resemblance to a domain present in the low density lipoprotein (LDL) receptor and epidermal growth factor precursor, respectively. Antibodies were raised against a peptide corresponding to the most conserved region of the LDL receptor/C9-homology segment. The antibodies were shown by immunoblotting to bind specifically to C9 but also to crossreact with C8 alpha, the alpha subunit of complement component C8. Moreover, a monoclonal antibody to a neoantigen present in polymerized C9 bound to an epitope exposed on C8 within the C5b-8 complex but buried in monomeric C8, suggesting that C8 and C9 undergo similar conformational changes during membrane-attack-complex assembly. Isolated C8 alpha-gamma exhibited the propensity to polymerize in the presence of Zn2+ and urea, as already demonstrated for C9. These data indicate that C8 alpha is closely related, both structurally and functionally, to C9.
Steady-state ATP synthesis by bacteriorhodopsin and chloroplast coupling factor co-reconstituted into asolectin vesicles.
Authors: Krupinski J , Hammes G G
Published: May 2006

A method was developed for the co-reconstitution of bacteriorhodopsin and chloroplast coupling factor in asolectin vesicles. First, bacteriorhodopsin was reconstituted from a mixture of octyl glucoside, asolectin, and protein in the presence of ethylenediaminetetraacetic acid by passage through a Sephadex G-50 centrifuge column. Then, the purified coupling factor was reconstituted from a mixture of sodium cholate, bacteriorhodopsin vesicles, and coupling factor in the presence of Mg2+ by passage through the centrifuge column. Sucrose density-gradient centrifugation indicated a band of vesicles with slightly different positions in the gradient for maximum vesicle concentration, bacteriorhodopsin vesicle concentration, ATP synthesis, and ATP hydrolysis. The rate of light-driven ATP synthesis reaches a limiting value as the concentration of bacteriorhodopsin and the light intensity are increased. A steady-state rate of ATP synthesis of 1 mumol per mg of coupling factor X min-1 has been achieved. Apparently this rate is limited by the heterogeneity within the vesicle population and by the ability of bacteriorhodopsin to form a sufficiently large pH gradient.
Autophosphorylation reversibly regulates the Ca2+/calmodulin-dependence of Ca2+/calmodulin-dependent protein kinase II.
Authors: Lai Y , Nairn A C , Greengard P
Published: May 2006

Ca2+/calmodulin-dependent protein kinase II contains two subunits, alpha (Mr 50,000) and beta (Mr 60,000/58,000), both of which undergo Ca2+/calmodulin-dependent autophosphorylation. In the present study, we have studied the mechanism of this autophosphorylation reaction and its effect on the activity of the enzyme. Both subunits are autophosphorylated through an intramolecular mechanism. Using synapsin I as substrate, Ca2+/calmodulin-dependent protein kinase II, in its unphosphorylated form, was totally dependent on Ca2+ and calmodulin for its activity. Preincubation of the enzyme with Ca2+, calmodulin, and ATP, under conditions where autophosphorylation of both subunits occurred, converted the enzyme to one that was only partially dependent on Ca2+ and calmodulin for its activity. No change in the total activity, measured in the presence of Ca2+ and calmodulin, was observed. The nonhydrolyzable ATP analog adenosine 5'-[beta, gamma-imido] triphosphate did not substitute for ATP in the preincubation. Moreover, dephosphorylation of autophosphorylated Ca2+/calmodulin-dependent protein kinase II with protein phosphatase 2A resulted in an enzyme that was again totally dependent on Ca2+ and calmodulin for its activity. We propose that autophosphorylation and dephosphorylation reversibly regulate the Ca2+ and calmodulin requirement of Ca2+/calmodulin-dependent protein kinase II.
Recombinant retroviruses that transduce individual polyoma tumor antigens: effects on growth and differentiation.
Authors: Cherington V , Morgan B , Spiegelman B M , Roberts T M
Published: May 2006

