bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.
Authors: Battey J F , Way J M , Corjay M H , Shapira H , Kusano K , Harkins R , Wu J M , Slattery T , Mann E , Feldman R I
Published: May 2006

The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B regulate numerous and varied cell physiologic processes in various cell types and have also been implicated as autocrine growth factors influencing the pathogenesis and progression of human small cell lung carcinomas. We report here the molecular characterization of the bombesin/GRP receptor. Structural analysis of cDNA clones isolated from Swiss 3T3 murine embryonal fibroblasts shows that the GRP receptor is a member of the guanine nucleotide binding protein-coupled receptor superfamily with seven predicted hydrophobic transmembrane domains. In vitro transcripts from cloned cDNA templates encompassing the predicted protein coding domain, when injected into Xenopus oocytes, resulted in expression of functional GRP receptors. The predicted amino acid sequence of the open reading frame in cDNA clones matches the amino-terminal sequence as well as the sequence of four tryptic fragments isolated from the purified protein. Expression of the GRP receptor cDNA in model systems potentially provides a powerful assay for the development of subtype-specific receptor antagonists that may prove to be of therapeutic importance in human small cell lung carcinoma.
Location of the active site for enzyme-adenylate formation in DNA ligases.
Authors: Tomkinson A E , Totty N F , Ginsburg M , Lindahl T
Published: May 2006

The enzyme-AMP reaction intermediate of the 102-kDa bovine DNA ligase I was digested with trypsin, and the adenylylated peptide was isolated by chromatography under conditions that maintain the acid-labile phosphoramidate bond. Microsequencing of the peptide showed that it contains an internal trypsin-resistant lysine residue, as expected for the site of adenylylation. Inhibition of DNA ligase I activity by pyridoxal 5'-phosphate also indicated the presence of a reactive lysine residue in the catalytic domain of the enzyme. Comparison of the known primary structures of several other DNA ligases with the adenylylated region of mammalian DNA ligase I allows their active sites to be tentatively assigned by sequence homology. The ATP-dependent DNA ligases of mammalian cells, fission yeast, budding yeast, vaccinia virus, and bacteriophages T3, T4, and T7 contain the active site motif Lys-Tyr/Ala-Asp-Gly-(Xaa)-Arg, with the reactive lysine residue flanked by hydrophobic amino acids. The distance between the postulated adenylylation site and the carboxyl terminus of the polypeptide is very similar in these ATP-dependent DNA ligases, whereas the size of the amino-terminal region is highly variable.
Corticostatic peptides cause nifedipine-sensitive volume reduction in jejunal villus enterocytes.
Authors: MacLeod R J , Hamilton J R , Bateman A , Belcourt D , Hu J , Bennett H P , Solomon S
Published: May 2006

We studied cell-volume changes caused by adding corticostatin (CS) or defensin-like peptides to villus enterocytes isolated in suspension from guinea pig jejunum. Guinea pig CS (10(-9) M) added to villus cells in Na(+)-containing medium reduced volume, but immediate cell swelling was caused by 10(-6) M guinea pig CS. In Na(+)-free N-methyl-D-glucamine-containing medium 10(-9) M guinea pig CS accelerated the initial rate of shrinkage compared with cells in N-methyl-D-glucamine-containing medium alone as well as causing greater cell shrinkage. Guinea pig CS-stimulated cell shrinkage was prevented by a Ca2(+)-channel blocker--5 microM nifedipine, by chelation of extracellular Ca2+ with 100 microM EGTA, or by omega-conotoxin (10(-9) M). The Ca2+ ionophore A23187 (2.5 microM) reduced volume when added to villus cells in N-methyl-D-glucamine-containing medium; this action was prevented by EGTA, or quinine--an inhibitor of K+ conductance, or 9-anthracenecarboxylic acid--a Cl- channel blocker, suggesting that the volume reduction occurred because K+ and Cl- conductances were activated. Guinea pig CS-stimulated volume reduction was also prevented by 100 microM quinine or 9-anthracenecarboxylic acid. We conclude that jejunal villus enterocytes possess a Ca2(+)-activated Cl- conductance and a K+ conductance that need not be stretch-activated. Corticostatic peptides cause volume reduction in villus cells by activating L-type Ca2+ channels; other defensin-like peptides were without effect.
A cyclic AMP-activated K+ channel in Drosophila larval muscle is persistently activated in dunce.
Authors: Delgado R , Hidalgo P , Diaz F , Latorre R , Labarca P
Published: May 2006

Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the presence of a potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2+. Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an increased probability of opening.
Overexpression of protein kinase C beta 1 enhances phospholipase D activity and diacylglycerol formation in phorbol ester-stimulated rat fibroblasts.
Authors: Pai J K , Pachter J A , Weinstein I B , Bishop W R
Published: May 2006

