bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletions of a DNA sequence in retinoblastomas and mesenchymal tumors: organization of the sequence and its encoded protein.
Authors: Friend S H , Horowitz J M , Gerber M R , Wang X F , Bogenmann E , Li F P , Weinberg R A
Published: May 2006

Retinoblastoma is a childhood tumor that can arise because of mutant alleles acquired as somatic or germinal mutations. The mutant allele can be carried in the germ line. The mutations creating these alleles act by inactivating copies of a recessive oncogene located within band q14 of chromosome 13 and termed the RB1 locus. We have reported isolation of a cDNA fragment that recognizes chromosomal sequences possessing many of the attributes of the retinoblastoma gene associated with the RB1 locus. We now report that this segment is additionally the target of somatic mutations in mesenchymal tumors among patients having no apparent predisposition to retinoblastoma and no previous evidence of retinoblastoma. These tumors provide additional evidence that the cloned sequences are representative of a gene that is a frequent target of inactivation during tumorigenesis. Sequence analysis of this cDNA provides little insight into its normal functional role.
The breakpoint of an inversion of chromosome 14 in a T-cell leukemia: sequences downstream of the immunoglobulin heavy chain locus are implicated in tumorigenesis.
Authors: Baer R , Heppell A , Taylor A M , Rabbitts P H , Boullier B , Rabbitts T H
Published: May 2006

T-cell tumors are characterized by inversions or translocations of chromosome 14. The breakpoints of these karyotypic abnormalities occur in chromosome bands 14q11 and 14q32--the same bands in which the T-cell receptor (TCR) alpha-chain and immunoglobulin heavy chain genes have been mapped, respectively. Patients with ataxia-telangiectasia are particularly prone to development of T-cell chronic lymphocytic leukemia with such chromosomal abnormalities. We now describe DNA rearrangements of the TCR alpha-chain gene in an ataxia-telangiectasia-associated leukemia containing both a normal and an inverted chromosome 14. The normal chromosome 14 has undergone a productive join of TCR alpha-chain variable (V alpha) and joining (J alpha) gene segments. The other allele of the TCR alpha-chain gene features a DNA rearrangement, about 50 kilobases from the TCR alpha-chain constant (C alpha) gene, that represents the breakpoint of the chromosome 14 inversion; this breakpoint is comprised of a TCR J alpha segment (from 14q11) fused to sequences derived from 14q32 but on the centromeric side of C mu. These results imply that 14q32 sequences located at an undetermined distance downstream of the immunoglobulin C mu locus can contribute to the development of T-cell tumors.
Identification, purification, and characterization of a mast cell-associated cytolytic factor related to tumor necrosis factor.
Authors: Young J D , Liu C C , Butler G , Cohn Z A , Galli S J
Published: May 2006

The role of mast cells and mast-cell-derived factors in natural cytotoxic reactions was investigated. Cultured and freshly isolated murine mast cells are shown to be cytotoxic to WEHI-164 and YAC-1 targets in 18-hr viability assays but not in 4-hr assays. Here, we describe a cytotoxic factor in murine mast cells that is immunologically related to tumor necrosis factor (TNF). This TNF-like factor lyses WEHI-164 cells with a slow time course requiring 16-20 hr for the lytic reaction to complete. Antibodies specific for human and murine TNF and human lymphotoxin partially block mast cell lysis of WEHI-164 cells. These antibodies react on immunoblots with one major mast cell protein band of 50 kDa. Immunoblot analysis shows this factor in cloned and uncloned cultured mouse mast cells and in mature "connective tissue-type" mast cells freshly purified from rat or mouse peritoneal cavities. The amount of this factor is greatly enhanced in cells that have been stimulated with a combination of phorbol ester/concanavalin A or bacterial lipopolysaccharide. Subcellular fractionation analysis of mast cells with Percoll gradients reveals two pools of TNF-related cytotoxic activity that are associated with free cytosolic material and granule fractions. In contrast to cytotoxic T lymphocytes and natural killer cells, granule-enriched fractions of mast cells do not contain any hemolytic activity. The localization of the TNF-like molecule in mast cell granules may play a strategical role in the rapid delivery of this mediator to the target cell membrane following cell surface stimulation and degranulation.
Coaggregation of the T-cell receptor with CD4 and other T-cell surface molecules enhances T-cell activation.
Authors: Owens T , Fazekas de St Groth B , Miller J F
Published: May 2006