We have constructed infectious retroviral vectors, derived from Moloney murine leukemia virus, that efficiently transduce the polyoma virus tumor (T) antigens individually. The parental vector we have chosen [pZIP-NeoSV(X)1] expresses a dominant selectable marker for neomycin resistance and is a shuttle vector capable of propagation in both eukaryotic and prokaryotic cells, thus facilitating its use in structure-function studies. To address the relationship between polyoma T-antigen tumorigenesis and the effects of individual T antigens on growth control and differentiation, we used these vectors to introduce and stably express large, middle-sized, or small T antigens into mouse fibroblasts and preadipocytes. All cDNAs introduced into the vector are expressed stably even in the absence of selective pressure. The stable expression of small T antigen is noted particularly because cell lines expressing small T antigen have not been readily available prior to the use of retroviral vectors. Small T antigen-induced increase in saturation density of NIH 3T3 cells can be scored on the basis of the morphology of drug-resistant colonies. Middle-sized T antigen eliminates the growth requirement of NIH 3T3 cells for epidermal growth factor in a defined medium and permits growth in platelet-poor plasma, indicating elimination of the platelet-derived growth factor requirement as well. Large T antigen suppresses mouse preadipocyte (3T3-F442A) differentiation. These vectors and these functional assays of T-antigen activity permit genetic analysis of the relationship between tumorigenesis by T antigens and the alteration of cellular growth and differentiation.
Structure of the human Ia-associated invariant (gamma)-chain gene: identification of 5' sequences shared with major histocompatibility complex class II genes.
Authors: O'Sullivan D M , Larhammar D , Wilson M C , Peterson P A , Quaranta V
Published: May 2006

The human gene encoding the Ia-associated gamma (or invariant) chain was isolated by screening a genomic library in phage lambda with cDNA probes. The frequency of positive clones in the library, the overlapping restriction maps of the cloned fragments, and the patterns of genomic hybridization suggested that the gamma-chain gene exists as a single copy per haploid genome. The gene consists of 8 exons, spanning approximately 12 kilobases of DNA. All exon sequences were in an open reading frame, contained appropriate splice junction sequences, and encompassed the entire sequence of full-length gamma-chain mRNA, suggesting that the gene we isolated is most likely functional. Furthermore, "CAAT"-type and "TATA"-type promoter sequences were found at the expected positions upstream from the proposed cap site. The organization of the gamma-chain gene has none of the distinctive features of the immunoglobulin superfamily of genes, of which Ia alpha and beta chains are members. Therefore, the evolutionary origins, and perhaps the functions, of the Ia gamma chains are distinct from those of the other two Ia subunits alpha and beta. Despite the unrelatedness of these genes, consensus sequences found approximately 150 base pairs upstream from all the Ia alpha- and beta-chain genes sequenced to date were also found in analogous positions in the gamma-chain gene, suggesting a possible role in the coregulation of expression of these genes.
Adult T-cell leukemia/lymphoma not associated with human T-cell leukemia virus type I.
Authors: Shimoyama M , Kagami Y , Shimotohno K , Miwa M , Minato K , Tobinai K , Suemasu K , Sugimura T
Published: May 2006

We describe five patients with adult T-cell leukemia/lymphoma (ATL) with neither integration of human T-cell leukemia virus type I (HTLV-I) into their leukemia cells nor anti-HTLV-I antibody in their sera. These findings indicate that HTLV-I may not have been involved in leukemogenesis in these patients. The clinicohematological, cytopathological, and immunological features of HTLV-I-negative ATL were exactly the same as those of HTLV-I-associated ATL. Leukemia cells with pleomorphic nuclei, generalized lymphadenopathy, hepatosplenomegaly, skin lesions, hypercalcemia, and elevated lactate dehydrogenase levels, all of which are characteristic features of typical ATL, were also seen in these patients with HTLV-I-negative ATL. Leukemia cells expressed T3, T4, and pan-T-cell antigens in three cases, and T3 and pan-T-cell antigens in two. All five patients had lived in ATL-nonendemic areas. The finding of HTLV-I-negative ATL suggests that factor(s) other than HTLV-I infection may be involved in ATL leukemogenesis.
Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: evidence for bypass of pyrimidine photodimers.
Authors: Livneh Z
Published: May 2006

Replication of UV-irradiated circular single-stranded phage M13 DNA by Escherichia coli RNA polymerase (EC 2.7.7.6) and DNA polymerase III holoenzyme (EC 2.7.7.7) in the presence of single-stranded DNA binding protein yielded full-length as well as partially replicated products. A similar result was obtained with phage G4 DNA primed with E. coli DNA primase, and phage phi X174 DNA primed with a synthetic oligonucleotide. The fraction of full-length DNA was several orders of magnitude higher than predicted if pyrimidine photodimers were to constitute absolute blocks to DNA replication. Recent models have suggested that pyrimidine photodimers are absolute blocks to DNA replication and that SOS-induced proteins are required to allow their bypass. Our results demonstrate that, under in vitro replication conditions, E. coli DNA polymerase III holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins.
Isolation of a Drosophila genomic sequence homologous to the kinase domain of the human insulin receptor and detection of the phosphorylated Drosophila receptor with an anti-peptide antibody.
Authors: Petruzzelli L , Herrera R , Arenas-Garcia R , Fernandez R , Birnbaum M J , Rosen O M
Published: May 2006