We are using a Rat-6 fibroblast cell line that stably overexpresses the beta 1 isozyme of protein kinase C (PKC) to study regulation of phospholipid hydrolysis by PKC. Stimulation of control (R6-C1) or overexpressing (R6-PKC3) cells with phorbol ester results in an increase in diacylglycerol (DAG) mass with no increase in inositol phosphates, indicating that DAG is not formed by inositol phospholipid breakdown. A more dramatic DAG increase occurs in R6-PKC3 cells (4.0-fold over basal) compared to R6-C1 cells (1.5-fold over basal). To further define the source of DAG, phosphatidylcholine (PC) pools were labeled with [3H]myristic acid or with [3H]- or [32P]alkyllyso-PC and formation of labeled phosphatidylethanol, an unambiguous marker of phospholipase D activation, was monitored. Phorbol ester-stimulated phosphatidylethanol formation is 5-fold greater in the R6-PKC3 cell line. Formation of radiolabeled phosphatidic acid (PA) is also enhanced by PKC overexpression. In cells double-labeled with [3H]- and [32P]-alkyl-lysoPC, the 3H/32P ratio of PA and PC are identical 15 min after stimulation, suggesting that a phospholipase D mechanism predominates. In support of this, the PA phosphohydrolase inhibitor propranolol decreased phorbol 12-myristate 13-acetate-stimulated DAG formation by 72%. Increases in DAG and phosphatidylethanol were inhibited by the PKC inhibitors K252a and staurosporine. These results indicate that phospholipase D is regulated by the action of PKC. Enhanced phospholipase D activity may contribute to the growth abnormalities seen in PKC-overexpressing cells.
The noncatalytic src homology region 2 segment of abl tyrosine kinase binds to tyrosine-phosphorylated cellular proteins with high affinity.
Authors: Mayer B J , Jackson P K , Baltimore D
Published: May 2006

Several proteins implicated in the regulation of cell proliferation contain a common noncatalytic domain, src homology region 2 (SH2). We have used the bacterially expressed SH2 domain of abl protein-tyrosine kinase to evaluate the ability of this domain to bind to cellular proteins. ablSH2 specifically bound to a number of tyrosine-phosphorylated proteins from cells transformed by tyrosine kinase oncogenes in a filter-binding assay and to a subset of those proteins in solution. The SH2 probe bound almost exclusively to tyrosine-phosphorylated proteins, and binding was eliminated by dephosphorylation of cell proteins. Free phosphotyrosine could partially disrupt SH2 binding, suggesting that phosphotyrosine is directly involved in the binding interaction. These results demonstrate that an SH2 domain is sufficient to confer direct, high-affinity phosphotyrosine-dependent binding to proteins and suggest a general role for SH2 domains in cellular signaling pathways.
Intracellular Ca2+ buffers disrupt muscarinic suppression of Ca2+ current and M current in rat sympathetic neurons.
Authors: Beech D J , Bernheim L , Mathie A , Hille B
Published: May 2006

The role of intracellular Ca2+ concentration ([Ca2+]i) in the muscarinic suppression of Ca2+ current and M-type K+ current has been investigated in isolated rat sympathetic neurons using the whole-cell patch-clamp technique and fura-2 fluorescence measurements. Muscarinic stimulation suppressed currents without raising [Ca2+]i. Nonetheless, intracellular bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) (11-12 mM), a Ca2+ chelator, reduced Ca2(+)-current suppression from 82 to 15%. For the latter, we explain the BAPTA action by a requirement for a certain minimum [Ca2+]i for continued operation of the pathway coupling muscarinic receptors to M-type K+ channels. The pathway coupling muscarinic receptors to Ca channels also showed some dependence on [Ca2+]i, but there may also be a blocking action of BAPTA that is independent of Ca2+ chelation.
Bleomycin-resistance gene derived from the transposon Tn5 confers selective advantage to Escherichia coli K-12.
Authors: Blot M , Meyer J , Arber W
Published: May 2006

The plasmid pRAB2 contains a silent operon derived from the transposon Tn5 and carrying the gene neo for neomycin-kanamycin resistance and a truncated ble gene (ble333) for bleomycin resistance. Spontaneous mutants that express the two resistances provide Escherichia coli cells an improved fitness during the phase of decline in the absence of the antibiotics. It is shown that the ble333 gene product is responsible for this better fitness. These results can explain a previously described selective advantage attributed to the presence of Tn5. The improved fitness of bleomycin-resistant bacteria is proposed to relate to DNA repair by the ble gene product. The consequences of the presence of an accessory gene improving fitness are discussed in terms of evolutionary stable strategy of a transposon in populations of E. coli.
Fyn tyrosine kinase associated with Fc epsilon RII/CD23: possible multiple roles in lymphocyte activation.
Authors: Sugie K , Kawakami T , Maeda Y , Kawabe T , Uchida A , Yodoi J
Published: May 2006

Expression of low-affinity Fc receptor for IgE (Fc epsilon RII), which is identical to the lymphocyte differentiation antigen CD23, is associated with activation of lymphoid cells. The mechanism of signal transduction through Fc epsilon RII/CD23 was dissected by transfection of cDNA coding for Fc epsilon RII to the YT human natural killer-like cell line, activation of which was easily detected by the induction of interleukin 2 receptor/p55(Tac). Cross-linking of Fc epsilon RII/CD23 with H107 anti-Fc epsilon RII monoclonal antibody markedly enhanced interleukin 2 receptor/p55 expression on the YT cells transfected with Fc epsilon RII cDNA (YTSER cells). Similar induction of interleukin 2 receptor/p55 by the cross-linking of Fc epsilon RII was observed on an Epstein-Barr virus-transformed B-cell line, 3B6, and fresh leukemic cells isolated from a patient with B-cell chronic lymphoblastic leukemia. Thus Fc epsilon RII/CD23 provides activation signals not only in YTSER cells but also in activated B cells. A possible involvement of protein-tyrosine kinase in the Fc epsilon RII-mediated signal transduction was studied. Fc epsilon RII was physically associated with a src family tyrosine kinase p59fyn and not with p56lck, which was also found in YT cells. Recently it was reported that p59fyn was associated with T-cell antigen receptor. Our results collectively suggest the multiple functions of p59fyn that may be implicated in Fc epsilon RII-mediated activation signal in YT cells.
Formation of phosphatidylinositol 3-phosphate by isomerization from phosphatidylinositol 4-phosphate.
Authors: Walsh J P , Caldwell K K , Majerus P W
Published: May 2006