The CD4 molecule, expressed by T cells restricted by class II major histocompatibility complex (MHC) molecules, is believed to play a role in T-cell activation. We have previously suggested that CD4 interacts with the T-cell receptor for antigen (TCR) and with class II MHC and that this dual interaction stabilizes the bond between the TCR and antigen in association with MHC. To investigate the contribution of CD4-TCR interaction, we have used the murine monoclonal anti-TCR V beta 8 antibody F23.1 to activate cloned T cells. Weak activation by soluble biotinylated F23.1 was markedly enhanced by crosslinking with either avidin or with anti-immunoglobulin (anti-Ig). The monoclonal anti-L3T4 antibody GK1.5, which normally inhibits the activation induced by F23.1, did not inhibit when GK1.5 and F23.1 were coaggregated on T cells by anti-Ig, and in many experiments activation was enhanced. Coaggregation of anti-Thy-1.2, anti-H-2Kk, or anti-LFA-1 with F23.1 also enhanced T-cell activation, although, unlike GK1.5, these antibodies in soluble form had no effect on the response to F23.1. These results are consistent with a model for T-cell activation that proposes a primary interaction between L3T4 and the TCR to stabilize TCR complexes and so to enhance T-cell activation. A related but less specific accessory role for other T-cell surface molecules is also suggested. We propose that the cellular interaction that leads to physiological T-cell activation not only achieves TCR ligation but also promotes through their ligation or redistribution the interaction of other T-cell surface molecules, all of which contribute to the overall strength of the activation signal.
Antibodies in cerebrospinal fluid of some Alzheimer disease patients recognize cholinergic neurons in the rat central nervous system.
Authors: McRae-Degueurce A , Booj S , Haglid K , Rosengren L , Karlsson J E , Karlsson I , Wallin A , Svennerholm L , Gottfries C G , Dahlstrom A
Published: May 2006

The etiology of Alzheimer disease is unclear. However, immunological aberrations have been suggested to be critical factors in the pathogenesis of this neurodegenerative disease. This study was carried out to investigate if cerebrospinal fluid (CSF) from Alzheimer disease patients contains antibodies that recognize specific neuronal populations in the rat central nervous system. The results indicate that in a subgroup of patients this is indeed the case. The antibodies reported in this study have the following properties: (i) they recognize neuronal populations and components in the medial septum and spinal motor neurons in rats perfused with a mixture that fixes small neurotransmitter molecules; (ii) adsorption of the patient CSF with staphylococcal protein A-Sepharose and using a polyclonal antiserum against human IgG3 indicates that the immunocytochemical reaction in these brain regions is mainly due to the subclass IgG3; and (iii) the CSF immunocytochemical reaction is blocked by preincubation of the sections with a rabbit anti-acetylcholine antiserum. These results provide evidence that antibodies in the CSF of some, but not all, Alzheimer disease patients recognize acetylcholine-like epitopes in cholinergic neurons in the rat central nervous system.
3' homologous free ends are required for stable joint molecule formation by the RecA and single-stranded binding proteins of Escherichia coli.
Authors: Konforti B B , Davis R W
Published: May 2006

The RecA protein of Escherichia coli is important for genetic recombination in vivo and can promote synapsis and strand exchange in vitro. The DNA pairing and strand exchange reactions have been well characterized in reactions with circular single strands and linear duplexes, but little is known about these two processes using substrates more characteristic of those likely to exist in the cell. Single-stranded linear DNAs were prepared by separating strands of duplex molecules or by cleaving single-stranded circles at a unique restriction site created by annealing a short defined oligonucleotide to the circle. Analysis by gel electrophoresis and electron microscopy revealed that, in the presence of RecA and single-stranded binding proteins, a free 3' homologous end is essential for stable joint molecule formation between linear single-stranded and circular duplex DNA.
Cloning and sequencing of human intestinal alkaline phosphatase cDNA.
Authors: Berger J , Garattini E , Hua J C , Udenfriend S
Published: May 2006

Partial protein sequence data obtained on intestinal alkaline phosphatase indicated a high degree of homology with the reported sequence of the placental isoenzyme. Accordingly, placental alkaline phosphatase cDNA was cloned and used as a probe to clone intestinal alkaline phosphatase cDNA. The latter is somewhat larger (3.1 kilobases) than the cDNA for the placental isozyme (2.8 kilobases). Although the 3' untranslated regions are quite different, there is almost 90% homology in the translated regions of the two isozymes. There are, however, significant differences at their amino and carboxyl termini and a substitution of an alanine in intestinal alkaline phosphatase for a glycine in the active site of the placental isozyme.
Incorporation of a retinal rod cGMP-dependent conductance into planar bilayers.
Authors: Tanaka J C , Furman R E , Cobbs W H , Mueller P
Published: May 2006

The light-modulated current of vertebrate retinal rods flows through a 3',5'-cyclic GMP-dependent conductance located in the outer segment plasma membrane. We report the incorporation into planar bilayers of a conductance derived from vertebrate rod outer segment membranes specifically activated by cGMP but not by cAMP, 5'-GMP, GTP, or 5'-AMP. When the mean currents were measured as a function of increasing cGMP concentration, maximal activation occurred at concentrations less than 50 microM. Washout of cGMP rapidly reversed the effect. The apparent half-saturating concentrations were between 12 and 27 microM. Sodium, lithium, cesium, and potassium supported current in the presence of low concentrations of Ca2+, Mg2+, and 100 microM cGMP; choline did not. Removal of the divalent cations reversibly increased the currents. When calcium was the only current-carrying cation, attenuated currents were seen. These experiments support the hypothesis that calcium is a permeant blocker of the conductance. At low concentrations of cGMP in solutions also containing 0.5 mM EDTA, brief current spikes occurred with amplitudes from 0.5 to 4 pA at 50 mV. These spikes differed from the well-defined, unitary conductance steps usually associated with the opening and closing of ion channels. Occasionally we saw longer-lasting channel-like events; however, amplitude histograms did not resolve discrete conductance levels.
Activation of growth factor secretion in tumorigenic states of breast cancer induced by 17 beta-estradiol or v-Ha-ras oncogene.
Authors: Dickson R B , Kasid A , Huff K K , Bates S E , Knabbe C , Bronzert D , Gelmann E P , Lippman M E
Published: May 2006

