bluuebunny/crossref_metadata_2025 huggingface dataset:
Dihydropyridine receptor of L-type Ca2+ channels: identification of binding domains for [3H](+)-PN200-110 and [3H]azidopine within the alpha 1 subunit.
Authors: Striessnig J , Murphy B J , Catterall W A
Published: May 2006

To identify the binding domain for dihydropyridine Ca2+ antagonists, skeletal muscle Ca2+ channels were photolabeled with [3H](+)-PN200-110 and [3H]azidopine. Regions of alpha 1 photolabeled by these ligands were then identified by antibody mapping of proteolytic fragments. Approximately 50% of the specific labeling by both ligands was incorporated in domain III. [3H]Azidopine labeled peptide Gln-989-Arg-1022, which contains a portion of the connecting loop between transmembrane segments IIIS5 and IIIS6 (IIIS5/S6), and peptide Ala-1023-Lys-1077, which contains IIIS6 itself and some adjacent amino acid residues. In contrast, [3H](+)-PN200-110 labeling occurred almost exclusively in the fragment containing IIIS6. A second site labeled by both ligands was identified in transmembrane segment S6 of domain IV and adjacent residues. In contrast to azidopine, the photoreactive benzofurazane group of (+)-PN200-110 is located in close proximity to the essential dihydropyridine ring. Therefore, the regions photolabeled by [3H](+)-PN200-110 within or adjacent to transmembrane segments IIIS6 and IVS6 must participate in the formation of the dihydropyridine binding site. As IIIS5/S6 is preferentially labeled by [3H]azidopine, it may contribute to drug binding by interaction with the long side chain of some dihydropyridines like azidopine. It is proposed, based on physiological studies, that these three peptide segments interact to form a receptor site accessible from the extracellular surface of the Ca2+ channel.
Antiviral activity of 1-docosanol, an inhibitor of lipid-enveloped viruses including herpes simplex.
Authors: Katz D H , Marcelletti J F , Khalil M H , Pope L E , Katz L R
Published: May 2006

This article reports that 1-docosanol, a 22-carbon-long saturated alcohol, exerts a substantial inhibitory effect on replication of certain viruses (e.g., herpes simplex virus and respiratory syncytial virus) within primary target cells in vitro. To study the basis for its viral inhibitory activity, a suspension of 1-docosanol was formulated in an inert and nontoxic surfactant, Pluronic F-68; this suspension exerted potent inhibitory activity on the ability of susceptible viruses to infect cultured target cells. Susceptible viruses included wild-type herpes simplex viruses 1 and 2 as well as acyclovir-resistant herpes simplex virus 2 and also respiratory syncytial virus--all of which are lipid-enveloped. In contrast, nonenveloped poliovirus was not susceptible to the inhibitory action of 1-docosanol. Although the precise mechanism has yet to be defined, current evidence suggests that 1-docosanol inhibits viral replication by interfering with the early intracellular events surrounding viral entry into target cells. It is possible that interaction between the highly lipophilic compound and components of target cell membranes renders such target cells less susceptible to viral fusion and/or entry. If this mechanism proves to be correct, 1-docosanol may provide a broad spectrum activity against many different viruses, especially those with lipid-containing envelopes.
Simultaneous visualization of chromosome bands and hybridization signal using colloidal-gold labeling in electron microscopy.
Authors: Fetni R , Drouin R , Lemieux N , Messier P E , Richer C L
Published: May 2006

Electron microscopy (EM) is seldom used with in situ hybridization to localize DNA sequences because banding methods for chromosome identification could not be coupled to EM techniques. We have applied an immunochemical replication-banding method specific for EM to solve this problem. A thymidine synchronization/BrdUrd release protocol allows BrdUrd incorporation only into late replicating bands. A biotinylated DNA probe is hybridized in situ to its complementary sequence. The biotinylated probe and the BrdUrd-substituted DNA are simultaneously localized by different reporter/detection systems using different-sized colloidal gold particles as electron-dense tags. We demonstrate the high precision of this mapping procedure by localizing on long prophase chromosomes (greater than 1000 bands per haploid set) the pXBR-1 sequence to a small subregion of the centromeric subband Xp11.1-Xq11.1. This localization to a part of an individual prophase subband is the most precise localization ever reported on human banded mitotic chromosomes.
T cells expressing the V beta 1 T-cell receptor are required for IgA production in the chicken.
Authors: Cihak J , Hoffmann-Fezer G , Ziegler-Heibrock H W , Stein H , Kaspers B , Chen C H , Cooper M D , Lösch U
Published: May 2006

While alpha beta T cells in mammals may express one of many variable (V) gene families in the beta locus, chickens have only two V beta gene families. The avian V beta 2+ T cells are recognized by the T-cell receptor 3 (TCR3) monoclonal antibody and V beta 1+ T cells are recognized by the TCR2 antibody, which we used to selectively suppress development of V beta 1+ T cells in order to examine their functional role. Suppression was accomplished by multiple injections of anti-TCR2 antibodies beginning in embryonic life and perpetuated by thymectomy 8 days after hatching. Young birds thus depleted of V beta 1+ T cells had greater than normal numbers of V beta 2+ T cells and appeared as healthy as thymectomized and untreated controls. While production of IgM and IgG antibodies was unimpaired, IgA antibody production was severely compromised in the V beta 1-depleted birds. The levels of secretory IgA in bile and lung lavage fluid were reduced 1000- to 10,000-fold and secretory IgA antibodies were not produced in response to mucosal immunization. B-cell production of IgA antibodies thus appears to require T cells expressing the V beta 1 genes, whereas T cells that express the V beta 2 genes lack this capacity.
Isolation of Drosophila genes encoding G protein-coupled receptor kinases.
Authors: Cassill J A , Whitney M , Joazeiro C A , Becker A , Zuker C S
Published: May 2006

