bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and sequence analysis of rabbit progesterone-receptor complementary DNA.
Authors: Loosfelt H , Atger M , Misrahi M , Guiochon-Mantel A , Meriel C , Logeat F , Benarous R , Milgrom E
Published: May 2006

Two lambda gt11 clones containing fragments of cDNA encoding the rabbit progesterone receptor were isolated with the aid of monoclonal and monospecific polyclonal antireceptor antibodies. RNA gel blot analysis showed that the corresponding mRNA was approximately equal to 5900 nucleotides in size and present in the uterus, where its concentration was increased by estrogen treatment, and in the vagina. This mRNA was not detected in liver, in spleen, in intestine, and in kidney where the receptor protein is known to be absent or present in very small concentration. Cross-hybridizing clones were isolated from a lambda 10 library. The DNA was sequenced, and the primary structure of the progesterone receptor was deduced. It consists of 930 amino acids and contains a basic, cysteine-rich region (residues 568-645) with extensive homology to the glucocorticoid and estrogen receptors and the v-erbA oncogene protein. This region is followed by a C-terminal domain that is similar in size to the corresponding domains of the other steroid receptors and v-erbA and shows striking amino acid homology with the glucocorticoid receptor and significant homology with the estrogen receptor. In contrast, the region extending from the cysteine-rich segment toward the N terminus differed in size and amino acid sequence from that of the other receptors and v-erbA. This region had a high proline content in the progesterone receptor.
A method for identifying the viral genes required for herpesvirus DNA replication.
Authors: Challberg M D
Published: May 2006

Several laboratories have shown that transfected plasmid DNAs containing either of the two known origins of herpes simplex virus (HSV) DNA replication, oriS or oriL, are replicated in HSV-1-infected cells or in cells cotransfected with virion DNA. I have found that HSV-1 (KOS) DNA digested to completion with the restriction enzyme Xba I is as efficient as intact viral DNA in supporting the in vivo replication of cotransfected plasmids containing oriS. On the basis of this result, several of the Xba I restriction fragments of HSV-1 DNA were cloned into the plasmid vector pUC19, and combinations of cloned DNAs were tested for their ability to supply the trans-acting functions required for HSV origin-dependent replication. A combination of five cloned fragments of HSV-1 can supply all of the necessary functions: Xba I C (coordinates 0.074-0.294), Xba I F (coordinates 0.294-0.453), Xba I E (coordinates 0.453-0.641), Xba I D (coordinates 0.641-0.830), and EcoRI JK (coordinates 0.0-0.086; 0.830-0.865). Transient plasmid replication in this system is dependent on the presence of either oriS or oriL in cis. The plasmid containing Xba I F can be replaced by two smaller plasmids, one of which contains only the gene for the HSV-encoded DNA polymerase, and the other of which contains only the gene for the major DNA binding protein (ICP8). Thus, plasmid DNA replication in this system depends on two of the genes known from genetic studies to be essential for viral DNA replication in infected cells. This system defines a simple complementation assay for cloned fragments of HSV DNA that contain other genes involved in viral DNA replication and should lead to the rapid identification of all such genes.
Mechanism of action of some inhibitors of endothelium-derived relaxing factor.
Authors: Moncada S , Palmer R M , Gryglewski R J
Published: May 2006

The mechanism of the inhibitory action of phenidone, 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW 755C), dithiothreitol, hydroquinone, and pyrogallol on the vascular relaxation induced by endothelium-derived relaxing factor (EDRF) was investigated. EDRF was released from porcine aortic endothelial cells in culture and bioassayed on a cascade of superfused rabbit aortic strips. These compounds inhibited EDRF-induced relaxation of vascular strips, without affecting the relaxation induced by glyceryl trinitrate, and their inhibitory potency was markedly attenuated (by more than 1 order of magnitude) by the addition of superoxide dismutase (5-15 units/ml) or oxidized cytochrome c (20-40 microM) but not by catalase (30 units/ml) or heat-inactivated superoxide dismutase. These data indicate that the above five inhibitors inactivate EDRF through the formation of superoxide ions, which have recently been shown to destroy EDRF. The inhibition of EDRF by these compounds is therefore attributable to their redox properties rather than to any specific biological action.
An Epstein-Barr virus transcript from a latently infected, growth-transformed B-cell line encodes a highly repetitive polypeptide.
Authors: Speck S H , Pfitzner A , Strominger J L
Published: May 2006

By screening a cDNA library prepared from poly(A)+ RNA isolated from an Epstein-Barr virus latently infected, growth-transformed human B-lymphoblastoid cell line, we have recovered a clone corresponding to a highly spliced viral transcript encoded largely by the major internal repeat (IR1). The 5' region contains one copy of a 26-base-pair (bp) exon (W0) [which is 28 bp downstream from a CAATT-(N)34TATAAA sequence (N, unspecified base)] and seven copies of two small exons (W1, 66 bp; W2, 132 bp). In addition, there are three exons from the "unique" region of the BamHI Y fragment of the viral genome. Two other cDNA clones that have been described, corresponding to latent viral transcripts, share homology in their 5' regions with this clone and are clearly divergent at their 3' ends. The cDNA clone described in this paper contains one long open reading frame that extends through the repeat element. In vitro transcription and translation of this open reading frame yielded a 62-kDa polypeptide that could be immunoprecipitated by an Epstein-Barr virus-positive human serum.
Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae.
Authors: Sass P , Field J , Nikawa J , Toda T , Wigler M
Published: May 2006