The MCF-7 human breast cancer cell line responds to estrogen stimulation in vitro by increased secretion of growth factors and proliferation and in vivo by tumor formation in the nude mouse. To test a possible role of growth factor secretion in expression of the tumorigenic phenotype, we stably transfected MCF-7 cells with the v-Ha-ras oncogene to produce the MCF-7ras cell line. The MCF-7ras cell line was tumorigenic in the absence of estrogens and secreted 3- to 5-fold elevated levels of a high molecular weight form of a type alpha transforming growth factor-like growth factor, type beta transforming growth factor, and insulin-like growth factor I. MCF-7ras cells, in contrast to MCF-7, were less sensitive to further growth stimulation by estrogen, type alpha transforming growth factor, and insulin-like growth factor I and showed little change in receptor levels for these hormones. Conditioned medium from MCF-7ras cells as well as two of its component growth factors (insulin-like growth factor I and type alpha transforming growth factor) replaced estrogen in stimulating MCF-7 colony formation in vitro. A coordinate increase in growth factor secretion by human breast cancer may contribute to its escape from estrogen dependence.
Quantitative autoradiography of brain binding sites for the vesicular acetylcholine transport blocker 2-(4-phenylpiperidino)cyclohexanol (AH5183).
Authors: Marien M R , Parsons S M , Altar C A
Published: May 2006

2-(4-Phenylpiperidino)cyclohexanol (AH5183) is a noncompetitive and potent inhibitor of high-affinity acetylcholine transport into cholinergic vesicles. It is reported here that [3H]AH5183 binds specifically and saturably to slide-mounted sections of the rat forebrain (Kd = 1.1 to 2.2 X 10(-8) M; Bmax = 286 to 399 fmol/mg of protein). The association and dissociation rate constants for [3H]AH5183 binding are 8.6 X 10(6) M-1 X min-1 and 0.18 min-1, respectively. Bound [3H]AH5183 can be displaced by nonradioactive AH5183 and by the structural analog (2 alpha,3 beta,4A beta,8A alpha)-decahydro-3-(4-phenyl-1-piperidinyl)-2- naphthalenol but not by 10 microM concentrations of the cholinergic drugs acetylcholine, choline, atropine, hexamethonium, eserine, or hemicholinium-3 or by the structurally related compounds 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-phenylpyridine, (+/-)-N-allylnormetazocine (SKF 10,047), levoxadrol, or dexoxadrol. Quantitative autoradiography reveals that [3H]AH5183 binding sites are distributed heterogenously throughout the rat forebrain and are highly localized to cholinergic nerve terminal regions. At the level of the caudate nucleus-putamen, the highest concentrations of saturable [3H]AH5183 binding (713-751 fmol/mg of protein) are found in the vertical limb of the diagonal band and the olfactory tubercle, with lesser amounts (334-516 fmol/mg of protein) in the caudate-putamen, nucleus accumbens, superficial layers of the cerebral cortex, and the primary olfactory cortex. At day 7 after transsection of the left fimbria, [3H]AH5183 binding and choline acetyltransferase activity in the left hippocampus were reduced by 33 +/- 6% and 61 +/- 7%, respectively. These findings indicate that [3H]AH5183 binds to a unique recognition site in rat brain that is topographically associated with cholinergic nerve terminals.
Papillomavirus sequences integrate near cellular oncogenes in some cervical carcinomas.
Authors: Dürst M , Croce C M , Gissmann L , Schwarz E , Huebner K
Published: May 2006

The chromosomal locations of cellular sequences flanking integrated papillomavirus DNA in four cervical carcinoma cell lines and a primary cervical carcinoma have been determined. The two human papillomavirus (HPV) 16 flanking sequences derived from the tumor were localized to chromosome regions 20pter----20q13 and 3p25----3qter, regions that also contain the protooncogenes c-src-1 and c-raf-1, respectively. The HPV 16 integration site in the SiHa cervical carcinoma-derived cell line is in chromosome region 13q14----13q32. The HPV 18 integration site in SW756 cervical carcinoma cells is in chromosome 12 but is not closely linked to the Ki-ras2 gene. Finally, in two cervical carcinoma cell lines, HeLa and C4-I, HPV 18 DNA is integrated in chromosome 8, 5' of the c-myc gene. The HeLa HPV 18 integration site is within 40 kilobases 5' of the c-myc gene, inside the HL60 amplification unit surrounding and including the c-myc gene. Additionally, steady-state levels of c-myc mRNA are elevated in HeLa and C4-I cells relative to other cervical carcinoma cell lines. Thus, in at least some genital tumors, cis-activation of cellular oncogenes by HPV may be involved in malignant transformation of cervical cells.
Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: close homology to placental alkaline phosphatase.
Authors: Henthorn P S , Raducha M , Edwards Y H , Weiss M J , Slaughter C , Lafferty M A , Harris H
Published: May 2006