A Drosophila genomic fragment has been isolated with a deduced amino acid sequence that is strikingly homologous to that of the kinase domain of the human insulin receptor. The Drosophila DNA hybridizes with an 11-kilobase mRNA that is most prominent in 8- to 12-hr embryos. An anti-peptide antibody prepared to a sequence in the human insulin receptor kinase domain that is conserved in the Drosophila sequence immunoprecipitates a single 95-kDa Drosophila protein whose phosphorylation on tyrosine residues is dependent on insulin. We conclude that the DNA sequence is that of the kinase domain of the Drosophila insulin receptor and that the 95-kDa phosphoprotein is the autophosphorylated beta subunit of that receptor. The results are compatible with our previous reports demonstrating a specific insulin-binding Drosophila glycoprotein and an insulin-dependent tyrosine protein kinase whose activity is greatest during embryogenesis. The observations suggest a role for insulin-dependent protein tyrosine phosphorylation during embryogenesis.
Purification of a membrane protein distributed in a topographic gradient in chicken retina.
Authors: Moskal J R , Trisler D , Schneider M D , Nirenberg M
Published: May 2006

Antigenic molecules termed TOP, which are distributed in a dorsal greater than ventral concentration gradient in chicken retina, are expressed early in development (by 48 hr after fertilization) in the optic cup of chicken embryos and continue to be expressed in retina thereafter. 35S-labeled-TOP-antibody complexes were purified by protein A-Sepharose column chromatography and subjected to NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. TOP also was purified from dorsal retina by anti-TOP IgG-Affi-Gel 10 affinity column chromatography. In both cases, one major band of protein at Mr approximately 47,000 was obtained. A protein of Mr approximately 47,000 also was purified from chicken embryo brain. Cultured cells dissociated from 8-day chicken embryo retinas accumulated the amount of TOP expected of cells in the intact retina, depending on the position of the cells in the retina. TOP accumulations by cells dissociated from dorsal or ventral retina, mixed in different proportions, and cocultured were additive. These results show that TOP is a protein, that the gradient of TOP is established early in development, and that perpetuation of the gradient does not depend on the continuous presence of an extracellular gradient of diffusable molecules or on maintenance of interactions between cells.
A long interspersed (LINE) DNA exhibiting polymorphic patterns in human genomes.
Authors: Musich P R , Dykes R J
Published: May 2006

Several human DNAs digested with Kpn I restriction endonuclease released a 0.6-kilobase (kb) segment that varied in its intensity among human samples. A recombinant DNA clone (N6.4) of these 0.6-kb Kpn I segments was isolated and used to probe the genomic content and restriction cleavage pattern of homologous sequences. The hybridization patterns revealed a previously undescribed, moderately repetitive long interspersed (LINE) sequence family, which we have termed L2Hs (second LINE family in Homo sapiens). This LINE family exhibits both quantitative and qualitative polymorphisms in the human population. The content of L2Hs sequences in human genomes varies over a 5-fold range. Relative to the value for a human placental DNA, sequences homologous to the L2Hs family occur in lower amounts in gorilla DNA (approximately 20%) and even less in DNA from chimpanzees and other primates (less than 1%). Thus, the L2Hs sequences appear to have emerged only recently as a moderately repetitive sequence family in primate evolution. The observed restriction fragment length polymorphism of the L2Hs family members may reflect patterns of sequence rearrangements, amplifications, and/or deletions in human genomes.
Flow-induced anisotropy in the susceptibility of a particle suspension
Authors: Karis Thomas E. , Jhon Myung S.
Published: May 2006

The initial susceptibility, or low-field high-frequency permeability, of a rod-like particle suspension or polymer solution can become anisotropic when the particles or molecules interact with a magnetic field in a directional manner and are nonrandomly oriented by certain types of flow field. This is the alignment that leads to thixotropy and deviatoric stresses during rheological measurements on these fluids. A general expression for the particle order parameter and the fluid permeability in terms of Legendre polynomials is derived by calculating the ensemble average over all possible orientation angles. This is an integral containing the orientation angle-dependent orientation probability function. The example case of single-domain magnetic particles suspended in extensional flow of a newtonian fluid is examined by using the following well-known models: chain-of-spheres model for the magnetic properties of a particle and the Kirkwood-Auer theory for the orientation probability function. From this, the particle orientation in the flow direction downstream from a sharp circular convergence is predicted to dramatically increase with an increase in the ratio of the extension rate to the particle rotary diffusion coefficient. Consequently, the measured permeability of the fluid is predicted to decrease parallel to, and to increase perpendicular to, the flow direction.
Nucleotide sequences of mRNAs encoding Epstein-Barr virus nuclear proteins: a probable transcriptional initiation site.
Authors: Sample J , Hummel M , Braun D , Birkenbach M , Kieff E
Published: May 2006