We have synthesized phosphatidylinositol 3-phosphate from phosphatidylinositol 4-phosphate by using diisopropylcarbodiimide to promote migration of the 4-phosphate via a cyclic phosphodiester intermediate. The product was isolated by a thin-layer chromatographic method that depends on the ability of phosphatidylinositol 4-phosphate, but not phosphatidylinositol 3-phosphate, to form complexes with boric acid. The final yield of the procedure was 8% phosphatidylinositol 3-phosphate, which was approximately 80% pure. The product was shown to be phosphatidylinositol 3-phosphate by the following criteria: (i) cochromatography with an authentic standard on borate thin-layer chromatography, (ii) cochromatography of the deacylated product with glycerophosphoinositol 3-phosphate on high-performance liquid chromatography, (iii) conversion of the product to phosphatidylinositol by homogeneous phosphatidylinositol 3-phosphate 3-phosphatase, and (iv) deacylation and deglyceration of the product to a compound that comigrates with inositol 1,3-bisphosphate on high-performance liquid chromatography. The availability of mass amounts of phosphatidylinositol 3-phosphate will allow further elaboration of reactions in this recently discovered pathway of phosphatidylinositol metabolism.
Pheromonal advertisement of a nuptial gift by a male moth (Utetheisa ornatrix).
Authors: Dussourd D E , Harvis C A , Meinwald J , Eisner T
Published: May 2006

Male Utetheisa ornatrix produce a courtship pheromone (hydroxydanaidal) that they derive from systemic pyrrolizidine alkaloid of plant origin. Pheromone titers in males correlate with systemic levels of alkaloid and with the quantity of alkaloid transmitted to the female at mating. The male's emission of pheromone during courtship may therefore communicate his possession of protective alkaloids and his capacity to provision the female. By mating preferentially with males endowed with hydroxydanaidal, females may ensure their acquisition of an alkaloidal gift for use in egg defense.
Evidence for increased in vitro recombination with insertion of human hepatitis B virus DNA.
Authors: Hino O , Tabata S , Hotta Y
Published: May 2006

Chromosomal translocation, deletion, and inversion/duplication directly linked to hepatitis B virus (HBV) DNA integration occur frequently in host DNA of human hepatocellular carcinomas. To test the possible recombinogenic effect of HBV DNA, we have utilized an in vitro recombination assay. Fragments containing the region spanning DR1, which is believed to be the origin of viral replication and a preferred site in the viral genome for integration, increased the recombination events reproducibly in the presence of extracts from actively dividing cells (e.g., hepatocellular carcinoma) but not resting cells (e.g., normal liver). Moreover, in these extracts we have found a protein(s) that specifically binds to these HBV DNA fragments. These results support the notion that in some instances integrated HBV DNAs cause further genomic instability, possibly involving specific cellular protein(s). The fact that extracts from nondividing, normal liver did not increase recombination events suggests that genomic instability depends upon active cellular growth, a feature more commonly found subsequent to HBV-induced hepatocellular injury than in healthy liver. Our results offer an explanation for the high incidence of liver cancer that accompanies chronic hepatitis and add HBV to the list of agents that can cause genetic recombination.
The two different receptors for tumor necrosis factor mediate distinct cellular responses.
Authors: Tartaglia L A , Weber R F , Figari I S , Reynolds C , Palladino M A , Goeddel D V
Published: May 2006

The individual roles of the murine type 1 and type 2 tumor necrosis factor (TNF) receptors (TNF-R1 and TNF-R2) were investigated utilizing (i) the strong species specificity of TNF-R2 for murine TNF compared to human TNF and (ii) agonistic rabbit polyclonal antibodies directed against the individual TNF receptors. Proliferation of mouse thymocytes and the murine cytotoxic T-cell line CT-6 is stimulated by murine TNF but not by human TNF. Consistent with this observation, polyclonal antibodies directed against TNF-R2 induced proliferation in both of these cell types, whereas polyclonal antibodies directed against TNF-R1 had no effect. In contrast, cytotoxicity in murine LM cells (which are sensitive to murine and human TNF) was induced by antibodies against TNF-R1 but not by antibodies against TNF-R2. Also, the steady-state level of manganous superoxide dismutase mRNA in the murine NIH 3T3 cell line was induced by murine TNF, human TNF, and anti-TNF-R1 but not by anti-TNF-R2. These results suggest that TNF-R2 initiates signals for the proliferation of thymocytes and cytotoxic T cells, whereas TNF-R1 initiates signals for cytotoxicity and the induction of the protective activity, manganous superoxide dismutase. The nonredundant signaling observed for the two TNF receptors cannot be explained simply by the differential expression of the two TNF receptors in the various cell types, because LM cells express on their surface higher levels of TNF-R2 than TNF-R1, and LM cells, NIH 3T3 cells, and thymus cells all express mRNA corresponding to both receptor types. It is therefore likely that the two receptors initiate distinct signaling pathways that result in the induction of different cellular responses.
Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function.
Authors: Hsieh J C , Jurutka P W , Galligan M A , Terpening C M , Haussler C A , Samuels D S , Shimizu Y , Shimizu N , Haussler M R
Published: May 2006