A cDNA clone for human adult intestinal alkaline phosphatase (ALP) [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1] was isolated from a lambda gt11 expression library. The cDNA insert of this clone is 2513 base pairs in length and contains an open reading frame that encodes a 528-amino acid polypeptide. This deduced polypeptide contains the first 40 amino acids of human intestinal ALP, as determined by direct protein sequencing. Intestinal ALP shows 86.5% amino acid identity to placental (type 1) ALP and 56.6% amino acid identity to liver/bone/kidney ALP. In the 3'-untranslated regions, intestinal and placental ALP cDNAs are 73.5% identical (excluding gaps). The evolution of this multigene enzyme family is discussed.
Alteration of the tumorigenic and metastatic properties of neoplastic cells is associated with the process of calcium phosphate-mediated DNA transfection.
Authors: Kerbel R S , Waghorne C , Man M S , Elliott B , Breitman M L
Published: May 2006

During the course of studies designed to assess the effect of human Ha-ras gene expression on the malignant behavior of transfected mouse tumor cells we noted that the process of Ca3(PO4)2-mediated DNA transfection was itself associated with profound alterations in tumorigenic or metastatic behavior. The cell line used as a recipient for these studies was a tumorigenic nonmetastatic CBA/J mouse mammary adenocarcinoma line called SP1. When cotransfected with plasmids containing the neo gene (pSV2neo) and the activated Ha-ras gene (pT24-c3), cells from the pooled (5-10 colonies) G418-resistant colonies gave rise to spontaneous lung metastases in 85% of mice after subcutaneous inoculation. However, we noted that 17% of control mice inoculated with G418-resistant pSV2neo-transfected SP1 cells also had lung metastases and that this number approached 100% as the inoculum comprised a greater pool size (50-100 colonies). When cell lines established from isolated pSV2neo-transfected colonies were examined, 3/16 were found to be metastatic. We also found that 3/16 clones grew slowly, or not at all, in CBA/J mice, whereas they grew readily in athymic (nude) mice. The increase in immunogenicity of two out of three of these latter clones was accompanied by expression of the class I H-2Dk major histocompatibility complex antigen that was not detectable in the parental SP1 cells. At least some of these results would appear to be due to exposure to Ca3(PO4)2 alone, as we found that it resulted in 5/20 (25%) clones manifesting metastatic properties. Our results suggest that heritable changes in malignant behavior of transfected tumor cells can be observed at high frequency subsequent to the process of Ca3(PO4)2-mediated DNA transfection, and these changes may be brought about in part by inherited disturbances in expression of recipient cell genes.
Cell-type-specific responses of RT4 neural cell lines to dibutyryl-cAMP: branch determination versus maturation.
Authors: Droms K , Sueoka N
Published: May 2006

This report describes the induction of cell-type-specific maturation, by dibutyryl-cAMP and testololactone, of neuronal and glial properties in a family of cell lines derived from a rat peripheral neurotumor, RT4. This maturation allows further understanding of the process of determination because of the close lineage relationship between the cell types of the RT4 family. The RT4 family is characterized by the spontaneous conversion of one of the cell types, RT4-AC (stem-cell type), to any of three derivative cell types, RT4-B, RT4-D, or RT4-E, with a frequency of about 10(-5). The RT4-AC cells express some properties characteristic of both neuronal and glial cells. Of these neural properties expressed by RT4-AC cells, only the neuronal properties are expressed by the RT4-B and RT4-E cells, and only the glial properties are expressed by the RT4-D cells. This in vitro cell-type conversion of RT4-AC to three derivative cell types is a branch point for the coordinate regulation of several properties and seems to resemble determination in vivo. In our standard culture conditions, several other neuronal and glial properties are not expressed by these cell types. However, addition of dibutyryl-cAMP induces expression of additional properties, in a cell-type-specific manner: formation of long cellular processes in the RT4-B8 and RT4-E5 cell lines and expression of high-affinity uptake of gamma-aminobutyric acid, by a glial-cell-specific mechanism, in the RT4-D6-2 cell line. These new properties are maximally expressed 2-3 days after addition of dibutyryl-cAMP. This indicates that conversion of RT4-AC to the derivative cell types is also a branch point for the regulation of cell-type-specific properties whose expression is responsive to cAMP. Thus, the potential for maturation in response to increased cAMP is a property that segregates in a cell-type-specific manner and is activated at the determinational level in this system.
Bizarre tRNAs inferred from DNA sequences of mitochondrial genomes of nematode worms.
Authors: Wolstenholme D R , Macfarlane J L , Okimoto R , Clary D O , Wahleithner J A
Published: May 2006

The complete nucleotide sequence of the mitochondrial DNA (mtDNA) molecule of the parasitic nematode worm Ascaris suum has been determined. This molecule lacks genes for tRNAs of the standard form. Instead, 21 sequences are found that can be folded into structures that resemble tRNAs in which the T psi C arm and variable loop are missing and replaced with a single loop of between 4 and 12 nucleotides. Considerations of various properties of these sequences, including the number, predicted anticodons, conserved nucleotides, direction of transcription, base composition, and relative gene arrangements are consistent with the interpretation that they are genes for a different sort of tRNA. Transfer RNA genes with a similar potential secondary structure are found in mtDNA of the free-living nematode Caenorhabditis elegans, suggesting that this unusual form of tRNA is used by all nematode mitochondria.
Single cell studies on the role of B-cell stimulatory factor 1 in B-cell activation.
Authors: Alderson M R , Pike B L , Nossal G J
Published: May 2006