Three cDNA clones of the second Epstein-Barr virus nuclear antigen (EBNA2) mRNA and two of the EBNA1 mRNA were analyzed. Two EBNA2 clones begin 42 bases 3' to a promoter in the Epstein-Barr virus long internal repeat, which is likely to be the EBNA2 promoter. Surprisingly, the first splice creates an AUG at the beginning of the first of two nonoverlapping open reading frames. The second open reading frame encodes EBNA2. Two incomplete EBNA1 mRNA cDNA clones begin with parts of two of the EBNA2 exons and contain two other exons that map 19 and 59 kilobases 3' to the EBNA2 coding domain. The 3' exon of this mRNA encodes EBNA1. A model for regulation of transcription of these RNAs is presented.
Structure of rat DNA polymerase beta revealed by partial amino acid sequencing and cDNA cloning.
Authors: Zmudzka B Z , SenGupta D , Matsukage A , Cobianchi F , Kumar P , Wilson S H
Published: May 2006

A cDNA library of newborn rat brain poly(A)+ RNA in phage lambda gt11 was screened with a polyclonal antibody against chicken DNA polymerase beta. One positive phage was isolated and purified after testing 2 X 10(7) recombinants. This phage, designated lambda pol beta-10, contained an 1197-base-pair cDNA insert that corresponded to a mRNA with a poly(A) sequence at the 3' terminus and a single, long open-reading frame of 957 bases. The open-reading frame, starting 44 residues from the 5' end of the cDNA, predicted a 36,375-Da protein of 318 amino acids. Comparison of this deduced amino acid sequence with the partial sequence obtained with purified polymerase beta revealed a match of six tryptic peptides, involving a total of 47 amino acid residues. This confirmed the identity of the cDNA. Blot-hybridization analysis of newborn rat brain poly(A)+ RNA revealed a mRNA species of approximately the same size as the cDNA insert; in addition, a second mRNA species approximately equal to 4000 bases long was detected. Computer-derived secondary structure analysis of the enzyme predicted seven regions of alpha-helix distributed throughout and three regions of beta-sheet.
Complete cDNA sequence of a cytochrome P-450 inducible by glucocorticoids in human liver.
Authors: Molowa D T , Schuetz E G , Wrighton S A , Watkins P B , Kremers P , Mendez-Picon G , Parker G A , Guzelian P S
Published: May 2006

HLp is a human liver cytochrome P-450 that is immunochemically related to the glucocorticoid-inducible liver cytochrome P-450p in the rat and its homologue in the rabbit, P-450 LM3c. To investigate the structure and regulation of HLp, we used a monoclonal antibody that recognizes purified HLp to screen a human liver cDNA library in lambda gt11. We isolated and sequenced two overlapping cDNA clones that span the entire 2011 bases of an mRNA that codes for a protein of 504 amino acids. The predicted amino-terminal amino acid sequence of this protein is identical to the first 20 residues determined from purified HLp. HLp mRNA shares more than 70% sequence homology with related proteins from the rat and rabbit but less than 40% homology with other published cytochrome P-450 genes. Moreover, Southern blot analysis of human and rat genomic DNA revealed 50 and 60 kilobases of DNA, respectively, hybridizable to the HLp cDNAs. Blot analysis of human liver RNA from five patients revealed major (2.2 kilobase) and minor (3.0 kilobase) bands that hybridized to HLp cDNAs. The apparent concentration of these hybridizable mRNAs as well as the amounts of immunoreactive HLp protein in microsomes from the same liver were increased in a dose-dependent relationship in three patients who received dexamethasone, a potent glucocorticoid. Furthermore, in samples of RNA and of microsomes isolated from cultures of a human hepatoma cell line (Hep G2) incubated for 120 hr in medium containing dexamethasone, there was a 6-fold induction of the two mRNA species hybridizable to HLp cDNAs and a 3-fold induction of immunoreactive HLp protein as compared to the values for cultures incubated in steroid-free medium. We conclude that HLp is a human representative of a conserved glucocorticoid-inducible cytochrome P-450 gene family whose mechanism of induction involves accumulation of HLp mRNA.
Immunohistochemical demonstration of choline acetyltransferase-immunoreactive preganglionic nerve fibers in guinea pig autonomic ganglia.
Authors: Lindh B , Staines W , Hökfelt T , Terenius L , Salvaterra P M
Published: May 2006