G protein-coupled receptors are regulated via phosphorylation by a variety of protein kinases. Recently, termination of the active state of two such receptors, the beta-adrenergic receptor and rhodopsin, has been shown to be mediated by agonist- or light-dependent phosphorylation of the receptor by members of a family of protein-serine/threonine kinases (here referred to as G protein-coupled receptor kinases). We now report the isolation of a family of genes encoding a set of Drosophila protein kinases that appear to code for G protein-coupled receptor kinases. These proteins share a high degree of sequence homology with the bovine beta-adrenergic receptor kinase. The presence of a conserved family of G protein-coupled receptor kinases in vertebrates and invertebrates points to the central role of these kinases in signal transduction cascades.
The role of lactic acid in autocrine B-cell growth stimulation.
Authors: Pike S E , Markey S P , Ijames C , Jones K D , Tosato G
Published: May 2006

Growth and survival of Epstein-Barr virus (EBV)-immortalized B lymphocytes cultured at low cell densities require autocrine soluble factors. In this study, we have purified a low molecular weight autocrine soluble factor that promotes growth of EBV-immortalized B cells in serum-free conditions and identified it as lactic acid (LA). Synthetic LA stimulated growth in EBV-immortalized B cells at 1-10 mM, a concentration of LA measured in the culture supernatant of EBV-immortalized cell lines. LA alone was found to account for greater than 70% of the autocrine growth factor activity in serum-free supernatants of EBV-immortalized B cells. Aminooxyacetate, a glutamate-oxaloacetate transaminase inhibitor, specifically inhibited B-cell growth induced by LA, suggesting that this process requires mitochondrial-cytosol transfers. Thus, LA is an autocrine stimulatory molecule that in serum-free conditions is essential for the continuous proliferation of EBV-immortalized B cells. This represents an unexpected function for LA.
Response to erythropoietin in erythroid subclones of the factor-dependent cell line 32D is determined by translocation of the erythropoietin receptor to the cell surface.
Authors: Migliaccio A R , Migliaccio G , D'Andrea A , Baiocchi M , Crotta S , Nicolis S , Ottolenghi S , Adamson J W
Published: May 2006

Regulation of the expression of the erythropoietin (Epo) receptor (EpoR) gene is under the control of transcriptional regulatory factor GATA-1. GATA-1 is expressed widely among the nonerythroid, factor-dependent subclones of the interleukin 3-dependent mouse cell line 32D. Consequently, to determine whether GATA-1 and EpoR gene expression are linked even in nonerythroid cells, we have studied the correlation of GATA-1 expression with expression and function of EpoR in these cell lines. EpoR mRNA (by RNase protection analysis) and EpoR protein (by specific antibody immunoprecipitation of metabolically labeled EpoR protein) were detectable not only in 32D and 32D Epo (an Epo-dependent subclone) but also in 32D GM, a subclone dependent for growth on granulocyte/macrophage colony-stimulating factor. EpoR mRNA also was detectable by PCR in 32D G, a subclone dependent for growth on granulocyte colony-stimulating factor. However, only 32D Epo cells bound 125I-labeled Epo and expressed EpoR protein on the cell surface, as determined by immunoprecipitation of surface-labeled proteins. These results indicate that, in these factor-dependent cell lines, the major regulatory step determining the erythroid-specific response to Epo is the efficiency of EpoR protein translocation to the cell surface. Mechanisms that could affect lineage-specific translocation are the presence of a chaperone protein, erythroid-specific editing of EpoR mRNA, or altered processing of the EpoR protein to the cell surface. In this model, lineage-restricted responses to growth factors such as Epo are determined not by expression of the genes for growth factor receptors but, rather, by appropriate processing of the receptor protein.
Folding of group I introns from bacteriophage T4 involves internalization of the catalytic core.
Authors: Heuer T S , Chandry P S , Belfort M , Celander D W , Cech T R
Published: May 2006

Fe(II)-EDTA, a solvent-based cleavage reagent that distinguishes between the inside and outside surfaces of a folded RNA molecule, has revealed some of the higher-order folding of the group IB intron from Tetrahymena thermophila pre-rRNA. This reagent has now been used to analyze the bacteriophage T4 sunY and td introns, both of which are members of the group IA subclass. Significant portions of the phylogenetically conserved secondary structure are protected from Fe(II)-EDTA cleavage. However, the P4 secondary structure element, which is substantially protected in the Tetrahymena intron, is available for cleavage in the two T4 introns. We conclude that a family of catalytic RNAs (ribozymes) that possess similar secondary structures and have similar activities fold into similar but nonidentical tertiary structures that nevertheless serve to internalize portions of the catalytic center. Furthermore, comparison of cleavage patterns of the sunY and td intron RNAs indicates that conserved nucleotides outside as well as within the catalytic core participate in the tertiary structure.
Antibody redesign by chain shuffling from random combinatorial immunoglobulin libraries.
Authors: Kang A S , Jones T M , Burton D R
Published: May 2006