The vitamin D receptor (VDR) is known to be a phosphoprotein and inspection of the deduced amino acid sequence of human VDR (hVDR) reveals the conservation of three potential sites of phosphorylation by protein kinase C (PKC)--namely, Ser-51, Ser-119, and Ser-125. Immunoprecipitated extracts derived from a rat osteoblast-like osteosarcoma cell line that contains the VDR in high copy number were incubated with the alpha, beta, and gamma isozymes of PKC, and VDR proved to be an effective substrate for PKC-beta, in vitro. When hVDR cDNAs containing single, double, and triple mutations of Ser-51, Ser-119, and Ser-125 were expressed in CV-1 monkey kidney cells, immunoprecipitated and phosphorylated by PKC-beta, in vitro, the mutation of Ser-51 selectively abolished phosphorylation. Furthermore, when transfected CV-1 cells were treated with phorbol 12-myristate 13-acetate, a PKC activator, phosphorylation of wild-type hVDR was enhanced, whereas that of the Ser-51 mutant hVDR was unaffected. Therefore, Ser-51 is the site of hVDR phosphorylation by PKC, both in vitro and in vivo. To evaluate the functional role of Ser-51 and its potential phosphorylation, hVDR-mediated transcription was tested using cotransfection with expression plasmids and a reporter gene that contained a vitamin D response element. Mutation of Ser-51 markedly inhibited transcriptional activation by the vitamin D hormone, suggesting that phosphorylation of Ser-51 by PKC could play a significant role in vitamin D-dependent transcriptional activation. Therefore, the present results link the PKC signal transduction pathway of growth regulation and tumor promotion to the phosphorylation and function of VDR.
Models of human metastatic colon cancer in nude mice orthotopically constructed by using histologically intact patient specimens.
Authors: Fu X Y , Besterman J M , Monosov A , Hoffman R M
Published: May 2006

There is an important need for clinically relevant animal models for human cancers. Toward this goal, histologically intact human colon-cancer specimens derived surgically from patients were implanted orthotopically to the colon or cecum of nude mice. We have observed extensive orthotopic growth in 13 of 20 cases of implanted patient colon tumors. These showed various growth patterns with subsequent regional, lymph-node, and liver metastasis, as well as general abdominal carcinomatosis. Thus, models for human colon cancer have been developed that show (i) local growth, (ii) abdominal metastasis, (iii) general abdominal carcinomatosis with extensive peritoneal seeding, (iv) lymph-node metastasis, (v) liver metastasis, and (vi) colonic obstruction. These models permit the passage of the tumors to form large cohorts. They will facilitate research into the biology of colon cancer metastatic capability and the development of new drugs active against metastatic cancer. These models may also predict the clinical course and the in vivo response to drugs of the cancer of individual patients.
Noninvasive detection of changes in membrane potential in cultured neurons by light scattering.
Authors: Stepnoski R A , LaPorta A , Raccuia-Behling F , Blonder G E , Slusher R E , Kleinfeld D
Published: May 2006

We report a procedure to detect electrical activity in cultured neurons by changes in their intrinsic optical properties. Using dark-field microscopy to detect scattered light, we observe an optical signal that is linearly proportional to the change in the membrane potential. Action potentials can be recorded without signal averaging. We use the dark-field method to show that there are substantial time delays between activity in the soma and in fine distal processes of identified Aplysia neurons. The biophysical basis for the change in optical properties of the neuron was deduced from measurements of the angular distribution of scattered laser light. An analysis of the data indicates that the radial component of the index of refraction of the membrane increases and the tangential components decrease concomitant with an increase in membrane potential. This is suggestive of a rapid reorientation of dipoles in the membrane during an action potential.
Crystal structure of recombinant human T-cell cyclophilin A at 2.5 A resolution.
Authors: Ke H M , Zydowsky L D , Liu J , Walsh C T
Published: May 2006

The structure of the unligated human T-cell recombinant cyclophilin has been determined at 3 A resolution by multipole isomorphous replacement methods and refined at 2.5 A resolution to an R factor of 0.209. The root-mean-square errors of the bond lengths and bond angles are 0.013 A and 2.8 degrees from ideal geometry, respectively. The overall structure is a beta-barrel, consisting of eight antiparallel beta-strands wrapping around the barrel surface and two alpha-helices sitting on the top and the bottom closing the barrel. Inside the barrel, seven aromatic and other hydrophobic residues form a compact hydrophobic core. A loop of Lys-118 to His-126 and four beta-strands (B3-B6) constitute a pocket we speculate to be the binding site of cyclosporin A.
Positive DNA supercoiling generates a chromatin conformation characteristic of highly active genes.
Authors: Lee M S , Garrard W T
Published: May 2006