The role of the T-cell-derived lymphokine B-cell stimulatory factor 1 (BSF-1) in the early activation, proliferation, and antibody-forming cell (AFC) clone formation of single fluorescein-specific B lymphocytes isolated from normal mouse spleens by hapten-gelatin adherence has been studied in vitro. BSF-1 acting alone induced early B-cell activation, as assessed by a significant increase in cell diameter of single B cells cultured for 24 hr. A small but significant number of these B cells formed proliferating clones, some of which secreted antibody. When acting with the specific antigen fluorescein-polymerized flagellin, BSF-1 augmented early cell enlargement and markedly enhanced proliferation, but it did not increase the frequency of AFC clones stimulated by fluorescein-polymerized flagellin alone. The further addition of recombinant murine interleukin 1 (IL-1) marginally enhanced proliferation caused by antigen plus BSF-1. No synergy was observed between BSF-1 and IL-1 for antibody formation. In the presence of fibroblast filler cells, BSF-1 substantially inhibited AFC clone development achieved by antigen plus IL-1. BSF-1 was also found to be inhibitory to AFC clone development stimulated by specific antigen acting with either recombinant human interleukin 2 (IL-2) or with IL-2 plus IL-1, both in the presence or absence of filler cells. The results suggest that BSF-1 plays a complex role in the regulation of the B-cell activation pathway by enhancing early activation and antigen-specific proliferation as well as inhibiting the effects of other B-cell factors on antibody formation. BSF-1 is the only cytokine so far tested in the single B-cell system that acts with antigen to promote proliferation without concomitant antibody production.
Discrete wave mechanics: The hydrogen atom with angular momentum
Authors: Wall Frederick T.
Published: May 2006

A discrete wave mechanical treatment of the hydrogen atom is extended to deal with states involving nonzero angular momentum. Only the radial portions of the wave vectors are covered. It is predicted that there is a nonzero minimum distance between the electron and the nucleus; this threshold distance increases with increasing angular momentum. Appropriate finite difference equations are formulated. The states with angular momentum exhibit the same degeneracy as do corresponding energy levels obtained from solutions of Schrödinger's equation.
Differential regulation of a Thy-1 gene in transgenic mice.
Authors: Kollias G , Spanopoulou E , Grosveld F , Ritter M , Beech J , Morris R
Published: May 2006

We have generated Thy-1.1-transgenic Thy-1.2 mice to study the developmental expression of the Thy-1 gene in detail by transcriptional and immunological methods. In brain, the expression of the injected gene was identical to that of the endogenous gene in a tissue- and development-specific manner. In lymphoid tissue, the transferred gene was also expressed correctly in the early phases of T-cell lineage development; however, as the T cells matured, the transcription of the transferred gene, but not the endogenous gene, was suppressed. This result shows that different regulatory elements are used to express the Thy-1 gene in early and late lymphoid development.
Detection of transcription-pausing in vivo in the trp operon leader region.
Authors: Landick R , Carey J , Yanofsky C
Published: May 2006

To determine whether RNA polymerase pauses during transcription in vivo, we have examined transcripts of the trp operon leader regions of Serratia marcescens and Escherichia coli. Labeled RNAs synthesized in E. coli strains containing plasmids bearing wild-type or mutant trp leader regions of S. marcescens or E. coli were isolated by hybridization and analyzed by polyacrylamide gel electrophoresis. The labeled RNAs synthesized in vivo on the S. marcescens wild-type and deletion mutant plasmids were the same size as the in vitro pause and leader transcripts. Hybridization of the presumed in vivo pause RNAs, and control in vitro pause RNAs, to M13 phage DNA containing a trp leader region deletion followed by treatment with S1 nuclease produced identical protected RNA species, proving that the in vitro and in vivo RNAs were identical. The amount of labeled pause RNAs relative to leader RNAs decreased following a chase with unlabeled uridine. E. coli RNAs identical to the previously characterized in vitro pause and leader transcripts were demonstrated by electrophoretic band position and fingerprint analysis. The finding that transcription pausing occurs in vivo is consistent with the view that transcription pausing and ribosome release of paused transcription complexes are responsible for the coupling of translation with transcription that is crucial to attenuation.
Antenna size dependence of fluorescence decay in the core antenna of photosystem I: estimates of charge separation and energy transfer rates.
Authors: Owens T G , Webb S P , Mets L , Alberte R S , Fleming G R
Published: May 2006

We have examined the photophysics of energy migration and trapping in photosystem I by investigating the spectral and temporal properties of the fluorescence from the core antenna chlorophylls as a function of the antenna size. Time-correlated single photon counting was used to determine the fluorescence lifetimes in the isolated P700 chlorophyll a-protein complex and in a mutant of Chlamydomonas reinhardtii that lacks the photosystem II reaction center complex. The fluorescence decay in both types of sample is dominated by a fast (15-45 psec) component that is attributed to the lifetime of excitations in the photosystem I core antenna. These excitations decay primarily by an efficient photochemical quenching on P700. The measured lifetimes show a linear relationship to the core antenna size. A linear dependence of the excitation lifetime on antenna size was predicted previously in a lattice model for excitation migration and trapping in arrays of photosynthetic pigments [Pearlstein, R.M. (1982) Photochem. Photobiol. 35, 835-844]. Based on this model, our data predict a time constant for photochemical charge separation in the photosystem I reaction center of 2.8 +/- 0.7 or 3.4 +/- 0.7 psec, assuming monomeric or dimeric P700, respectively. The predicted average single-step transfer time for excitation transfer between core antenna pigments is 0.21 +/- 0.04 psec. Under these conditions, excitation migration in photosystem I is near the diffusion limit, with each excitation making an average of 2.4 visits to the reaction center before photoconversion.
Structural domains in phage Mu transposase: identification of the site-specific DNA-binding domain.
Authors: Nakayama C , Teplow D B , Harshey R M
Published: May 2006