The distribution of choline acetyltransferase (ChoAcTase)-like immunoreactivity in the superior cervical ganglion and the stellate ganglion was analyzed with immunohistochemistry. A dense network of ChoAcTase-immunoreactive nerve fibers was observed in both ganglia studied. The ChoAcTase-positive fibers were found in all parts of the ganglia but had a differential distribution, giving rise to a patchy staining pattern. After preganglionic denervation of the superior cervical ganglia, all ChoAcTase-positive fibers disappeared. For comparison, the distributions of enkephalin- and neuropeptide Y-like immunoreactivity were analyzed. In the stellate ganglion, the enkephalin-positive fibers had a marked regional distribution. Areas with the highest enkephalin-fiber density seemed to contain low numbers of ChoAcTase-positive fibers and vice versa. The findings give evidence for occurrence of acetylcholine synthesis in preganglionic fibers in paravertebral sympathetic ganglia.
Estradiol receptor has proteolytic activity that is responsible for its own transformation.
Authors: Puca G A , Abbondanza C , Nigro V , Armetta I , Medici N , Molinari A M
Published: May 2006

We have investigated the effect of various protease inhibitors and substrates on the hormone- and temperature-dependent binding of partially purified estradiol-receptor complex to isolated nuclei. Only serine protease substrates and inhibitors significantly depressed estradiol receptor transformation. At 20 degrees C, we observed 50% inhibition with about 3 microM aprotinin or with 1.4 mM diisopropyl fluorophosphate. Aprotinin also blocked those size and charge modifications of receptor that are characteristic of the transformation process. The estradiol receptor was able to bind to aprotinin-agarose only under transforming conditions; i.e., the interaction was hormone- and temperature-dependent and inhibited by molybdate. Diisopropyl fluorophosphate, a covalent reagent for serine esterases, competitively inhibited the binding and specifically eluted the estradiol-receptor complex that had been bound to aprotinin-agarose. These results indicate that estradiol receptor transformation is due to the effect of a serine protease and that the receptor itself is endowed with this catalytic activity, which is triggered by the steroid.
Bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania tropica: sequence homology with the corresponding monofunctional proteins.
Authors: Grumont R , Washtien W L , Caput D , Santi D V
Published: May 2006

In protozoa, thymidylate synthase (TS; EC 2.1.1.45) and dihydrofolate reductase (DHFR; EC 1.5.1.3) exist as a bifunctional protein. Full-length cDNA and genomic DNA clones encoding the bifunctional TS-DHFR from Leishmania tropica have been isolated, characterized, and sequenced. There are no intervening sequences within the coding region for TS-DHFR, and the G+C content is high, with a strong bias for codons possessing guanine or cytosine in the third base. From the predicted protein sequence for TS-DHFR, we conclude that the enzymes exist as separate structural domains with DHFR located at the amino terminus and TS at the carboxyl terminus; in between, there is a junctional peptide predicted to consist of a random coil flanked by two alpha-helices that corresponds to the site of a putative gene fusion. Comparison of the predicted amino acid sequence of L. tropica TS-DHFR with sequences of the monofunctional enzymes from other sources shows that the TS domain is much more conserved than is the DHFR domain and that both enzymes are more related to their monofunctional counterparts in vertebrates than in prokaryotes. Most of the recognized elements of secondary structure of other DHFRs, as ascertained by x-ray crystallography, are found in the predicted structure of the DHFR domain of the bifunctional protein.
Design and biological activity of analogs of growth hormone releasing factor with potential amphiphilic helical carboxyl termini.
Authors: Veliçelebi G , Patthi S , Kaiser E T
Published: May 2006

A 29-amino acid analog of growth hormone releasing factor (GHRF) was designed in which the sequence of the first six amino acids at the amino terminus was maintained while the postulated amphiphilic helical structure in the remainder of the molecule was optimized. The amino acid sequence of the analog differed from that of the first 29 residues of human GHRF by 13 residues. The peptide was synthesized by the solid-phase procedure in amide and free acid forms, both of which were tested for biological activity. When assayed for the ability to stimulate growth hormone secretion in primary cultures of rat anterior pituitary cells, the amide analog was 1.57 times as potent as GHRF-(1-40)-OH, which was used as the standard for comparison, while the free acid form was 1/6th as potent in the same assay. The two forms of the analog were also tested for stimulation of cAMP formation; they exhibited relative potencies similar to those observed for growth hormone secretion. The high activity of the analog provides good evidence for the importance of an amphiphilic helical structure in the carboxyl-terminal portion of the GHRF molecule.
Stimulation of vesicular stomatitis virus in vitro RNA synthesis by microtubule-associated proteins.
Authors: Hill V M , Harmon S A , Summers D F
Published: May 2006