During transcription, positive DNA supercoils generated ahead of RNA polymerase could theoretically uncoil the negative DNA supercoils associated with nucleosomes and thereby decondense the chromatin fiber in preparation for RNA polymerase passage. Here we examine the effect of positive DNA supercoiling on the structure of yeast 2-microns minichromosomes. We utilized a conditional topoisomerase mutant expressing Escherichia coli topoisomerase I to convert the DNA supercoiling state from negative to positive in vivo. Minichromosomes containing positively supercoiled DNA exhibited a striking increase in DNase I sensitivity. They also displayed additional micrococcal nuclease cleavage sites but yielded nearly typical nucleosomal ladders after extensive digestion. Upon in vitro relaxation with eukaryotic topoisomerase I, the minichromosomes remained DNase I sensitive but were converted to negative DNA supercoiling with a slightly increased linking number compared to typical minichromosomes, thus indicating the presence of bound histones. Therefore, positive DNA supercoiling provides a mechanism for generating, but is not required for maintaining, a conformation in chromatin characteristic of highly transcribed genes.
Genetic variants of serum albumin in Americans and Japanese.
Authors: Madison J , Arai K , Sakamoto Y , Feld R D , Kyle R A , Watkins S , Davis E , Matsuda Y , Amaki I , Putnam F W
Published: May 2006

A collaborative search for albumin genetic variants (alloalbumins) was undertaken by cellulose acetate and agarose electrophoresis at pH 8.6 of the sera of patients at two major medical centers in the United States and of nearly 20,000 blood donors in Japan. Seventeen instances of alloalbuminemia were ascertained, and seven different alloalbumin types were characterized by structural study. Two previously unreported alloalbumin types were identified. In one type, which was present in a Caucasian family and designated Iowa City-1, aspartic acid at position 365 was replaced by valine (365 Asp----Val); this is the second reported mutation at this position. The other type present in a Japanese blood donor had the mutation 128 His----Arg. An unexpected finding was the presence in a single Japanese of a Naskapi-type alloalbumin (372 Lys----Glu), a variant that had previously been described only for certain Amerindian tribes in whom it occurs with a polymorphic frequency (greater than 1%) and in Eti Turks. An arginyl-albumin (-1 Arg, 1 Asp----Val) occurred in an American family. The other alloalbumin types identified were proalbumins Lille and Christchurch and albumin B that have a cumulative frequency of about 1:3500 in Caucasians probably because of the hypermutability of CpG dinucleotides at the mutated sites. All of the variants characterized in this study are point mutants, and the sites are spread throughout the albumin gene. However, about one-fourth of all known albumin mutations are clustered in the sequence segment from position 354 through 382.
Amino acids 367-376 of the Gs alpha subunit induce membrane association when fused to soluble amino-terminal deleted Gi1 alpha subunit.
Authors: Journot L , Pantaloni C , Poul M A , Mazarguil H , Bockaert J , Audigier Y
Published: May 2006

Signal transduction GTP-binding proteins are tightly associated with plasma membrane. In the resting state, the anchorage of the alpha subunit could be indirect by means of the other beta gamma subunits or polydisperse multimers. In the activated state, although the alpha subunit is dissociated from other subunits, it is not released from the membrane and therefore is likely to contain information necessary to remain associated with the plasma membrane. Previous proteolytic experiments suggested that, in contrast to other G proteins alpha subunits, the C-terminal domain of Gs alpha (the G protein involved in adenylate cyclase stimulation) is essential for membrane association of the activated form. To better define the crucial residues involved in membrane attachment, we constructed chimeras between a soluble core and various parts of the Gs alpha C-terminal domain. We first deleted codons 2-6 of Gi1 alpha (the inhibitory G protein of the i1 subtype) to generate a soluble GTP-binding protein, delta N-Gi1 alpha. We then replaced the last 14 C-terminal codons of delta N-Gi1 alpha by different domains of the Gs alpha C terminus and looked for the membrane association of chimeric proteins after in vitro transcription, in vitro translation, and interaction with S49 cyc- membranes (obtained from a mutant cell line that does not express Gs alpha). Our results showed that addition of amino acids 367-376 of Gs alpha is sufficient to promote membrane association of the soluble N-terminal deleted Gi1 alpha.
Trans splicing in trypanosomes requires methylation of the 5' end of the spliced leader RNA.
Authors: Ullu E , Tschudi C
Published: May 2006

Trypanosoma brucei spliced leader (SL) RNA contains an unusual cap 4 structure consisting of 7-methylguanosine linked to four modified nucleosides. During RNA maturation, trans splicing transfers the first 39 nucleotides of the SL RNA including the cap structure to the 5' end of all mRNAs. Here we show that exposure of permeable trypanosome cells to S-adenosyl-L-homocysteine inhibits methylation of the nucleosides adjacent to 7-methylguanosine of newly synthesized SL RNA and prevents utilization of the SL RNA in trans splicing. However, trans splicing of the SL RNA preexisting in the cells is not inhibited by S-adenosyl-L-homocysteine as shown by the observation that newly synthesized alpha-tubulin RNA is trans spliced at the same level as in control cells. Therefore, it appears that the newly synthesized SL RNA is the only known component of the trans-splicing machinery that is impaired in its function by inhibition of methylation. Undermethylation does not alter either the stability of the SL RNA or the electrophoretic mobility and chromatographic behavior of the core SL ribonucleoprotein particle. Taken together, our data suggest that the cap 4 structure of the SL RNA plays an essential role in the trans-splicing process.
Mouse retinoic acid receptor alpha 2 isoform is transcribed from a promoter that contains a retinoic acid response element.
Authors: Leroy P , Nakshatri H , Chambon P
Published: May 2006