A number of experiments on the shuffling of heavy and light chains from antibodies of defined specificity for the transition-state analogue hapten nitrophenyl phosphonamidate are described. The experiments report on the promiscuity of heavy and light chains in binding antigen and the feasibility of antibody redesign by this shuffling process. The concepts of incestuous and extraclonal promiscuous association are described. Shuffling opens the possibility of generating panels of antibodies with related specificity but of distinct idiotypic composition that may have significance in the use of human monoclonal antibodies in therapy.
Cloning and characterization of a receptor-class phosphotyrosine phosphatase gene expressed on central nervous system axons in Drosophila melanogaster.
Authors: Hariharan I K , Chuang P T , Rubin G M
Published: May 2006

We have cloned and characterized cDNAs coding for a receptor-class phosphotyrosine phosphatase gene from Drosophila melanogaster. The gene maps to the polytene chromosome bands 99A7-8. The cDNA clones code for a polypeptide of 1301 amino acids with a predicted molecular mass of 145 kDa. The extracellular domain includes two fibronectin-type III-like domains. The cytoplasmic region contains two tandemly repeated phosphotyrosine phosphatase-like domains. Residues shown crucial for catalytic activity are absent in the second domain. This Drosophila receptor-class phosphotyrosine phosphatase polypeptide is expressed on axons of the embryonic central nervous system.
Domain exchange: characterization of a chimeric lipase of hepatic lipase and lipoprotein lipase.
Authors: Wong H , Davis R C , Nikazy J , Seebart K E , Schotz M C
Published: May 2006

Hepatic lipase and lipoprotein lipase hydrolyze fatty acids from triacylglycerols and are critical in the metabolism of circulating lipoproteins. The two lipases are similar in size and amino acid sequence but are distinguished by functional differences in substrate preference and cofactor requirement. Presumably, these distinctions result from structural differences in functional domains. To begin localization of these domains, a chimeric lipase was constructed composed of the N-terminal 329 residues of rat hepatic lipase linked to the C-terminal 136 residues of human lipoprotein lipase. The chimera hydrolyzed both monodisperse short-chain (esterase) and emulsified long-chain (lipase) triacylglycerol substrates with catalytic and kinetic properties closely resembling those of native hepatic lipase. However, monoclonal antibodies to lipoprotein lipase inhibited the lipase activity, but not the esterase function, of the chimera. Therefore, the chimeric molecule is a functional lipase and contains elements and characteristics from both parental enzymes. It is proposed that the N-terminal domain, containing the active center from hepatic lipase, governs the catalytic character of the chimera, and the C-terminal domain is essential for hydrolysis of long-chain substrates.
Synaptic background activity influences spatiotemporal integration in single pyramidal cells.
Authors: Bernander O , Douglas R J , Martin K A , Koch C
Published: May 2006

The standard one-dimensional Rall cable model assumes that the electrotonic structure of neurons does not change in response to synaptic input. This model is used in a great number of both theoretical and anatomical-physiological structure-function studies. In particular, the membrane time constant, tau m, the somatic input resistance, Rin, and the electrotonic length are used to characterize single cells. However, these studies do not take into account that neurons are embedded in a network of spontaneously active cells. Synapses from these cells will contribute significantly to the membrane conductance, especially if recent evidence of very high specific membrane resistance, Rm = 100 k omega.cm2, is taken into account. We numerically simulated the electrical behavior of an anatomically reconstructed layer V cortical pyramidal cell receiving input from 4000 excitatory and 1000 inhibitory cells firing spontaneously at 0-7 Hz. We found that, over this range of synaptic background activity, tau m and Rin change by a factor of 10 (80-7 msec, 110-14 M omega) and the electrotonic length of the cell changes by a factor of 3. We show that this significantly changes the response of the cell to temporal desynchronized versus temporal synchronized synaptic input distributed throughout the neuron. Thus, the global activity of the network can control how individual cells perform spatial and temporal integration.
Cloning and expression of a rat brain alpha 2B-adrenergic receptor.
Authors: Flordellis C S , Handy D E , Bresnahan M R , Zannis V I , Gavras H
Published: May 2006

We have isolated a cDNA clone (RB alpha 2B) and its homologous gene (GR alpha 2B) encoding an alpha 2B-adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor (alpha 2-C4) and divergent from the rat kidney nonglycosylated alpha 2B subtype (RNG alpha 2). Transient expression of RB alpha 2B in COS-7 cells resulted in high-affinity saturable binding (Kd = 0.25 nM) for [3H]rauwolscine and a high receptor number (Bmax = 7.7 pmol/mg of protein) in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine greater than yohimbine greater than prazosin greater than oxymetazoline, with a prazosin-to-oxymetazoline Ki ratio of 0.34. This profile is characteristic of the alpha 2B-adrenergic receptor subtype. Blotting analysis of rat brain mRNA gave one major (3.0-kilobase) and two minor (4.6- and 2.3-kilobase) mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GR alpha 2B may be transcriptionally active. These findings show that rat brain expresses an alpha 2B-adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated alpha 2B subtype. Thus the rat expresses at least two divergent alpha 2B-adrenergic receptors.
CD11c/CD18 on neutrophils recognizes a domain at the N terminus of the A alpha chain of fibrinogen.
Authors: Loike J D , Sodeik B , Cao L , Leucona S , Weitz J I , Detmers P A , Wright S D , Silverstein S C
Published: May 2006