Microtubule-associated proteins purified from bovine brains stimulated the in vitro transcription and replication reactions of vesicular stomatitis virus. The products of these reactions were intact messenger or genome-sized RNA species. A preparation from HeLa cells containing tubulin and microtubule-associated proteins also stimulated vesicular stomatitis virus transcription in vitro. This observation is in accord with previous studies, which suggested that a host cell factor was involved with the function of the vesicular stomatitis virus RNA polymerase, and others that indicated that several animal viruses displayed an association with host cell cytoskeletal elements during their replication cycles. We show evidence in this report of a host cell protein that seems to have a functional role in interacting with the virion polymerase.
Thermodynamics of the B to Z transition in poly(m5dG-dC).
Authors: Chaires J B , Sturtevant J M
Published: May 2006

Differential scanning calorimetry, temperature-dependent absorbance, and circular dichroic spectroscopy were used to study the thermodynamics of the B-Z transition in poly(m5dG-dC). In sodium phosphate buffer (pH 7.0) containing 50 mM NaCl and 1.0 mM MgCl2, the reversible B-Z transition of the polymer is centered at 38.2 degrees C and is characterized by delta Hcal = 0.61 +/- 0.07 kcal/(mol X base pair) (1 cal = 4.184 J), delta HvH = 68 +/- 7 kcal/mol, and a cooperative unit of length 110 +/- 20 base pairs. A second transition centered at 53.6 degrees C is observed and represents a conformational change of poly(m5dG-dC) from the Z to an alternate left-handed form of unknown structure. This transition does not arise from aggregation to the Z form, or from the helix-coil transition. The left-handed helix-coil transition is observed at 120.9 +/- 0.3 degrees C and is characterized by delta Hcal = 9.1 +/- 1.0 kcal/(mol X base pair). These results indicate a substantial enthalpic contribution to the B-Z transition, in contrast to previous assertions that the process is entropically driven.
Sequence-specific interaction between the replication initiator protein of plasmid pT181 and its origin of replication.
Authors: Koepsel R R , Murray R W , Khan S A
Published: May 2006

The replication of the pT181 plasmid is dependent on the plasmid-encoded initiator protein RepC. We have previously shown that RepC protein has sequence-specific endonuclease and topoisomerase-like activities. In this paper we demonstrate that this initiator protein has sequence-specific DNA-binding properties. Based on filter binding of plasmid restriction fragments, RepC protein specifically recognizes only the pT181 origin region. Using DNase I and neocarzinostatin "footprinting" techniques, we show that RepC protein specifically binds to a 32-base-pair sequence within the origin that is part of the initiator cistron. Using dimethyl sulfate as a chemical probe, we have identified the purine residues that interact with the initiator protein. The features of the DNA region that interacts with RepC protein include sequences with the potential to form Z DNA and/or hairpin structures. The specific DNA-protein interaction at the origin may be critical in the initiation of pT181 DNA replication by RepC protein in association with other host initiation proteins.
Talin is phosphorylated on tyrosine in chicken embryo fibroblasts transformed by Rous sarcoma virus.
Authors: Pasquale E B , Maher P A , Singer S J
Published: May 2006

We have examined the extent of tyrosine phosphorylation of talin, a component of the cytoskeleton localized in the focal adhesions and, therefore, a potential substrate of p60v-src, the transforming protein of Rous sarcoma virus. p60v-src is a tyrosine kinase that induces high levels of phosphotyrosine and the disorganization of the cytoskeleton in transformed cells. With a polyclonal antibody utilized in a previous study [Maher, P. A., Pasquale, E. B., Wang, J. Y. J. & Singer, S. J. (1985) Proc. Natl. Acad. Sci. USA 82, 6576-6580] for the detection of tyrosine-phosphorylated proteins, we have detected phosphotyrosine residues in talin molecules immunoprecipitated from Rous sarcoma virus-transformed, but not normal, chicken embryo fibroblasts. Phospho amino acid analysis of talin from the infected cells confirmed the presence of phosphotyrosine, in addition to phosphoserine and phosphothreonine. The extent of tyrosine modification in talin was compared to that in vinculin, the other focal adhesion component previously found to contain enhanced levels of phosphotyrosine in various retrovirus-transformed cells. A considerably (3 times) larger fraction of the talin than of the vinculin molecules was found to be phosphorylated on tyrosine. The phosphorylation of talin on tyrosine may be crucial for the expression of the abnormal morphology characteristic of cells transformed by Rous sarcoma virus.
Products of two common alleles at the locus for human placental alkaline phosphatase differ by seven amino acids.
Authors: Henthorn P S , Knoll B J , Raducha M , Rothblum K N , Slaughter C , Weiss M , Lafferty M A , Fischer T , Harris H
Published: May 2006