We have characterized the promoter of the mouse retinoic acid receptor alpha 2 (mRAR-alpha 2) isoform. This promoter contains a retinoic acid response element (RARE) that closely resembles the RARE that is present in the RAR-beta 2 promoter. Moreover, RAR-alpha 2 and RAR-beta 2 proximal promoter sequences are similar to each other and generate transcripts whose respective start sites are located at similar positions. The RAR-alpha 2 RARE consists of two directly repeated 5'-GTTCA-3' motifs to which all three RARs (alpha, beta, and gamma) bind in vitro.
Centrosomes competent for parthenogenesis in Xenopus eggs support procentriole budding in cell-free extracts.
Authors: Tournier F , Cyrklaff M , Karsenti E , Bornens M
Published: May 2006

Heterologous centrosomes from diversed species including humans promote egg cleavage when injected into metaphase-arrested Xenopus eggs. We have recently isolated centrosomes from calf thymocytes and shown that they were unable to induce egg cleavage, an inability that was apparently correlated with the peculiar structure of these centrosomes rather than with a lack of microtubule-nucleating activity: the two centrioles were associated in a colinear orientation by their proximal ends. To promote cleavage, a heterologous centrosome probably is required to duplicate, although this has not yet been demonstrated. Therefore, we designed an in vitro assay that would enable us to directly observe the duplication process. We show that competent centrosomes from KE37 cells synchronized in G1 phase initiate procentriole budding in interphasic extracts from Xenopus eggs in the absence of protein synthesis, whereas calf thymocyte centrosomes do not. Since calf thymocyte centrosomes do not support parthenogenesis, the present results suggest that duplication of the foreign centrosome is required for centrosome-induced parthenogenesis. Furthermore, procentriole budding takes place in the absence of protein synthesis in egg extracts arrested in S phase. This in vitro assay should contribute to the identification of molecular mechanisms involved in the initiation of centrosome duplication.
virF, the host-range-determining virulence gene of Agrobacterium tumefaciens, affects T-DNA transfer to Zea mays.
Authors: Jarchow E , Grimsley N H , Hohn B
Published: May 2006

The monocotyledonous plant Zea mays does not develop tumors after inoculation with Agrobacterium tumefaciens and is thus defined as nonhost. Agroinfection, Agrobacterium-mediated delivery of maize streak virus, demonstrates that transferred DNA (T-DNA) transfer to the plant does occur. Nopaline-type Agrobacterium strains such as C58 are efficient in the transfer process whereas the octopine-type strain A6 is unable to transfer T-DNA to maize. This phenotypic difference maps to the tumor-inducing (Ti) plasmid but not to the T-DNA. Steps preceding T-DNA transfer, such as attachment and induction of the virulence genes, were shown to take place in the octopine strain. The nopaline-plasmid-specific locus tzs and the octopine-plasmid-specific locus pinF (virH) are not involved in the strain specificity. However, mutations in the virF locus rendered the octopine strain agroinfectious on maize, whereas such virF-defective octopine strains, when complemented by virF on a plasmid, completely lost their agroinfectivity. We propose that VirF, known to increase the host range of the bacteria in other systems, acts as an inhibitor of T-DNA transfer to maize.
GAL4 is phosphorylated as a consequence of transcriptional activation.
Authors: Sadowski I , Niedbala D , Wood K , Ptashne M
Published: May 2006

GAL4 protein isolated from yeast in which it is active is phosphorylated predominantly on two different serine residues. One of these was identified as Ser-837; substitution of this residue for alanine has no detectable effect on transcriptional activation by GAL4. Phosphorylation at Ser-837 requires that both the DNA binding and transcriptional activation functions be intact. We propose that some phosphorylations of GAL4, including that at Ser-837, occur concomitantly with activation of transcription.
Ring fission of anthracene by a eukaryote.
Authors: Hammel K E , Green B , Gai W Z
Published: May 2006

Ligninolytic fungi are unique among eukaryotes in their ability to degrade polycyclic aromatic hydrocarbons (PAHs), but the mechanism for this process is unknown. Although certain PAHs are oxidized in vitro by the fungal lignin peroxidases (LiPs) that catalyze ligninolysis, it has never been shown that LiPs initiate PAH degradation in vivo. To address these problems, the metabolism of anthracene (AC) and its in vitro oxidation product, 9,10-anthraquinone (AQ), was examined by chromatographic and isotope dilution techniques in Phanerochaete chrysosporium. The fungal oxidation of AC to AQ was rapid, and both AC and AQ were significantly mineralized. Both compounds were cleaved by the fungus to give the same ring-fission metabolite, phthalic acid, and phthalate production from AQ was shown to occur only under ligninolytic culture conditions. These results show that the major pathway for AC degradation in Phanerochaete proceeds AC----AQ----phthalate + CO2 and that it is probably mediated by LiPs and other enzymes of ligninolytic metabolism.
Functional expression cloning and characterization of the hepatocyte Na+/bile acid cotransport system.
Authors: Hagenbuch B , Stieger B , Foguet M , Lübbert H , Meier P J
Published: May 2006