A gene, PDE2, has been cloned from the yeast Saccharomyces cerevisiae that, when present in high copy, reverses the phenotypic effects of RAS2Val19, a mutant form of the RAS2 gene that renders yeast cells sensitive to heat shock and starvation. It has previously been shown that the RAS proteins are potent activators of yeast adenylate cyclase. We report here that PDE2 encodes a high-affinity cAMP phosphodiesterase that shares sequence homology with animal cell phosphodiesterases. These results therefore imply that the effects of RAS2Val19 are mediated through its changes in cAMP concentration.
Polarized expression of a chimeric protein in which the transmembrane and cytoplasmic domains of the influenza virus hemagglutinin have been replaced by those of the vesicular stomatitis virus G protein.
Authors: McQueen N , Nayak D P , Stephens E B , Compans R W
Published: May 2006

In polarized epithelial cells, influenza virus buds exclusively from the apical domain of the plasma membrane, whereas vesicular stomatitis virus (VSV) buds exclusively from the basolateral domain. In virus-infected cells, the envelope proteins, influenza hemagglutinin (HA) and vesicular stomatitis virus G (VSV G), are likewise transported to and localized in the same domain of the plasma membrane from which the viruses bud. Previous studies have shown that influenza HA and VSV G proteins, when expressed from cloned cDNAs, are accumulated preferentially on the proper domains (apical and basolateral, respectively), indicating that the signal(s) for polarized transport resides in the polypeptide backbone of the proteins. To further elucidate the structural features required for apical vs. basolateral transport, we have constructed a gene that encodes a chimeric protein (H1GA) containing the external domain of HA and the transmembrane and cytoplasmic domains of VSV G. When the chimeric protein (H1GA) is expressed in CV1 cells using a simian virus 40 late expression vector, it is transported to the cell surface with kinetics similar to that of the native HA protein. Further, the chimeric protein, when expressed in polarized MDCK cells using a vaccinia virus early expression vector, is transported only to the apical surface, suggesting that the ectodomain of HA contains a signal for apical transport.
Phosphatidylinositol from ethanol-fed rats confers membrane tolerance to ethanol.
Authors: Taraschi T F , Ellingson J S , Wu A , Zimmerman R , Rubin E
Published: May 2006

The presence of ethanol disorders (fluidizes) biological membranes, but its chronic administration confers resistance to this perturbation (membrane tolerance). The latter effect has been invoked as an explanation for behavioral tolerance in alcoholics, but the molecular basis for membrane tolerance is obscure. To study the molecular mechanisms of this acquired resistance to disordering, we fed rats ethanol (36% of total calories) for 35 days, after which we quantitatively separated the phospholipids of hepatic microsomal membranes by high-performance liquid chromatography. Multilamellar vesicles were prepared from the recombined phospholipid classes, and their physical properties were examined by electron spin resonance. Vesicles composed of phospholipids from untreated rats were disordered (fluidized) in the presence of ethanol, whereas those made from phospholipids of ethanol-fed rats were resistant to this effect. When phosphatidylcholine (66.5 mol %), phosphatidylethanolamine (21 mol %), or phosphatidylserine (4.0 mol %) from ethanol-fed rats replaced their corresponding phospholipids in vesicles prepared from microsomal phospholipids from untreated rats, the membranes were still disordered by ethanol. In contrast, when 2.5-8.5 mol % phosphatidylinositol from ethanol-fed rats replaced phosphatidylinositol from untreated rats, the reconstituted membranes were rendered resistant to ethanol-induced disordering. Liver microsomal phosphatidylinositol (2.5-8.5 mol %) from ethanol-fed rats also conferred membrane tolerance to vesicles composed of bovine liver and brain phospholipids, an effect which demonstrates that the ability of phosphatidylinositol to confer membrane tolerance is not restricted to the microsomal membrane.
Genetically engineered mutant of the cyanobacterium
Synechocystis
6803 lacks the photosystem II chlorophyll-binding protein CP-47
Authors: Vermaas Wim F. J. , Williams John G. K. , Rutherford A. William , Mathis Paul , Arntzen Charles J.
Published: May 2006