Fibrinogen and fibrin serve as adhesive substrates for a variety of cells including platelets, endothelial cells, and leukocytes. Previously, we identified the C terminus of the gamma chain of fibrinogen as the region of the fibrinogen molecule that contains a ligand for CD11b/CD18 (complement receptor 3) on phorbol ester-stimulated polymorphonuclear leukocytes. In contrast, we report here that neutrophils stimulated with tumor necrosis factor adhere to fibrinogen-coated surfaces, but not to human serum albumin-coated surfaces, via the integrin CD11c/CD18 (p150/95). Monoclonal antibodies LeuM5 and 3.9, which are directed against the alpha subunit of CD11c/CD18, but not monoclonal antibodies OKM10 and OKM1, which are directed against the alpha subunit of CD11b/CD18, inhibit the adhesion of tumor necrosis factor-stimulated neutrophils to fibrinogen-coated surfaces. To identify the site on fibrinogen recognized by CD11c/CD18, we have examined the adhesion of tumor necrosis factor-stimulated neutrophils to surfaces coated with various fibrinogen fragments. Stimulated neutrophils adhere to surfaces coated with the N-terminal disulfide knot fragment of fibrinogen or fibrinogen fragment E. Moreover, peptides containing the sequence Gly-Pro-Arg (which corresponds to amino acids 17-19 of the N-terminal region of the A alpha chain of fibrinogen), and monoclonal antibody LeuM5, block tumor necrosis factor-stimulated neutrophil adhesion to fibrinogen and to the N-terminal disulfide knot fragment of fibrinogen. Thus, CD11c/CD18 on tumor necrosis factor-stimulated neutrophils functions as a fibrinogen receptor that recognizes the sequence Gly-Pro-Arg in the N-terminal domain of the A alpha chain of fibrinogen.
Electron probe microanalysis of calcium release and magnesium uptake by endoplasmic reticulum in bee photoreceptors.
Authors: Baumann O , Walz B , Somlyo A V , Somlyo A P
Published: May 2006

Honey bee photoreceptors contain large sacs of endoplasmic reticulum (ER) that can be located unequivocally in freeze-dried cryosections. The elemental composition of the ER was determined by electron probe x-ray microanalysis and was visualized in high-resolution x-ray maps. In the ER of dark-adapted photoreceptors, the Ca concentration was 47.5 +/- 1.1 mmol/kg (dry weight) (mean +/- SEM). During a 3-sec nonsaturating light stimulus, approximately 50% of the Ca content was released from the ER. Light stimulation also caused a highly significant increase in the Mg content of the ER; the ratio of Mg uptake to Ca released was approximately 0.7. Our results show unambiguously that the ER is the source of Ca2+ release during cell stimulation and suggest that Mg2+ can nearly balance the charge movement of Ca2+.
Conversion of human choriogonadotropin into a follitropin by protein engineering.
Authors: Campbell R K , Dean-Emig D M , Moyle W R
Published: May 2006

Human reproduction is dependent upon the actions of follicle-stimulating hormone (hFSH), luteinizing hormone (hLH), and chorionic gonadotropin (hCG). While the alpha subunits of these heterodimeric proteins can be interchanged without effect on receptor-binding specificity, their beta subunits differ and direct hormone binding to either LH/CG or FSH receptors. Previous studies employing chemical modifications of the hormones, monoclonal antibodies, or synthetic peptides have implicated hCG beta-subunit residues between Cys-38 and Cys-57 and corresponding regions of hLH beta and hFSH beta in receptor recognition and activation. Since the beta subunits of hCG or hLH and hFSH exhibit very little sequence similarity in this region, we postulated that these residues might contribute to hormone specificity. To test this hypothesis we constructed chimeric hCG/hFSH beta subunits, coexpressed them with the human alpha subunit, and examined their ability to interact with LH and FSH receptors and hormone-specific monoclonal antibodies. Surprisingly, substitution of hFSH beta residues 33-52 for hCG beta residues 39-58 had no effect on receptor binding or stimulation. However, substitution of hFSH beta residues 88-108 in place of the carboxyl terminus of hCG beta (residues 94-145) resulted in a hormone analog identical to hFSH in its ability to bind and stimulate FSH receptors. The altered binding specificity displayed by this analog is not attributable solely to the replacement of hCG beta residues 108-145 or substitution of residues in the "determinant loop" located between hCG beta residues 93 and 100.
Glutamate and 2-amino-4-phosphonobutyrate evoke an increase in potassium conductance in retinal bipolar cells.
Authors: Hirano A A , MacLeish P R
Published: May 2006

Although there is general agreement that L-glutamate can produce a depolarizing inward current to account for the hyperpolarizing (OFF) bipolar cell response, the conductance mechanism underlying the depolarizing (ON) response has been difficult to establish satisfactorily. To investigate the ionic bases of the center responses, we studied the whole-cell currents controlled by L-glutamate and its analogues in solitary bipolar cells from salamander retina. We report here two groups of isolated bipolar cells: one group responded to L-glutamate with the previously described inward current [Attwell, D., Mobbs, P., Tessier-Lavigne, M. & Wilson, M. (1987) J. Physiol. (London) 387, 125-161] and a second group showed an outward current that reversed at about -70 mV. Both were associated with an increase in membrane conductance. In addition, DL-2-amino-4-phosphonobutyrate, a compound diagnostic for ON-bipolar cell activity [Slaughter, M. M. & Miller, R. F. (1981) Science 211, 182-185], elicited outward currents that closely resembled those seen in response to L-glutamate and, furthermore, that were shown to arise from an increase in conductance to potassium ions. Thus the presence of two distinct conductances controlled by L-glutamate in solitary cells would provide one mechanism for generating the ON and OFF light responses at the bipolar cell level in the intact retina.
Cloning of murine ferrochelatase.
Authors: Brenner D A , Frasier F
Published: May 2006

Ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) catalyzes the last step in the heme biosynthetic pathway, the chelation of ferrous iron and protoporphyrin to form heme. The activity of ferrochelatase is deficient in the inherited disease protoporphyria. In this study, murine ferrochelatase cDNAs were obtained by screening cDNA libraries with an oligonucleotide probe. The derived amino acid sequence of murine ferrochelatase has 47% identity with the recently cloned Saccharomyces cerevisiae ferrochelatase, but it is not significantly similar to other published sequences. Results of Southern blotting are consistent with a single murine ferrochelatase gene, while Northern blotting demonstrates two ferrochelatase transcripts in all tissues examined. The ferrochelatase protein and mRNAs have different relative concentrations in different tissues. The cloning of murine ferrochelatase cDNAs provides the basis for future studies on ferrochelatase gene expression and on the identification of the molecular defect in protoporphyria.
CGG: an unassigned or nonsense codon in Mycoplasma capricolum.
Authors: Oba T , Andachi Y , Muto A , Osawa S
Published: May 2006

CGG is an arginine codon in the universal genetic code. We previously reported that in Mycoplasma capricolum, a relative of Gram-positive eubacteria, codon CGG did not appear in coding frames, including termination sites, and tRNA(ArgCCG) pairing with codon CGG, was not detected. These facts suggest that CGG is a nonsense (unassigned and untranslatable) codon--i.e., not assigned to arginine or to any other amino acid. We have investigated whether CGG is really an unassigned codon by using a cell-free translation system prepared from M. capricolum. Translation of synthetic mRNA containing in-frame CGG codons does not result in "read-through" to codons beyond the CGG codons--i.e., translation ceases just before CGG. Sucrose-gradient centrifugation profiles of the reaction mixture have shown that the bulk of peptide that has been synthesized is attached to 70S ribosomes and is released upon further incubation with puromycin. The result suggests that the peptide is in the P site of ribosome in the form of peptidyl-tRNA, leaving the A site empty. When in-frame CGG codons are replaced by UAA codons in mRNA, no read-through occurs beyond UAA, just as in the case of CGG. However, the synthesized peptide is released from 70S ribosomes, presumably by release factor 1. These data suggest strongly that CGG is an unassigned codon and differs from UAA in that CGG is not used for termination.
Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes.
Authors: Kuppuswamy M N , Hoffmann J W , Kasper C K , Spitzer S G , Groce S L , Bajaj S P
Published: May 2006

In this report, we describe an approach to detect the presence of abnormal alleles in those genetic diseases in which frequency of occurrence of the same mutation is high (e.g., cystic fibrosis and sickle cell disease), and in others in which multiple mutations cause the disease and the sequence variation in an affected member of a given family is known (e.g., hemophilia B). Initially, from each subject, the DNA fragment containing the putative mutation site is amplified by the polymerase chain reaction. For each fragment two reaction mixtures are then prepared. Each contains the amplified fragment, a primer (18-mer or longer) whose sequence is identical to the coding sequence of the normal gene immediately flanking the 5' end of the mutation site, and either an alpha-32P-labeled nucleotide corresponding to the normal coding sequence at the mutation site or an alpha-32P-labeled nucleotide corresponding to the mutant sequence. Single nucleotide primer extensions are then carried out and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. As predicted by the Watson-Crick base-pair rule, in the wild type only the normal base, in an affected member only the mutant base, and in carriers both the normal and the mutant base are incorporated into the primer. Thus, an essential feature of the present methodology is that the base immediately 3' to the template-bound primer is one of those altered in the mutant, since in this way an extension of the primer by a single base will give an extended molecule characteristic of either the mutant or the wild type. The method is rapid and should be useful in carrier detection and prenatal diagnosis of every genetic disease with a known sequence variation.
Sexual transmission of human T-cell leukemia virus type I associated with the presence of anti-Tax antibody.
Authors: Chen Y M , Okayama A , Lee T H , Tachibana N , Mueller N , Essex M
Published: May 2006

The tax gene product (Tax protein) of human T-cell leukemia virus type I (HTLV-I) is a specific transcriptional activator of the viral long terminal repeat sequence and is essential for the replication cycle of the virus. To elucidate the relationship between the presence of anti-Tax antibody and the transmission of the viral infection, annual consecutive serum samples from married couples serologically discordant or concordant for HTLV-I were examined. These included 5 individuals whose spouses seroconverted during this 5-year follow-up study period. The samples were tested by a Western blot assay using a recombinant Tax protein as the antigen. The results showed that 24 of 32 (75%) men in the concordant couples (both husband and wife were HTLV-I carriers) had anti-Tax antibody, while only 5 of 18 (27.8%) men in the discordant couples (husband was carrier and wife was seronegative to HTLV-I) were positive for anti-Tax antibody (P = 0.0012). Furthermore, all spouses of the 5 seroconverters (4 women and 1 man) had anti-Tax antibody, while only 23 of 46 (50%) age-matched randomly selected HTLV-I carriers from the discordant-couple group had anti-Tax antibody. When the data were analyzed by gender, all husbands of the female seroconverters had anti-Tax antibodies, which was significantly higher than the prevalence of anti-Tax antibodies in men who did not transmit the virus to their spouses during the follow-up period (P = 0.017). In addition, antibody reactivity to other HTLV-I antigens (including Env gp46, transmembrane protein gp21, and Gag p19 and p24) were examined. The results indicated no significant differences between the prevalence of antibody reactivity to any of the antigens in the spouses of the seroconverters and the reference group. We conclude that the presence of anti-Tax antibody in men may indicate a high risk of viral transmission to their wives via heterosexual routes.
Phased cis-acting promoter elements interact at short distances to direct avian skeletal alpha-actin gene transcription.
Authors: Chow K L , Hogan M E , Schwartz R J
Published: May 2006