Liver parenchymal cells continuously extract high amounts of bile acids from portal blood plasma. This uptake process is mediated by a Na+/bile acid cotransport system. A cDNA encoding the rat liver bile acid uptake system has been isolated by expression cloning in Xenopus laevis oocytes. The cloned transporter is strictly sodium-dependent and can be inhibited by various non-bile-acid organic compounds. Sequence analysis of the cDNA revealed an open reading frame of 1086 nucleotides coding for a protein of 362 amino acids (calculated molecular mass 39 kDa) with five possible N-linked glycosylation sites and seven putative transmembrane domains. Translation experiments in vitro and in oocytes indicate that the transporter is indeed glycosylated and that its polypeptide backbone has an apparent molecular mass of 33-35 kDa. Northern blot analysis with the cloned probe revealed crossreactivity with mRNA species from rat kidney and intestine as well as from liver tissues of mouse, guinea pig, rabbit, and man.
CD10/neutral endopeptidase 24.11 hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung.
Authors: Shipp M A , Tarr G E , Chen C Y , Switzer S N , Hersh L B , Stein H , Sunday M E , Reinherz E L
Published: May 2006

Bombesin-like peptides are essential autocrine growth factors for many small cell carcinomas (SCCas) of the lung. Herein, we demonstrate that these malignant pulmonary neuroendocrine cells express low levels of the cell surface metalloendopeptidase CD10/neutral endopeptidase 24.11 (CD10/NEP, common acute lymphoblastic leukemia antigen) and that this enzyme hydrolyzes bombesin-like peptides. The growth of bombesin-like peptide-dependent SCC as is inhibited by CD10/NEP and potentiated by CD10/NEP inhibition. The results provide evidence that CD10/NEP is involved in the regulation of tumor cell proliferation. Since SCCa of the lung occurs almost exclusively in cigarette smokers and cigarette smoke inactivates CD10/NEP, decreased cell surface CD10/NEP enzymatic activity may be causally related to the development of SCCa of the lung.
Characterization of human mineralocorticosteroid receptor expressed in the baculovirus system.
Authors: Binart N , Lombes M , Rafestin-Oblin M E , Baulieu E E
Published: May 2006

To investigate the structure and function of the mineralocorticosteroid receptor (MR), one has to circumvent the major difficulty related to its very low abundance. For this purpose, the full-length human MR (hMR) has been produced using the efficient baculovirus system. The recombinant protein is overexpressed in Sf9 insect cells at a concentration of approximately 2 pmol/mg of protein, which is 50-100 times more than the concentration in aldosterone target tissues. It binds aldosterone with high affinity (Kd approximately 1 nM) and clearly displays a mineralocorticosteroid specificity as evidenced by competition studies with steroid ligands and by the monoclonal anti-idiotypic antibody H10E interacting with the steroid-binding domain of MR. After [35S]methionine labeling, a single polypeptide band at approximately 120 kDa is detected and further identified as hMR by immunoblotting with A4, an anti-peptide antibody. Sedimentation analyses show that the native form of MR is recognized by A4 and B174, an antibody to the 90-kDa heat shock protein, since they both induce a shift of the receptor from 9S to 11S sedimentation coefficient. These results clearly demonstrate that MR is a heterooligomer containing the insect equivalent of the 90-kDa heat shock protein. This 9S receptor complex is converted to a 4S form during high-salt gradient ultracentrifugation, suggesting that MR can undergo a complete in vitro transformation. Immunofluorescence studies indicate that hMR, which is almost exclusively a cytoplasmic protein in Sf9 cells, is translocated to the nucleus after aldosterone exposure. Therefore, the recombinant hMR seems to behave as the native receptor. Given the possibility of large-scale protein production, the baculovirus system should prove useful in studies of the molecular basis of MR function as a transcription factor.
Involvement of gap junctions in tumorigenesis: transfection of tumor cells with connexin 32 cDNA retards growth in vivo.
Authors: Eghbali B , Kessler J A , Reid L M , Roy C , Spray D C
Published: May 2006

Gap junction channels provide a pathway for exchange of ions and small molecules between coupled cells, and this exchange is believed to be critical for normal tissue growth and development. As a test for a role of gap junction-mediated intercellular communication in control of cell growth, we have compared growth rates of communication-deficient human tumor cells (SKHep1) with clones stably transfected with cDNA encoding the rat liver gap junction protein connexin 32. In culture, growth rates for parental and transfected clones were similar. However, when sizes of tumors were evaluated following injection of these clones into athymic nude mice, growth rates for two well-coupled clones were significantly lower than for communication-deficient or poorly coupled clones. This study demonstrates that growth rate of these tumor cells in situ is negatively correlated with strength of intercellular communication.
A herpesvirus maturational proteinase, assemblin: identification of its gene, putative active site domain, and cleavage site.
Authors: Welch A R , Woods A S , McNally L M , Cotter R J , Gibson W
Published: May 2006

A herpesvirus proteinase activity has been identified and partially characterized by using the cloned enzyme and substrate genes in transient transfection assays. Evidence is presented that the proteinase gene of cytomegalovirus strain Colburn encodes a 590-amino acid protein whose N-terminal 249 residues contain the proteolytic activity and two domains that are highly conserved in the homologous protein of other herpesviruses. Insertion of a short amino acid sequence between these domains abolished proteinase activity, suggesting that this region constitutes part or all of the enzyme active site. Plasma desorption mass spectrometry was used to identify the C terminus of the mature assembly protein as alanine, enabling the recognition of a consensus proteinase cleavage sequence of V/L-X-A decreases S/V, near the C-terminal end of all herpesvirus assembly protein homologs. Interestingly, the proteinase and its substrate, the assembly protein precursor, are encoded by opposite halves of the same open reading frame.
Both activation and repression of a-mating-type-specific genes in yeast require transcription factor Mcm1.
Authors: Elble R , Tye B K
Published: May 2006