CP-47 is absent in a genetically engineered mutant of cyanobacterium
Synechocystis
6803, in which the
psbB
gene [encoding the chlorophyll-binding photosystem II (PSII) protein CP-47] was interrupted. Another chlorophyll-binding PSII protein, CP-43, is present in the mutant, and functionally inactive PSII-enriched particles can be isolated from mutant thylakoids. We interpret these data as indicating that the PSII core complex of the mutant still assembles in the absence of CP-47. The mutant lacks a 77 K fluorescence emission maximum at 695 nm, suggesting that the PSII reaction center is not functional. The absence of primary photochemistry was indicated by EPR and optical measurements: no chlorophyll triplet originating from charge recombination between P680
+
and Pheo
-
was observed in the mutant, and there were no flash-induced absorption changes at 820 nm attributable to chlorophyll P680 oxidation. These observations lead us to conclude that CP-47 plays an essential role in the activity of the PSII reaction center.
Regulation of pulmonary surfactant apoprotein SP 28-36 gene in fetal human lung.
Authors: Ballard P L , Hawgood S , Liley H , Wellenstein G , Gonzales L W , Benson B , Cordell B , White R T
Published: May 2006

Pulmonary surfactant stabilizes lung alveoli, preventing respiratory failure and hyaline membrane disease in premature infants. In addition to lipids, surfactant contains apoproteins that are thought to be critical for normal surfactant function. We have examined the ontogeny and regulation of the major surfactant-associated protein of molecular mass 28-36 kDa (SP 28-36) in human fetal lung. SP 28-36 was not detected in tissue from second trimester abortuses by either immunoblot analysis or enzyme-linked immunosorbent assay (less than 0.02 microgram per mg of DNA). Levels of mRNA for SP 28-36, assayed by cDNA hybridization, were low or undetectable in all preculture specimens. The concentration of saturated phosphatidylcholine in lung tissue was 30% of the adult value with no apparent increase between 15 and 24 weeks gestation. SP 28-36 content increased during explant culture in the absence of serum and hormones, exceeding adult levels (3.2 +/- 1.0 micrograms per mg of DNA) after 5 days. In cultures treated with triiodothyronine (2 nM) and dexamethasone (10 nM), hormones that regulate phosphatidylcholine synthesis, the increase in SP 28-36 was accelerated (treated/control ratio was 7.1 and 3.4 at 3 and 5 days, respectively). Levels of mRNA for SP 28-36 also increased during culture and were stimulated by hormones (treated/control = 8.6 and 1.9 at 3 and 5 days, respectively). SP 28-36 and its mRNA increased similarly in the presence of dexamethasone alone, whereas triiodothyronine alone had no apparent effect. The molecular weight and charge pattern was similar for SP 28-36 of adult and cultured fetal tissue. These findings indicate that expression of the SP 28-36 gene is low during the second trimester, increases during explant culture, and is accelerated by glucocorticoid treatment.
Isolation of a cDNA for human acid alpha-glucosidase and detection of genetic heterogeneity for mRNA in three alpha-glucosidase-deficient patients.
Authors: Martiniuk F , Mehler M , Pellicer A , Tzall S , La Badie G , Hobart C , Ellenbogen A , Hirschhorn R
Published: May 2006

Lysosomal acid alpha-glucosidase (EC 3.2.1.3) hydrolyzes 1,4-linked alpha-D-glucose polymers present in glycogen. Genetic deficiency of acid alpha-glucosidase results in glycogen-storage disease type II, encompassing a spectrum of disorders of varying severity. To study the molecular basis for this heterogeneity, we sought to clone the coding sequence for human acid alpha-glucosidase. We screened 10(6) recombinant phage from a human liver cDNA expression library with an affinity-purified polyclonal antibody to human acid alpha-glucosidase. When we retested positive phage for reactivity to monoclonal antibodies, we identified a single phage, containing a 2-kilobase (kb) cDNA insert, that reacted with both polyclonal and monoclonal antibodies. The 2-kb cDNA hybridized to a 20-kb EcoRI fragment of human genomic DNA. This 20-kb EcoRI fragment was present only in DNA from somatic cell hybrids that retained the human chromosome 17 segment q21-q23, which contains the gene for human acid alpha-glucosidase. The cDNA also hybridized to a 3.4-kb mRNA, consistent with the size (approximately 105 kDa) of the acid alpha-glucosidase protein. Finally, in one of two infantile-onset acid alpha-glucosidase-deficient cell lines tested, the 3.4-kb mRNA was not detectable, whereas in an adult-onset cell line, an mRNA of reduced size and amount was found. Examination of DNA digested with restriction enzymes did not reveal any major deletions in the genomic DNA of these patients.
HLA antigens expressed on murine cells are preferentially recognized by murine cytotoxic T cells in the context of the H-2 major histocompatibility complex.
Authors: Holterman M J , Engelhard V H
Published: May 2006

The frequency of murine cytotoxic T lymphocytes (CTL) capable of responding to HLA antigens expressed on human or murine cells was determined by limiting dilution analysis. HLA antigens expressed on human cells stimulated CTL with a precursor frequency of about 1 in 2 X 10(5) spleen cells in primed mice, over two orders of magnitude weaker than a primary allogeneic response. There was a 10-fold increase in the frequency of precursors responding to HLA antigens when they were expressed on murine cells. It was determined that the increased frequency of responders was due to CTL that could only recognize HLA antigens on the syngeneic murine line to which they had been stimulated and that these CTL could not lyse any other HLA expressing murine cells of different H-2 haplotypes. The lytic activity of these CTL was inhibited by H-2b-specific antibodies. These results indicate that such CTL recognize HLA antigens in the context of the H-2 major histocompatibility complex. The magnitude and specificity of CTL responses to xenoantigens are discussed in the context of a model for T-cell interactions with major histocompatibility antigens.
Block of calcium channels by enkephalin and somatostatin in neuroblastoma-glioma hybrid NG108-15 cells.
Authors: Tsunoo A , Yoshii M , Narahashi T
Published: May 2006