Limited proteolysis of phage Mu transposase with three proteases of differing specificities produced a common pattern of fragmentation. The fragments were mapped by using a combination of immunoblotting and amino acid sequence analysis. Our results suggest that the transposase molecule is organized principally into three domains: an amino-terminal domain of molecular mass 30 kDa, a core region of approximately 35 kDa, and a carboxyl-terminal domain of approximately 10 kDa. The amino-terminal domain has at least two additional sites that are partially accessible to proteases. Filter binding and nuclease protection studies were done to determine the functions of the isolated domains. Site-specific binding to Mu DNA was localized to the amino-terminal domain. The core domain showed nonspecific DNA-binding activity.
GTP-binding domain: three consensus sequence elements with distinct spacing.
Authors: Dever T E , Glynias M J , Merrick W C
Published: May 2006

A sequence comparison of nine functionally different GTP-binding protein families has yielded further information on the general characterization of the conservation and importance of amino acid sequences in the GTP-binding domain, including a consensus sequence composed of three consensus elements GXXXXGK, DXXG, and NKXD with consensus spacings of either 40-80 or approximately equal to 130-170 amino acid residues between the first and second elements and approximately 40-80 amino acid residues between the second and third sequence elements; the sequence NKXW in place of NKXD in the sequence element responsible for base specificity allows the use of ITP as well as GTP; dGTP can be used with essentially the same efficiency as GTP; signal transducing proteins and enzymes have been identified in the nine families; and family conservations allow the identification of the most probable consensus sequence element when more than one is present. Employing these features we have screened the protein sequence data base of the Protein Identification Resource and have identified only known GTP-binding proteins with the exception of protein 2C from foot-and-mouth disease virus as matching the consensus sequence. Based on this finding we predict that foot-and-mouth disease virus protein 2C binds GTP and, by analogy, that protein 2C from several related viruses (polio, rhino, encephalomyocarditis, and cowpea mosaic) will bind a nucleotide as part of its biologic activity.
Multiple forms of basic fibroblast growth factor: amino-terminal cleavages by tumor cell- and brain cell-derived acid proteinases.
Authors: Klagsbrun M , Smith S , Sullivan R , Shing Y , Davidson S , Smith J A , Sasse J
Published: May 2006

Basic fibroblast growth factor (FGF) was purified by heparin-Sepharose chromatography from two sources, brain and hepatoma cells. Brain cell-derived basic FGF (brFGF) and hepatoma cell-derived basic FGF (heFGF) were found to exist in multiple forms whose molecular weights depended on whether they were extracted from their respective tissue or cells at neutral or acid pH. When extracted at pH 7.0 brFGF and heFGF comigrated on NaDodSO4/PAGE with a Mr of approximately 18,400. When extracted at pHs 3.5-4.5, acid proteinases cleaved brFGF and heFGF to lower molecular weight forms but to different extents. brFGF was cleaved to a Mr 18,000 form at acid pH by a brain-derived acid proteinase that could be inhibited by pepstatin. heFGF was cleaved mostly to a Mr 16,500 form at acid pH by a hepatoma cell-derived acid proteinase that was inhibited by leupeptin. Electrophoretic transfer blot analysis using site-specific anti-FGF antibodies suggested that the cleavages occurred at the amino-terminal ends of brFGF and heFGF. Cleavage to lower molecular weight forms of brFGF and heFGF did not affect growth factor activity or chromatographic behavior on heparin-Sepharose columns.
Macromolecular crowding increases binding of DNA polymerase to DNA: an adaptive effect.
Authors: Zimmerman S B , Harrison B
Published: May 2006

Macromolecular crowding extends the range of ionic conditions supporting high DNA polymerase reaction rates. Reactions tested were nick-translation and gap-filling by DNA polymerase I of Escherichia coli, nuclease and polymerase activities of the large fragment of that polymerase, and polymerization by the T4 DNA polymerase. For all of these reactions, high concentrations of nonspecific polymers increased enzymatic activity under otherwise inhibitory conditions resulting from relatively high ionic strength. The primary mechanism of the polymer effect seems to be to increase the binding of polymerase to DNA. We suggest that this effect on protein-DNA complexes is only one example of a general "metabolic buffering" action of crowded solutions on a variety of macromolecular interactions.
Cloning and analysis of the promotor region of the human fibronectin gene.
Authors: Dean D C , Bowlus C L , Bourgeois S
Published: May 2006