Recently, site-directed mutagenesis uncovered four positive cis-acting elements in the 5' promoter region of the chicken skeletal alpha-actin gene that directs myogenic tissue-restricted expression. In this study, interactions between the four promoter sites were examined by means of a series of insertion mutations that increased the linker region between adjacent elements by roughly half or complete DNA helical turns. Unexpectedly, transcriptional activity for all three sets of linker mutants, as assayed with a chloramphenicol acetyltransferase reporter gene, was found to vary in a fashion resembling a damped sinusoid with a period of roughly 10 base pairs, where the sinusoidal maxima appeared when length was increased by half-integral number of helix turns. We present a model which states that in the undistorted wild-type 5' flanking sequence, linker domains position each of the four promoter sites on the helix face opposite that of its immediate neighbors; when any of the three linkers is increased by approximately a half-integral number of helix turns, pairs of neighboring promoter sites are brought into alignment. We propose that this is the required orientation for inducing skeletal muscle-specific promoter activity, achieved in the wild-type promoter as a result of protein-induced torsional deformation.
Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: intracellular targeting to the amyloplast.
Authors: Singh M B , Hough T , Theerakulpisut P , Avjioglu A , Davies S , Smith P M , Taylor P , Simpson R J , Ward L D , McCluskey J
Published: May 2006

We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain.
Characterization of anti-peptide antibodies for the localization of D2 dopamine receptors in rat striatum.
Authors: McVittie L D , Ariano M A , Sibley D R
Published: May 2006

Seven different peptides of 14-23 residues in length based on the predicted amino acid sequence of the cloned rat D2 receptor cDNA were used as immunogens to develop antibodies in rabbits. Two of these peptides were derived from the amino terminus and four were from the third cytoplasmic loop, including one to the splice variant insertion sequence and one to the carboxyl terminus of the receptor protein. These peptides were conjugated to bovine thyroglobulin prior to rabbit immunization. Antibody production was monitored by a solid-phase ELISA. With the exception of the carboxyl-terminal peptide, all of the peptide immunogens produced antiserum of high titer ranging from 1:10(4) to 1:10(6) on ELISA. Specificity of the reaction was demonstrated by the absence of a response in the preimmune serum and by the absence of cross-reactivity between the various antisera and the nonimmunization peptides. Moreover, preincubation of the antiserum with the immunization peptide, but not other peptides, blocked the subsequent ELISA reactions. Some of the antisera were additionally characterized by immunodot assays using solubilized rat striatal membranes blotted onto nitrocellulose. Positive reactions with antiserum dilutions of 1:500 were observed that were dependent on the presence and concentration of membrane protein and were not observed using preimmune serum. Additionally, immunofluorescent staining by the D2 receptor antiserum was observed on cells that had been transfected with the D2 receptor cDNA but not on untransfected cells. Immunoprecipitation of the photoaffinity-labelled and solubilized D2 receptor also suggested that the antisera were able to directly recognize the native receptor protein. Immunohistochemical localization of the D2 receptor in slices of fresh frozen and perfusion-fixed rat brain was performed using these antisera. Within the striatum, about 50% of the medium-sized neurons were labeled as well as large, putatively cholinergic interneurons.
The conserved GTPase center and variable region V9 from Saccharomyces cerevisiae 26S rRNA can be replaced by their equivalents from other prokaryotes or eukaryotes without detectable loss of ribosomal function.
Authors: Musters W , Conçalves P M , Boon K , Raué H A , van Heerikhuizen H , Planta R J
Published: May 2006

Using the "tagged" rRNA gene system, which allows in vivo mutational analysis of Saccharomyces cerevisiae rRNA, we studied the role of two distinct structural elements of 26S rRNA in ribosome biogenesis and function--namely, the evolutionarily highly conserved "GTPase center" located in domain II and the eukaroyote-specific variable region V9 in domain III. Replacement of the S. cerevisiae GTPase center with its counterpart from Escherichia coli did not affect the assembly of the mutant 26S rRNA into functional (as judged by their polysomal distribution) 60S subunits, indicating that the E. coli GTPase center functions efficiently in the context of the heterologous rRNA. Removal of most of the S. cerevisiae V9 region or replacement of this segment by the equivalent segment from mouse 28S rRNA also did not affect the formation of functional 60S subunits carrying the mutant 26S rRNA. Therefore, the V9 region does not seem to play a role in the biological functioning of the yeast 60S subunits, and these subunits appear to be able to accommodate V9 regions of various size and secondary structure without apparent loss of function.
Independently arising macrophage mutants dissociate growth factor-regulated survival and proliferation.
Authors: Pollard J W , Morgan C J , Dello Sbarba P , Cheers C , Stanley E R
Published: May 2006