Leucine-enkephalin, methionine-enkephalin, and morphine caused a reversible block of Ca2+ channel currents in neuroblastoma-glioma hybrid cells (NG108-15). The long-lasting (type 2) component of the Ca2+ channel current was blocked by leucine-enkephalin, while the transient (type 1) component was not affected. The enkephalin-induced blocking action was antagonized by naloxone and appears to be mediated by delta-opiate receptors. Two different aspects of the blocking effect were detected, a resting block and a recovery from block during prolonged depolarizing pulses. Recovery from block was more complete, and its time course was more rapid, with depolarization to more positive potentials. The dose dependence of the type 2 channel block at rest indicated a one-to-one binding stoichiometry, with an apparent dissociation constant of 8.8 nM. Somatostatin exerted a similar selective blocking action on the type 2 Ca2+ channel. The time- and voltage-dependent block of type 2 Ca2+ channels may provide a mechanism underlying the enkephalinergic presynaptic inhibition of transmitter release and the somatostatin block of pituitary growth hormone release.
High-affinity binding of granulocyte-macrophage colony-stimulating factor to normal and leukemic human myeloid cells.
Authors: Gasson J C , Kaufman S E , Weisbart R H , Tomonaga M , Golde D W
Published: May 2006

Purified natural and biosynthetic (recombinant) human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate colony formation by myeloid progenitor cells and enhance the function of mature neutrophils. Both of these actions occur at concentrations between 1 and 100 pM, with half-maximal stimulation at 10-20 pM. We have examined specific binding of 125I-labeled GM-CSF to responsive target cells in this range of concentrations. The results show a low number (50-250) of high-affinity (15-30 pM) binding sites on GM-CSF-responsive leukemic cells (KG-1, HL-60), as well as on peripheral blood neutrophils from normal donors. This high-affinity binding component was absent from unresponsive cell lines (KG-1a, K562). These results suggest that this binding site mediates the biological activities of GM-CSF on both proliferation and function of myeloid cells.
Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, Hirudo medicinalis.
Authors: Harvey R P , Degryse E , Stefani L , Schamber F , Cazenave J P , Courtney M , Tolstoshev P , Lecocq J P
Published: May 2006

Cloned cDNAs have been isolated that encode a variant of hirudin, a potent thrombin inhibitor that is secreted by the salivary glands of the medicinal leech, Hirudo medicinalis. This variant probably corresponds to a form that has been purified from leech heads but differs in amino acid sequence from the hirudin purified from whole leeches. There are at least three hirudin transcripts detectable in leech RNAs that are different in size, site of synthesis, inducibility by starvation, and relationship to hirudin activity. The new hirudin variant predicted by the cDNA and the heterodisperse transcription products suggest a hirudin protein family. The hirudin cDNA was expressed in Escherichia coli under the control of the bacteriophage lambda PL promoter. The recombinant product is biologically active, inhibiting the cleavage by thrombin of fibrinogen and a synthetic tripeptide substrate.
A sialic acid-specific O-acetylesterase in human erythrocytes: possible identity with esterase D, the genetic marker of retinoblastomas and Wilson disease.
Authors: Varki A , Muchmore E , Diaz S
Published: May 2006

The "nonspecific" esterases are a family of enzymes that were originally identified because of their reaction with synthetic O-acetyl ester substrates. While the electrophoretic polymorphisms of these enzymes have been extremely useful for genetic studies, their biological functions have remained completely unknown. Esterase D is characterized by its reactivity with 4-methylumbelliferyl acetate. This enzyme has recently been of particular interest because of its tight linkage to the putative recessive gene causing retinoblastomas, and to the recessive gene causing Wilson disease. We describe here the partial purification of a human erythrocyte esterase that appears to be highly specific for O-acetylated sialic acids. We next present evidence that suggests that esterase D is identical to this sialic acid-specific O-acetylesterase. First, both activities copurify from human erythrocyte lysates through several different purification steps, each of which use different principles of separation. Second, both activities show a remarkably similar profile of inhibition with a variety of different agents. Third, they both show a nearly identical heat-inactivation profile. This cytosolic sialic acid-specific O-acetylesterase appears to be involved in the "recycling" of O-acetylated sialic acid molecules. Thus, esterase D may be the first nonspecific esterase for which a specific biological role can be predicted.
Identification of a thrombin sequence with growth factor activity on macrophages.
Authors: Bar-Shavit R , Kahn A J , Mann K G , Wilner G D
Published: May 2006