Mcm1 is a yeast transcription factor with homologs throughout the metazoa. MCM1 was first identified as a gene involved in maintenance of artificial minichromosomes in yeast. More recently Mcm1 has been shown to serve as a transcriptional regulator of mating-type-specific genes. Biochemical data suggest that Mcm1 coactivates alpha-specific genes and corepresses a-specific genes by binding to a 10-base-pair dyad symmetry element in their upstream regions. We reported previously that an mcm1 point mutation reduced activation of alpha-specific genes but had little effect on the expression of a-specific genes. We now show that another mcm1 allele, which depletes the Mcm1 protein, affects both activation and repression of a-specific genes. The mutant strain remains capable of high levels of pheromone induction of a-specific genes, although with retarded kinetics. Mcm1 joins an increasing number of transcription factors involved in both positive and negative regulation of gene expression.
In vivo biodistribution of a radiolabeled blood substitute: 99mTc-labeled liposome-encapsulated hemoglobin in an anesthetized rabbit.
Authors: Rudolph A S , Klipper R W , Goins B , Phillips W T
Published: May 2006

Liposome-encapsulated hemoglobin (LEH) is an erythrocyte substitute that is a potential resuscitative fluid for the in vivo delivery of oxygen. We have noninvasively imaged radiolabeled LEH in vivo with technetium-99m (99mTc) to study the biodistribution in an anesthetized rabbit. Rabbits (2.5 kg, n = 8) were infused with 30 ml of LEH (200 mg of phospholipid, 2.5 g of hemoglobin per kg of body weight) and imaged with a gamma camera continuously for 2 hr. At 20 hr postinfusion, the animals were imaged again and sacrificed; the organs were weighted and their radioactivity was determined for autopsy organ distribution. Organ uptake from the images was corrected for organ-associated blood pool, which was determined by infusion of 99mTc-labeled rabbit erythrocytes. Blood pool and decay-corrected biodistribution data reveal the kinetics of LEH distribution, with an initial rapid uptake by the liver, 8% at 30 min and 15% at 2 hr. The spleen accumulates less LEH initially, 3% at 30 min and 7% at 2 hr, with an apparent linear uptake of LEH over this time period. Image biodistribution data was also validated at 20 hr by tissue sampling. At 20 hr postinfusion, autopsy biodistribution data reveals approximately 42.6% of the total counts remaining in the blood, 15.4% in the liver, 18.1% in spleen, 3.2% in the lungs, 2.4% in muscle, 1.6% in urine, and trace levels in the kidney, brain, and heart (less than 1%). There is no evidence of hemoglobin release from LEH or kidney dysfunction (normal creatinine and blood urea nitrogen) at any time over the course of the study.
Completely functional double-barreled chloride channel expressed from a single Torpedo cDNA.
Authors: Bauer C K , Steinmeyer K , Schwarz J R , Jentsch T J
Published: May 2006

We have performed an electrophysiological analysis of the recently cloned Torpedo marmorata Cl- channel. Functional expression of Cl- channels in oocytes of Xenopus laevis previously injected with cRNA yielded an outward-rectifying current activated by hyperpolarization. Replacement of Cl- with other anions significantly reduced or inhibited the current. Single-channel recordings from cell-attached patches exhibited burst-like Cl- channel activity with rapid fluctuations between three equally spaced substates (0 pS, 9 pS, and 18 pS). The properties of the cloned Cl- channel were almost identical to those of the reconstituted native T. californica Cl- channel and were in full agreement with the predictions of the double-barreled channel model [Hanke, W. & Miller, C. (1983) J. Gen. Physiol. 82, 25-45]. Our results imply that the cloned cDNA codes for the completely functional Torpedo electroplax Cl- channel.
Uncoupling thermotolerance from the induction of heat shock proteins.
Authors: Smith B J , Yaffe M P
Published: May 2006

Exposure of cells to elevated temperatures causes a rapid increase in the synthesis of heat shock proteins (hsps) and induces thermotolerance, the increased ability of cells to survive exposure to lethal temperatures; however, the connection between hsp induction and the acquisition of thermotolerance is unclear. hsp induction in the yeast Saccharomyces cerevisiae is mediated by the activation of heat-shock transcription factor, and recently we have described a mutation, hsf1-m3, in heat-shock transcription factor that prevents the factor's activation. We now demonstrate that this mutation results in a general block in heat-shock induction but does not affect the acquisition of thermotolerance. Our results indicate that high-level induction of the major hsps is not required for cells to acquire thermotolerance.
Predicting chaos for infinite dimensional dynamical systems: the Kuramoto-Sivashinsky equation, a case study.
Authors: Smyrlis Y S , Papageorgiou D T
Published: May 2006

The results of extensive computations are presented to accurately characterize transitions to chaos for the Kuramoto-Sivashinsky equation. In particular we follow the oscillatory dynamics in a window that supports a complete sequence of period doubling bifurcations preceding chaos. As many as 13 period doublings are followed and used to compute the Feigenbaum number for the cascade and so enable an accurate numerical evaluation of the theory of universal behavior of nonlinear systems, for an infinite dimensional dynamical system. Furthermore, the dynamics at the threshold of chaos exhibit a self-similar behavior that is demonstrated and used to compute a universal scaling factor, which arises also from the theory of nonlinear maps and can enable continuation of the solution into a chaotic regime. Aperiodic solutions alternate with periodic ones after chaos sets in, and we show the existence of a period six solution separated by chaotic regions.