Analysis of a simian virus 40-immortalized colony-stimulating factor 1 (CSF-1) -dependent macrophage cell line (BAC1.2F5) and independently arising autonomous mutants derived from it (aut4A, aut4A.1, aut2A, and aut2A.1) revealed distinct phenotypes. The parental line, BAC1.2F5, is dependent on CSF-1 for survival and growth. Of the mutants derived from BAC1.2F5, aut4A has lost the requirement of CSF-1 for survival; aut4A.1 (derived from aut4A) and aut2A grow in the absence of growth factor but proliferate more rapidly in its presence, and aut2A.1 (derived from aut2A) produces CSF-1 and proliferates as rapidly in the presence as in the absence of exogeneous CSF-1. The separation of the CSF-1 requirement for survival and proliferation observed in aut4A is also observed in a temperature-sensitive (ts) mutant tsgro1. At the nonpermissive temperature, tsgro1 cell proliferation is arrested, but the cells survive provided CSF-1 is present. The four cellular phenotypes observed--immortalization, loss of growth factor requirement for survival, loss of growth factor requirement for proliferation, and loss of growth factor-stimulated proliferation--indicate a divergence of the pathways of growth factor-regulated survival and proliferation and may represent phenotypes occurring at intermediate stages in tumor-cell progression.
Interactive effects of neurohypophyseal neuropeptides with receptor antagonists on passive avoidance behavior: mediation by a cerebral neurohypophyseal hormone receptor?
Authors: de Wied D , Elands J , Kovács G
Published: May 2006

The neurohypophyseal neuropeptides (Arg8)-vasopressin (AVP) and [pGlu4,Cyt6]AVP-(4-8) (where pGlu is pyroglutamic acid and Cyt is cystine) facilitate the retention of one-trial-learning passive avoidance behavior in rats when administered into the cerebral ventricle immediately after the learning trial. The fragment [pGlu4,Cyt6]AVP-(4-8) was considerably more effective than AVP. Oxytocin (OXT) and [pGlu4,Cyt6]OXT-(4-8) have the opposite effect and attenuate passive avoidance behavior also when administered into the cerebral ventricle after the learning trial. Again the fragment was more active than the parent molecule. The ancient arginine-containing neurohypophyseal hormone vasotocin in "high" doses (10ng) had a vasopressin-like effect and in "low" doses (0.1 ng) had an OXT-like effect on passive avoidance behavior. Because both vasopressinergic (V1) and oxytocinergic receptors have been demonstrated in the central nervous system, we asked whether specific antagonists of the V1, V2, and OXT receptor could antagonize the effects of these neuropeptides on passive avoidance behavior. The three antagonists were approximately equally active in blocking the effect of vasopressin, whereas the fragment [pGlu4]AVP-(4-8) and the high dose of vasotocin were more readily blocked by the OXT antagonist. The attenuating effect of OXT, the fragment [pGlu4,Cyt6]OXT-(4-8), and the low dose of vasotocin was markedly reduced by the OXT antagonist. This effect could also be reduced by pretreatment with the V1 antagonist but not with the V2 antagonist. These results suggest the existence of a separate neurohypophyseal hormone receptor complex in the brain affecting memory processes that differs from the peripheral V1, V2, and OXT receptor.
ToxR regulates the production of lipoproteins and the expression of serum resistance in Vibrio cholerae.
Authors: Parsot C , Taxman E , Mekalanos J J
Published: May 2006

The genes encoding three lipoproteins of Vibrio cholerae were identified by a combination of DNA sequence analysis and [3H]palmitate labeling of hybrid proteins encoded by TnphoA gene fusions. The expression of these three lipoproteins, TagA, AcfD, and TcpC, was controlled by ToxR, the cholera toxin transcriptional activator. The involvement of other bacterial lipoproteins in conferring resistance to the bactericidal effects of complement prompted us to examine this possibility in V. cholerae. Remarkably, mutations in toxR and tcp genes (including tcpC), involved in the biogenesis of the toxin coregulated pili, rendered V. cholerae about 10(4)-10(6) times more sensitive to the vibriocidal activity of antibody and complement. Since V. cholerae is a noninvasive organism and toxR and tcp mutants are highly defective in intestinal colonization in animals and humans, these results raise the possibility that resistance to a gut-associated, "complement-like" bactericidal activity may be a major virulence determinant of V. cholerae and other enterobacterial species.
Promoter region of the human platelet-derived growth factor A-chain gene.
Authors: Takimoto Y , Wang Z Y , Kobler K , Deuel T F
Published: May 2006