In contrast to fibroblasts, the exposure of G0/G1-arrested J774 cells, a murine macrophage-like tumor cell line, with either active or esterolytically inactive diisopropyl phosphorofluoridate-conjugated alpha-thrombin (the enzymatically active form of thrombin, EC 3.4.21.5) results in a mitogenic response as measured by increased [3H]thymidine incorporation. This response to thrombin is optimal at 10 nM and is specifically blocked by hirudin, a high-affinity thrombin inhibitor. When prethrombin 1 [a single-chain prothrombin derivative lacking fragment 1, resulting from the action of thrombin on prothrombin] is cleaved with cyanogen bromide, a fragment (peptide CB67-129) is produced that, like the parent thrombin molecule, is mitogenic for J774 cells but not for fibroblasts. Limited tryptic digests of this fragment retain the ability to stimulate macrophages--a function that can be mimicked by a synthetic tetradecapeptide homologue of CB67-129 (representing residues 367-380 of the human thrombin B chain sequence) but not by any of a series of well-known growth promoters, including platelet-derived growth factor, epidermal growth factor, nerve growth factor, and fibroblast epidermal growth factor, nerve growth factor, and fibroblast growth factor. The mitogenic effects of this peptide are not limited to J774 cells but can be expressed in other macrophage-like tumor cell lines, including P388D1, RAW, and PU5. In addition to increased [3H]thymidine incorporation, the synthetic B chain peptide stimulates cell proliferation as evidenced by a dose-dependent increase in total protein per culture well and cell number. We conclude that the thrombin molecule contains a macrophage growth factor domain that is separate and distinct from its active center. Thus, thrombin, in addition to its major role in hemostasis and thrombosis, may also have important functions in such basic processes as the inflammatory response and monocytopoiesis.
Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae.
Authors: Fujiyama A , Tamanoi F
Published: May 2006

We demonstrate the pathway for the biosynthesis of RAS1 and RAS2 gene products of Saccharomyces cerevisiae leading to their localization in membranes. The primary translation products of these genes are detected in a soluble fraction. Shortly after synthesis, these precursor molecules are converted to forms that migrate slightly faster than the precursor forms on a NaDodSO4/polyacrylamide gel. These processed proteins are further modified by fatty acid acylation, which is detected by [3H]palmitic acid labeling. The acylated derivatives are found exclusively in cell membranes, indicating the translocation of the RAS proteins from cytosol to membranes during maturation process. The attached fatty acids can be released by mild alkaline hydrolysis, suggesting that the linkage between the fatty acid and the protein is an ester bond. The site of the modification by fatty acid is presumably localized to the COOH-terminal portion of the RAS proteins. Fractionation of the membranes by sucrose gradient demonstrates that a majority of the fatty-acylated RAS proteins are localized in plasma membrane.
Hybrid hybridoma producing a bispecific monoclonal antibody that can focus effector T-cell activity.
Authors: Staerz U D , Bevan M J
Published: May 2006

Previous studies have shown that heteroconjugates of monoclonal antibodies in which one of the component antibodies is directed at the T-cell receptor and the other is directed against any chosen site can focus effector T cells to function at the targeted site. We report here the production of a hybrid hybridoma cell line, H1.10.1.6, which secretes large amounts of a bispecific hybrid antibody of the IgG2a class, that can focus T-cell activity. The parental hybridoma lines for the secondary fusion were F23.1, which secretes an antibody specific for an allotypic determinant on the T-cell receptor of most mouse strains, and 19E12, secreting an anti-Thy-1.1 antibody. The bispecific hybrid antibody was partially purified by hydroxylapatite chromatography and characterized by isoelectric focusing. It efficiently targets Thy-1.1-expressing tumor cells for lysis by F23.1 receptor-positive cytotoxic T-cell clones in vitro. Such hybrid antibodies produced by hybrid hybridoma cell lines may have application in the therapeutic targeting of tumors or sites of viral infections for attack by T cells.
Biogenesis of the platelet receptor for fibrinogen: evidence for separate precursors for glycoproteins IIb and IIIa.
Authors: Bray P F , Rosa J P , Lingappa V R , Kan Y W , McEver R P , Shuman M A
Published: May 2006

Congenital absence of platelet glycoproteins IIb and IIIa (GPIIb and GPIIIa) results in a severe bleeding disorder characterized by defective platelet aggregation and failure of fibrinogen to bind to platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIb and GPIIIa are present as a heterodimeric, noncovalent complex in the platelet plasma membrane and function as the fibrinogen receptor. To characterize synthesis of these two proteins, RNA isolated from a human leukemia cell line that contains GPIIb and GPIIIa was translated in a wheat germ cell-free system. Polyclonal antibodies specific for each protein immunoprecipitated distinct [35S]methionine-labeled precursors, indicating that GPIIb and GPIIIa are translated from separate mRNAs. Moreover, using specific antibodies against either intact unreduced GPIIb or the beta subunit, we obtained evidence for synthesis of a common polypeptide precursor for GPIIb alpha and GPIIb beta. Based on experiments using microsomal membranes, it appears that GPIIb is integrated into the platelet membrane with little or no cytoplasmic component. These results suggest that precursors of GPIIb and GPIIIa may be encoded by separate genes and that each precursor is processed before delivery to the plasma membrane.
Trans effect of the E1 region of adenoviruses on the expression of a prokaryotic gene in mammalian cells: resistance to 5' -CCGG- 3' methylation.
Authors: Langner K D , Weyer U , Doerfler W
Published: May 2006