Human fibronectin (FN) genomic clones were isolated by screening a human genomic library with a 75-base oligonucleotide. The sequence of the oligonucleotide corresponds to a region near the 5' end of the human FN cDNA clone pFH6 that contains the amino-terminal coding sequences but does not extend to the 5' end of the mRNA [Kornblihtt, A. R., Umezawa, K., Vibe-Pedersen, K. & Baralle, F. E. (1985) EMBO J. 4, 1755-1759]. The 5' end of the FN gene is found on a 3.7-kilobase-pair EcoRI fragment that contains about 2.7 kilobase pairs of flanking sequence. The first exon is 414 base pairs long, with a 5' untranslated region of 267 base pairs. As deduced on the basis of the position of the initiation codon, FN is synthesized with a 31-residue amino acid extension on the amino terminus that is not present in the mature polypeptide. This amino-terminal extension appears to contain both a signal peptide and a propeptide. The first 200 base pairs of 5'-flanking sequence is very G + C rich. Upstream of this the sequence becomes relatively A + T rich. The sequence ATATAA is found at -25 and the sequence CAAT is present at -150. The sequence GGGGCGGGGC at -102 exhibits homology to the binding site for the transcription factor SP1, and the sequence TGACGTCA at -173 exhibits homology to 5'-flanking sequences important for induction by cAMP.
Final steps in exocytosis observed in a cell with giant secretory granules.
Authors: Breckenridge L J , Almers W
Published: May 2006

Secretion by single mast cells was studied in normal and beige mice, a mutant with grossly enlarged secretory vesicles or granules. During degranulation, the membrane capacitance increased in steps, as single secretory vesicles fused with the cell membrane. The average step size was 10 times larger in beige than in normal mice, in agreement with the different granule sizes measured microscopically in the two preparations. Following individual capacitance steps in beige mice, individual granules of the appropriate size were observed to swell rapidly. Capacitance steps are frequently followed by the stepwise loss of a fluorescent dye loaded into the vesicles. Stepwise capacitance increases were occasionally intermittent before they became permanent, indicating the existence of an early, reversible, and incomplete state of vesicle fusion. During such "capacitance flicker," loss of fluorescent dye from vesicles did not occur, suggesting that the earliest aqueous connection between vesicle interior and cell exterior is a narrow channel. Our results support the view that the reversible formation of such a channel, which we term the fusion pore, is an early step in exocytosis.
C3bi receptor (complement receptor type 3) recognizes a region of complement protein C3 containing the sequence Arg-Gly-Asp.
Authors: Wright S D , Reddy P A , Jong M T , Erickson B W
Published: May 2006

Human phagocytes express a receptor termed complement receptor type 3 (CR3) that recognizes the complement protein fragment C3bi. We show here that CR3 recognizes a region of C3 that contains the sequence Arg-Gly-Asp (RGD). CR3 is down-modulated upon spreading of macrophages on surfaces coated with a synthetic 21-residue peptide from C3 (residues 1383-1403). This peptide was also attached to erythrocytes by coupling myristic acid to its amino terminus and allowing the myristoylated peptide to bind to erythrocytes through hydrophobic interactions. Erythrocytes coated with this RGD-containing segment of C3 were bound by macrophages, and binding could be blocked by specific monoclonal antibodies against CR3. Since CR3 recognizes a peptide sequence that contains the RGD triplet, it appears to be a member of a larger family of adhesion-promoting receptors that recognize RGD-containing proteins. However, since CR3 does not recognize a hexapeptide containing RGD, we presume that residues beyond the RGD triplet contribute to binding. We have compared the RGD-containing region of fibronectin and vitronectin, proteins known to be recognized by means of their RGD-containing regions, with those in human and murine C3. A striking homology is observed over an approximately equal to 50 amino acid sequence present in all four proteins. We suggest that this extended region of homology contains a structure recognized by adhesion-promoting receptors.
Neuropeptide Y inhibits cholinergic transmission in the isolated guinea pig colon: mediation through alpha-adrenergic receptors.
Authors: Wiley J , Owyang C
Published: May 2006

Neuropeptide Y, a member of the pancreatic polypeptide family, caused dose-dependent relaxation of guinea pig colon longitudinal muscle. This inhibitory effect was unaffected by hexamethonium but was abolished by atropine and tetrodotoxin, suggesting that neuropeptide Y is acting via postganglionic cholinergic neurons. Studies utilizing alpha-adrenergic and dopamine receptor antagonists revealed that neuropeptide Y-mediated relaxation was blocked only by the alpha 2 antagonist yohimbine. Neuropeptide Y also antagonized muscle response to electric field stimulation that was reversed by yohimbine. Additional studies with muscle slices incubated with [3H]norepinephrine or [3H]choline showed that neuropeptide Y stimulated norepinephrine release from sympathetic nerves, which, in turn, inhibited the release of acetylcholine via alpha 2 receptors located on postganglionic nerves. This pathway provides a mechanism for neuropeptide modulation of classical neurotransmitter function.
Specific binding of atrial natriuretic factor in brain microvessels.
Authors: Chabrier P E , Roubert P , Braquet P
Published: May 2006