The platelet-derived growth factor (PDGF) A- and B-chain genes are widely expressed in mammalian tissues and their homodimeric gene products appear to regulate the autocrine growth of both normal and transformed cells. In this study, we analyzed the 5' flanking sequences of the human PDGF A-chain gene to seek elements important to regulating its transcription. The promoter region was exceptionally G + C-rich and contained a "TATA box" but no "CAAT box." The transcription start site was identified 845 base pairs 5' to the translation initiation site by S1 nuclease mapping and by primer extension. Both in vitro transcription and transient expression of the chloramphenicol acetyltransferase gene linked to the PDGF A-chain 5' flanking sequences established that the putative promoter region was active, and RNase H mapping established that the three characteristic mRNAs (1.9, 2.3, and 2.8 kilobases) used the same transcription start site, which was used in normal endothelial cells and in two human tumor cell lines that express high levels of A-chain transcripts. The results established an exceptionally G + C-rich promoter region and a single transcription start site active for each of the three mRNAs of the PDGF A-chain gene. DNA sites of potential importance in mediating the activation of the PDGF A-chain gene in normal cells and in transformed cell lines expressing high levels of PDGF A chain were identified.
Stage transitions in B-lymphocyte differentiation correlate with limited variations in nuclear proteins.
Authors: Rabilloud T , Pennetier J L , Hibner U , Vincens P , Tarroux P , Rougeon F
Published: May 2006

Total nuclear proteins extracted from cell lines representing various stages of differentiation of mouse B lymphocytes were studied by computer analysis of two-dimensional gels. Of the 1438 spots present on the gels, 55 varied significantly in intensity during differentiation. The variations occurred most often in steps correlating with those classically defined for B-cell differentiation. Seventeen spots were not detectable in at least one of the stages (qualitative variations) and could represent switching on or off of genes coding for nuclear proteins. Detailed analysis of the 55 variable spots showed that they fall into small sets characterized by similar expression profiles, which argues for a combinatorial, multistep control mechanism of gene expression. In addition, analysis of the expression of all the nuclear proteins resolved on the gels clearly differentiated B-lineage cells from myeloid cells and suggested that the most important transition in B-cell differentiation occurs between the resting B cell and plasmocyte stages.
Primary structure of the 170-kDa surface lectin of pathogenic Entamoeba histolytica.
Authors: Tannich E , Ebert F , Horstmann R D
Published: May 2006

The adherence of Entamoeba histolytica to colonic mucins and to host cells appears to be predominantly mediated by a 170-kDa surface lectin of the amoebae. By using an antiserum to the purified lectin, the corresponding cDNA was isolated from an expression library of the pathogenic E. histolytica isolate HM-1:IMSS. Northern blot analysis indicated a transcript of approximately 4 kilobases, and Southern blot analyses suggested that multiple genes may encode the lectin or closely related proteins in HM-1:IMSS trophozoites. The cDNA-deduced amino acid sequence revealed an N-terminal signal peptide and a mature protein of 1270 amino acids corresponding to a molecular mass of 143 kDa, which comprises a short C-terminal cytoplasmic domain with potential phosphorylation sites, a transmembrane region, and a large extracellular portion with nine potential asparagine-linked glycosylation sites. The extracellular portion may be separated into a cysteine-poor domain and a cysteine-rich domain, the latter of which shows in part repetitive structural elements with a low degree of sequence homology to wheat germ agglutinin and to pDd63, a developmentally expressed protein of Dictyostelium discoideum.
Mutasynthesis of siderophore analogues by Pseudomonas aeruginosa.
Authors: Ankenbauer R G , Staley A L , Rinehart K L , Cox C D
Published: May 2006

The Gram-negative bacterium Pseudomonas aeruginosa produces the phenolic siderophore pyochelin. Salicylic acid is an intermediate in the pyochelin biosynthetic pathway, and mutants blocked in salicylic acid biosynthesis (Sal-) are able to incorporate exogenously supplied salicylic acid into pyochelin. A P. aeruginosa Sal- mutant was incubated with 13 salicylic acid analogues and was found to incorporate three (5-fluorosalicylic acid, 4-methylsalicylic acid, and 3-hydroxypicolinic acid) into pyochelin analogues, trivially designated as 5-fluoropyochelin, 4-methylpyochelin, and 6-azapyochelin. The structures of the mutasynthetic products were confirmed by 1H and 13C NMR and high-resolution fast atom bombardment mass spectrometry as being identical to pyochelin except for the expected changes in the aromatic ring. The biological activity of the three pyochelin analogues was determined in iron transport assays. In comparison to pyochelin, 4-methylpyochelin was more active in the assays whereas the activities of 5-fluoropyochelin and 6-azapyochelin were markedly decreased. In coincubation assays, 5-fluoropyochelin substantially inhibited iron transport by pyochelin; 4-methylpyochelin and 6-azapyochelin did not demonstrate this inhibitory effect.
Canonical ordered cosmid library of the symbiotic plasmid of Rhizobium species NGR234.
Authors: Perret X , Broughton W J , Brenner S
Published: May 2006

Many of the bacterial genes involved in nodulation (nod) and nitrogen fixation (nif) are dispersed over the 500-kilobase plasmid pNGR234a of the broad host-range Rhizobium species NGR234. As a first step toward generating a complete physical and genetic map of the plasmid, a full overlapping collection of cosmids was derived from a total genomic library. Clones were aligned by combining fingerprinting, hybridization, and pulsed-field gel electrophoresis data. Symbiotic loci were localized by probing a representative set of cosmids with both homologous and heterologous genes. nodABC, nodD1, nodD2, nodSU, nolB, and region II are widely dispersed over pNGR234a, while the two functional copies of nifKDH are separated by only 28 kilobases. Interestingly, sequences homologous to nodE, nodG, nodP, and nodQ have been assigned to another autonomously replicating element in Rhizobium species NGR234. Similarly one copy of the structural dctA gene is located on the symbiotic plasmid (dctA1) while the other is on what we assume to be the chromosome.