The plasmid construct pSVO-CAT has been used to test adenovirus promoter activities in the unmethylated or methylated state. We have now observed that the E2A late promoter of adenovirus type 2 (Ad2) DNA also activated the chloramphenicol acetyltransferase (CAT) gene upon transfection of the pAd2E2A-CAT construct into mammalian cells, and it was inactivated by specific methylations of three 5' -CCGG- 3' sites. Similar results had been reported previously after microinjecting promoter-methylated constructs into oocytes of Xenopus laevis. Surprisingly, it was found that the pSVO-CAT construct, which lacked eukaryotic promoter sequences, was able to express the CAT gene upon transfection into human or hamster cells that harbored and constitutively expressed the E1 region of Ad2 or Ad5 DNA. In these cells, the expression of the pAd2E2A-CAT construct was enhanced, but it was only partly sensitive to DNA methylation, possibly because DNA methylation was counteracted directly or indirectly by E1 functions. The pSVO-CAT construct was also expressed in HeLa or BHK21 cells upon cotransfection with a plasmid carrying the HindIII-G fragment of Ad2 DNA that contained the E1A region and part of the E1B region. By mapping pSVO-CAT-specific RNAs, we could demonstrate that pSVO-CAT activity in Ad2- or Ad5-transformed cells was mediated by prokaryotic promoter-like sequences in the pBR322 section of the construct, and it presumably functioned via trans-activation mediated by the E1 region. This trans-activation of pSVO-CAT in adenovirus-transformed cells was partly insensitive to DNA methylation.
Expression of a cDNA derived from the yeast killer preprotoxin gene: implications for processing and immunity.
Authors: Hanes S D , Burn V E , Sturley S L , Tipper D J , Bostian K A
Published: May 2006

The type I killer strains of Saccharomyces cerevisiae secrete a dimeric 19-kDa protein that kills sensitive cells by disrupting cytoplasmic membrane function. This toxin is encoded by the double-stranded RNA plasmid M1-dsRNA, which also determines specific immunity to toxin. A preprotoxin, the 35-kDA in vitro translation product of denatured M1-dsRNA, is presumed to be the primary in vivo gene product. To facilitate studies on preprotoxin structure and maturation, we have inserted a partial cDNA copy of M1-dsRNA into the yeast vector p1A1, bringing it under control of the phosphate-repressible PHO5 promoter. This in-frame gene fusion encodes all of the preprotoxin sequence except for its N-terminal secretion leader, which is replaced by the leader sequence of PHO5. Transformation of sensitive yeast strains lacking M1-dsRNA with such fusion plasmids converts them to phosphate-repressible, immune killers, demonstrating that both toxin and immunity determinants are contained within the preprotoxin molecule. L-1-Tosylamido-2-phenylethyl chloromethyl ketone retards glycosylation of preprotoxin to toxin, facilitating size comparisons and indicating that processing of the normal precursor involves three glycosylation events but does not involve cotranslational leader peptidase action. In contrast, the PHO5 leader is apparently removed from the fusion preprotoxin.
Identification and preferential expression in thymic and bursal lymphocytes of a c-ets oncogene-encoded Mr 54,000 cytoplasmic protein.
Authors: Ghysdael J , Gegonne A , Pognonec P , Dernis D , Leprince D , Stehelin D
Published: May 2006

The avian retrovirus E26 is unique among acute leukemia viruses in its ability to induce transformation of cells belonging to either the myeloid or erythroid lineage. The genome of E26 carries two oncogenes, v-myb and v-ets, that are derived from distinct cellular loci, c-myb and c-ets. We have constructed a plasmid vector that allows expression of part of the coding region of v-ets in a bacterial host. Antisera to the bacterially synthesized ets protein specifically precipitated the E26-encoded P135gag-myb-ets transforming protein. These antisera permitted us to identify a chicken c-ets-encoded protein of Mr 54,000 (P54c-ets) that shares 7 out of 10 of its major [35S]methionine-containing tryptic peptides with the v-ets-encoded domain of P135gag-myb-ets. Unlike P135gag-myb-ets and the Mr 75,000 translation product of c-myb (P75c-myb), which are nuclear proteins, P54c-ets was found to be predominantly cytoplasmic. P54c-ets is expressed at low levels in most cell lines and tissues tested, including bone marrow cells and circulating lymphocytes. P54c-ets, together with a minor but closely related Mr 56,000 protein, was found to be expressed at high levels in chicken thymocytes and bursal lymphocytes.
Translation of open reading frame E5 of bovine papillomavirus is required for its transforming activity.
Authors: DiMaio D , Guralski D , Schiller J T
Published: May 2006