Amino-terminal amino acid sequences (42 residues) were determined for the products of the three common alleles at the human placental alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] gene locus. The sequences differ at position 3, which is proline in types 1 and 2 but is leucine in type 3. cDNA libraries were constructed in phage lambda gt11 and used to isolate clones covering the coding regions of types 1 and 3 cDNAs. Comparison of the deduced amino acid sequences of the types 1 and 3 proteins showed 7 differences out of 513 amino acids, each due to a single base substitution. cDNA sequence comparisons showed three silent substitutions in the coding regions and three base differences in the greater than 1 kilobase pairs of 3' untranslated sequences.
Stable transformation of tobacco by electroporation: evidence for plasmid concatenation.
Authors: Riggs C D , Bates G W
Published: May 2006

Electroporation (electric field-mediated DNA transfer) of tobacco protoplasts in the presence of the linearized plasmid pMON200 has led to the formation of transgenic plants. Defined electric shocks were delivered by capacitive discharges with readily available, low-cost electrical components. This transformation procedure is simple and efficient and may suggest a quick method for determining the appropriate electric fields for new cell systems. An optimal transformation frequency of 2.2 X 10(-4) (based on the number of cells subjected to the shock) was obtained with a single 2000-V/cm, 250-microseconds-duration capacitive discharge. Calli transformed to kanamycin resistance have been regenerated into whole plants. Southern blots of DNA from the transgenic plants demonstrate the integration of the selectable marker gene (neomycin phosphotransferase) at single or multiple genomic sites. In some cases, the plasmid appears to be integrated intact; in others, it is rearranged. The blots also provide evidence of plasmid recircularization and/or the formation of head-to-head and head-to-tail concatemers in most of the plants analyzed. Although some plants apparently have multiple integration sites, analysis of progeny obtained by self-fertilization of the transgenic plants indicates that the kanamycin-resistance marker is inherited as a single dominant gene.
Extrachromosomal sequences of hepatitis B virus DNA in peripheral blood mononuclear cells of acquired immune deficiency syndrome patients.
Authors: Noonan C A , Yoffe B , Mansell P W , Melnick J L , Hollinger F B
Published: May 2006

The primary etiologic agent of the acquired immune deficiency syndrome (AIDS) is a human T-lymphotropic retrovirus (the AIDS virus). However, the pathogenesis of this virus suggests that other cofactors may contribute to the development of clinically overt disease. The hepatitis B virus (HBV) has been implicated as a potential cofactor because HBV and AIDS virus infections frequently coexist, striking similarities exist in their epidemiologic patterns, and recent data indicate that HBV is lymphotropic. To establish the prevalence of HBV infections in lymphoid cells from individuals with AIDS-related disorders, sera and peripheral blood mononuclear cells (PBMC) from 16 males with AIDS virus infections were examined for the presence of HBV DNA by DNA X DNA blot hybridization. Fifteen (94%) of these individuals had serologic evidence of a recent or prior HBV infection. HBV DNA was detected in the PBMC of all of these patients, regardless of existing HBV serology. Among the 36 control individuals without AIDS-related symptomatology, PBMC-associated HBV DNA was detected in 8 of 14 carriers of hepatitis B surface antigen (HBsAg) and in 3 of 10 individuals immune to HBV, but it was absent from the PBMC of 12 individuals without HBV infection. In all instances, the HBV DNA was extrachromosomal and existed as replicative intermediates or high molecular weight oligomers of the viral genome. Replicative intermediates and serum-associated HBV DNA were detected in all hepatitis B e antigen-positive carriers, regardless of their clinical status. In contrast, the high molecular weight oligomers of HBV DNA were detected in the PBMC of all of the AIDS virus-infected patients examined, but in only 33% of those in the control group who had evidence of HBV infection. This finding suggests that a unique and complex HBV-host-cell interaction exists in patients infected with the AIDS virus.
Substance P-containing ganglion cells become progressively less detectable during retinotectal development in the frog Rana pipiens.
Authors: Kuljis R O , Karten H J
Published: May 2006