Cerebral capillaries constitute the blood-brain barrier. Studies of specific receptors (neurotransmitters or hormones) located on this structure can be performed by means of radioligand-binding techniques on isolated brain microvessels. We examined on pure bovine cerebral microvessel preparations the binding of atrial natriuretic factor (ANF), using 125I-labeled ANF. Saturation and competition experiments demonstrated the presence of a single class of ANF-binding sites with high affinity (dissociation constant, approximately 10(-10) M) and with a binding capacity of 58 fmol/mg of protein. The binding of 125I-labeled ANF to brain microvessels is specific, reversible, and time dependent, as is shown by association-dissociation experiments. The demonstration of specific ANF-binding sites on brain microvessels supposes a physiological role of ANF on brain microvasculature. The coexistence of ANF and angiotensin II receptors on this cerebrovascular tissue suggests that the two circulating peptides may act as mutual antagonists in the regulation of brain microcirculation and/or blood-brain barrier function.
Castration reversibly alters levels of cholecystokinin immunoreactivity within cells of three interconnected sexually dimorphic forebrain nuclei in the rat.
Authors: Simerly R B , Swanson L W
Published: May 2006

Three sexually dimorphic cell groups in the forebrain of the rat--the central part of the medial preoptic nucleus, the encapsulated part of the bed nucleus of the stria terminalis, and the posterodorsal part of the medial nucleus of the amygdala--are larger in males, contain a high density of gonadal-steroid-concentrating cells, and are thought to play important roles in the control of reproductive behavior and physiology. Since each of these regions contains a large number of cholecystokinin-immunoreactive cells, we used an indirect immunohistochemical method to examine the possibility that levels of this peptide are modulated by circulating gonadal steroids in adult male rats. Rats were castrated at 60 days of age, and one group each was pretreated with colchicine and then killed 3, 7, and 14 days after gonadectomy. Castration clearly decreased CCK immunoreactivity within cells of each region, with the most dramatic effects occurring 7 and 14 days after gonadectomy, and these effects were reversed by treatment with testosterone over a 14-day period. The results suggest that CCK levels within individual cells in each of the interconnected sexually dimorphic nuclei examined here are regulated by circulating gonadal steroids and may be related to the hormonal modulation of reproductive functions thought to be mediated by these cell groups.
Expression of human class II major histocompatibility complex antigens using retrovirus vectors.
Authors: Korman A J , Frantz J D , Strominger J L , Mulligan R C
Published: May 2006

Retrovirus vectors [direct orientation (DO) vectors] that permit the simultaneous expression of an inserted protein-coding sequence and a dominant-acting selectable marker have been constructed. In these vectors, an internal simian virus 40 or human metallothionein promoter sequence serves to drive the expression of the bacterial neomycin phosphotransferase or guanine-xanthine phosphoribosyltransferase genes, whereas the viral long terminal repeat sequences are utilized to promote expression of inserted sequences. In some of the vectors, the viral 5' splice site, normally used in the biogenesis of the subgenomic env-encoding mRNA, has been eliminated. These vectors yield high transient and stable titers of virus after transfection of viral packaging cell lines, show little or no depression of virus titer with a variety of inserts, and faithfully transmit recombinant proviral sequences to recipient cells. To characterize the expression potential of these vectors, a variety of inserts encoding the alpha and beta subunits of the human major histocompatibility complex class II antigen HLA-DR have been introduced into these vectors. NIH 3T3 cells infected by viruses containing HLA-DR alpha or beta cDNAs express these proteins as shown by immunoprecipitation of metabolically labeled extracts. In addition, through the sequential infection of cells with retrovirus constructions expressing two different selectable markers, both subunits of the class II antigen have been introduced into NIH 3T3 cells. Such infected cells express HLA-DR molecules at the cell surface.
Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer.
Authors: Wahl G M , Lewis K A , Ruiz J C , Rothenberg B , Zhao J , Evans G A
Published: May 2006

We have designed cosmid vectors for rapid genomic "walking" and restriction mapping. These vectors contain the transcription promoters from either bacteriophage SP6, T7, or T3 flanking a unique BamHI cloning site. Mammalian expression modules encoding the dominant marker neomycin phosphotransferase or the amplifiable dihydrofolate reductase gene expressed from SV40 promoters were inserted for use in gene transfer studies. Restriction sites for the enzymes Not I and Sfi I, which cut mammalian DNA very infrequently, have been engineered near the transcriptional promoters to enable the excision of most inserts as single, full-length fragments. Genomic libraries representative of mouse, human, and hamster genomes were constructed by inserting 33- to 44-kilobase-pair (kbp) DNA fragments, generated by partial cleavage of genomic DNA with Mbo I or Sau3A, into the unique BamHI site. Digestion of recombinant cosmids with restriction enzymes that cleave frequently but do not disrupt the transcriptional promoters generates two small DNA templates for the synthesis of end-specific RNA probes to facilitate directional "walking." Cosmid restriction maps can be determined rapidly by one of several methods. The cosmids and methods we describe should have wide utility in determining the functional and structural organization of complex eukaryotic genomes and for physically linking distant genetic loci.
Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C.
Authors: Lapetina E G , Reep B R
Published: May 2006

We have assessed the binding of [alpha-32P]GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO4/PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with [alpha-32P]GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP and by 100 nM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) or GDP; binding was unaffected by 1 nM-1 microM ATP. One main GTP-binding protein (29.5 kDa) was detected in the membrane fraction, while three others (29, 27, and 21 kDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 kDa) were degraded by trypsin; another cytosolic protein (21 kDa) and the membrane-bound protein (29.5 kDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of [alpha-32P]GTP to the membrane-bound protein. GTP[gamma S] still stimulated phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTP[gamma S].