A series of mutations in open reading frame (ORF) E5 of bovine papillomavirus type 1 has been constructed to determine whether this putative gene is required for in vitro oncogenic transformation by viral DNA. Frameshift mutations at either of two different positions located exclusively in ORF E5 cause a substantial reduction in the ability of the cloned viral DNA to induce the appearance of transformed foci of mouse C127 cells. A genetic mapping experiment with one of the mutants indicates that this characteristic transformation defect is actually due to the constructed mutation in ORF E5. Analysis of 10 different mutants with sequence changes at a single position in the ORF showed that there is an exact correspondence between transformation-competence and the ability of the 3' half of ORF E5 to be correctly translated. The transformation defect of an ORF E5 frameshift mutant can be suppressed by a second mutation that restores the correct reading frame to most of the ORF, but not by one that restores the reading frame near the 3' end of the ORF. These results constitute strong genetic evidence that translation of ORF E5 is required for efficient transformation of mouse C127 cells by bovine papillomavirus DNA. Wild-type ORF E5 has the potential to encode a short hydrophobic protein or polypeptide domain.
Early transmembrane events in alloimmune cytotoxic T-lymphocyte activation as revealed by stopped-flow fluorometry.
Authors: Utsunomiya N , Tsuboi M , Nakanishi M
Published: May 2006

We have studied early transmembrane events in mouse alloimmune cytotoxic T-lymphocyte (LC7, H-2b) activation by specific target cells (mouse mastocytoma P815, H-2d) and a mitogenic lectin, Con A, by using stopped-flow fluorometry with three different fluorescent probes. After binding to target cells (P815), cytotoxic T lymphocytes (LC7) first increased their membrane fluidity and, then, calcium was released from intracellular stores. After that, there was a calcium influx from the external medium into the T lymphocytes. This calcium influx was blocked by calcium antagonists (verapamil or diltiazem). The same sequence of events was also observed in the activation of T lymphocytes (LC7) by Con A and in the response of specific target cells (P815) after cytotoxic T lymphocytes (LC7) binding. Nonspecific (syngeneic) target cells (mouse lymphoma EL-4, H-2b) did not cause any early transmembrane events in cytotoxic T lymphocytes (LC7, H-2b).
Bile acids regulate hepatic low density lipoprotein receptor activity in the hamster by altering cholesterol flux across the liver.
Authors: Spady D K , Stange E F , Bilhartz L E , Dietschy J M
Published: May 2006

The effect of different bile acids on receptor-dependent and receptor-independent low density lipoprotein (LDL) uptake in the liver and intestine was investigated. When fed at the 0.1% level for three weeks, cholic acid and chenodeoxycholic acid suppressed hepatic cholesterol synthesis in the rat by 80% and 50%, respectively, while ursodeoxycholic acid had no effect. In contrast, hepatic cholesteryl ester levels, rates of hepatic LDL transport, and concentrations of plasma LDL-cholesterol were not affected by bile acid feeding in this species. Cholic acid and chenodeoxycholic acid also suppressed hepatic cholesterol synthesis in the hamster. However, since basal rates of hepatic cholesterol synthesis in this species, as in man, are very low, the absolute reduction in hepatic synthesis could not compensate for the change in hepatic sterol balance induced by bile acid feeding. Hence, in the hamster the feeding of cholic acid and chenodeoxycholic acid increased hepatic cholesteryl ester levels 660% and 39%, respectively, reduced hepatic receptor-dependent LDL uptake by 50% and 32%, respectively, and elevated plasma LDL-cholesterol levels by 160% and 50%, respectively. Ursodeoxycholic acid feeding did not alter any of these processes, and none of the bile acids changed the rate of hepatic receptor-independent LDL transport. In the intestine, none of the bile acids altered rates of cholesterol synthesis or LDL uptake. When cholic acids, chenodeoxycholic acid, or ursodeoxycholic acid was infused continuously for 8 hr in supranormal amounts into control hamsters or rats or into animals pretreated with cholestyramine, there were no changes in LDL transport or any other parameter of hepatic cholesterol metabolism. Thus, these studies indicated that cholic acid and chenodeoxycholic acid have no acute, direct effect on rates of receptor-dependent LDL transport or cholesterol synthesis but do alter these processes indirectly by inducing changes in cholesterol balance across the liver. Ursodeoxycholic acid, in contrast, does not affect these processes either directly or indirectly and so causes no change in plasma LDL levels.
Distribution of corticotropin-releasing factor receptors in primate brain.
Authors: Millan M A , Jacobowitz D M , Hauger R L , Catt K J , Aguilera G
Published: May 2006