Substance P-like immunoreactivity (SPLI) was immunohistochemically analyzed in the retinae and optic tecta of Rana pipiens embryos and tadpoles between stages 25 of Shumway (S25) and XXV of Taylor and Kollros (TKXXV). A population of retinal ganglion cell (RGC) somata display SPLI. The number of labeled cell bodies increases in proportion and staining intensity between S25 and TKX and progressively decreases toward the end of metamorphosis. At TKXXV, only occasional cells in the periphery of the retina displaying SPLI can be observed in the RGC layer, heralding the adult condition, in which SPLI can only be seen rarely in occasional RGCs. An increasing proportion of optic nerve axons display SPLI from S25 through TKXVI, decreasing progressively thereafter toward the end of the larval period. Concurrently, SPLI appears for the first time in the superficial tectal neuropil between TKIII and TKV, with progressively increasing staining intensity and in a discrete lamina previously shown to contain retinofugal terminals in the adult. These observations corroborate inferences from previous studies indicating the existence of populations of peptidergic RGCs that terminate within precisely restricted synaptic loci in the tectum and presumably perform different functional operations in the adult. Previous observations, however, necessitated various experimental manipulations involving injuries to the visual system in order to demonstrate neuroactive peptide-like immunoreactivity in RGCs, thus allowing the possibility of posttraumatic expression of anomalous peptide phenotypes that may not reflect normal features of RGCs. The present study eliminates this variable and provides further evidence of the existence of peptidergic RGCs.
Persistent expression of v-mos oncogene in transformed cells that revert to nonmalignancy after prolonged treatment with interferon.
Authors: Sergiescu D , Gerfaux J , Joret A M , Chany C
Published: May 2006

BALB/c embryonic fibroblasts productively transformed by Moloney sarcoma virus and cultivated for over 600 generations in the presence of mouse alpha/beta interferon reverted to an apparently normal phenotype and were unable to produce tumors in nude mice. Nevertheless, the presence of an integrated Moloney sarcoma virus genome in the nonmalignant Moloney sarcoma virus-transformed interferon-treated cell DNA could be shown by focus formation upon transfection and by hybridization with a v-mos probe. After digestion with various restriction endonucleases, similar hybridization patterns of v-mos sequences were obtained with DNAs from both reverted and transformed cells. However, additional integration sites and at least twice as many copies of the oncogene were found in the nonmalignant Moloney sarcoma virus-transformed interferon-treated cell DNA. Polyadenylylated RNA extracted from reverted and control cells contained two mos-specific transcripts. Interestingly, the nonmalignant Moloney sarcoma virus-transformed interferon-treated cells produced helper virus, but no detectable mos-containing virions, suggesting that a posttranscriptional block in the v-mos gene expression had occurred in these cells. It should be stressed that, after up to 100 additional passages, cells cultured in the absence of interferon maintained their nontumorigenic character in spite of the persistent transcription of the mos oncogene.
Identification of the probable site of choleragen-catalyzed ADP-ribosylation in a Go alpha-like protein based on cDNA sequence.
Authors: Angus C W , Van Meurs K P , Tsai S C , Adamik R , Miedel M C , Pan Y C , Kung H F , Moss J , Vaughan M
Published: May 2006

Go alpha, a 39-kDa guanyl nucleotide-binding protein, is functionally and structurally similar to the alpha subunits of the stimulatory and inhibitory guanyl nucleotide-binding proteins (Gs alpha, Gi alpha) of adenylate cyclase and to the alpha subunit of transducin (T alpha), the guanyl nucleotide-binding protein of the retinal photon reception system. A cDNA clone was isolated from a bovine retinal lambda gt10 library by using oligonucleotide probes complementary to sequences in two putative T alpha clones. Partial sequence analysis revealed a deduced amino acid sequence identical to sequences of four tryptic peptides from bovine brain Go alpha. Gs alpha and T alpha are known to serve as substrates for ADP-ribosylation by choleragen. Other workers have established the sequence of the tetrapeptide in T alpha containing the arginine that is ADP-ribosylated and its location in the amino acid sequence deduced from T alpha cDNA. The Go alpha cDNA described here includes a region encoding an amino acid sequence very similar to that surrounding the ADP-ribosylation site in T alpha, consistent with observations that Go alpha can also be a substrate for choleragen. A corresponding sequence in the recently identified Gs alpha cDNA is less homologous to that in T alpha or Go alpha. The reported differences in conditions that promote choleragen-catalyzed ADP-ribosylation of Gs alpha vs. Go alpha could be related to differences in amino acid sequence in the region of the acceptor arginine.