The distribution and properties of receptors for corticotropin-releasing factor (CRF) were analyzed in the brain of cynomolgus monkeys. Binding of [125I]tyrosine-labeled ovine CRF to frontal cortex and amygdala membrane-rich fractions was saturable, specific, and time- and temperature-dependent, reaching equilibrium in 30 min at 23 degrees C. Scatchard analysis of the binding data indicated one class of high-affinity sites with a Kd of 1 nM and a concentration of 125 fmol/mg (approximately equal to 30% of the receptor number in monkey anterior pituitary membranes). As in the rat pituitary and brain, CRF receptors in monkey cerebral cortex and amygdala were coupled to adenylate cyclase. Autoradiographic analysis of specific CRF binding in brain sections revealed that the receptors were widely distributed in the cerebral cortex and limbic system. Receptor density was highest in the pars tuberalis of the pituitary and throughout the cerebral cortex, specifically in the prefrontal, frontal, orbital, cingulate, insular, and temporal areas, and in the cerebellar cortex. A very high binding density was also present in the hippocampus, mainly in the dentate gyrus, and in the arcuate nucleus and nucleus tuberis lateralis. A high binding density was present in the amygdaloid complex and mamillary bodies, olfactory tubercle, and medial portion of the dorsomedial nucleus of the thalamus. A moderate binding density was found in the nucleus accumbens, claustrum, caudate-putamen, paraventricular and posterior lateral nuclei of the thalamus, inferior colliculus, and dorsal parabrachial nucleus. A low binding density was present in the superior colliculus, locus coeruleus, substantia gelatinosa, preoptic area, septal area, and bed nucleus of the stria terminalis. These data demonstrate that receptors for CRF are present within the primate brain at areas related to the central control of visceral function and behavior, suggesting that brain CRF may serve as a neurotransmitter in the coordination of endocrine and neural mechanisms involved in the response to stress.
Isolation and characterization of a mouse interleukin cDNA clone that expresses B-cell stimulatory factor 1 activities and T-cell- and mast-cell-stimulating activities.
Authors: Lee F , Yokota T , Otsuka T , Meyerson P , Villaret D , Coffman R , Mosmann T , Rennick D , Roehm N , Smith C
Published: May 2006

A cDNA sequence coding for a unique mouse interleukin that expresses B-cell-, T-cell, and mast-cell-stimulating activities has been isolated from a mouse helper T-cell cDNA library. The library, constructed in the pcD expression vector, was screened by transfecting COS monkey cells with DNA pools to express the products encoded by full-length cDNA inserts. By assaying the transfected cell supernatants, we identified clones encoding a factor that stimulates T-cell and mast cell lines. This factor also induces Ia expression on resting B cells and enhances IgG1 and IgE production by B cells, two properties of B-cell-stimulatory factor 1. The DNA sequence codes for a polypeptide of 140 amino acid residues including a putative signal peptide. These results demonstrate that a single cDNA clone distinct from interleukin 2 and interleukin 3 encodes a polypeptide with multiple biological activities.
Primary structure of the (1→3,1→4)-β-D-glucan 4-glucohydrolase from barley aleurone
Authors: Fincher Geoffrey B. , Lock Peter A. , Morgan Margaret M. , Lingelbach Klaus , Wettenhall Richard E. H. , Mercer Julian F. B. , Brandt Anders , Thomsen Karl Kristian
Published: May 2006

During germination of barley grains, the cell walls of the starchy endosperm are degraded by (1→3,1→4)-β-glucanases (EC 3.2.1.73) secreted from the aleurone and scutellar tissues. The complete sequence of the aleurone (1→3,1→4)-β-glucanase isoenzyme II comprises 306 amino acids and was determined by sequencing nine tryptic peptides (110 residues) and aligning them with the amino acid sequence deduced from a cDNA clone encoding the 291 NH
2
-terminal residues. Although no amino acid sequence homology with a bacterial (1→3)(1→4)-β-glucanase is apparent, close to 50% homology is found with two large regions of a (1→3)-β-glucanase from tobacco pith tissue. The gene for barley (1→3,1→4)-β-glucanase isoenzyme II shares with that for the α-amylase isoenzyme 1 a strongly preferred use of codons with G and C in the wobble position (94% and 90%, respectively). Both enzymes are secreted from the aleurone cells during germination. Such one-sided codon usage is not characteristic for the gene encoding the (1→3)-β-glucanase of tobacco pith tissue or the
hor2-4
gene encoding the B
1
hordein storage protein in the endosperm.
Nerve growth factor modulates the drug sensitivity of neurotransmitter release from PC-12 cells.
Authors: Kongsamut S , Miller R J
Published: May 2006

The release of catecholamines from adrenal chromaffin cells is known to be blocked by dihydropyridines, such as nitrendipine, and enhanced by others, such as BAY K8644. On the other hand, release from sympathetic neurons is predominantly insensitive to these agents. Release of [3H]norepinephrine from undifferentiated PC-12 pheochromocytoma cells resembles that from chromaffin cells in that it is extremely sensitive to dihydropyridines. Following differentiation, however, release of catecholamine becomes predominantly insensitive to both nitrendipine and BAY K8644. Under both growth conditions, release remains completely blocked by 3 mM Co2+ or by removal of Ca2+ from the release media. Dose-response curves to K+ show that following differentiation, cells become more sensitive, releasing transmitter at lower K+ concentrations. In contrast, depolarization-induced uptake of 45Ca2+ remains sensitive to dihydropyridines and shows similar sensitivity to K+ stimulation in both growth conditions. These results can be explained by invoking a model involving dihydropyridine-sensitive and -insensitive types of voltage-sensitive calcium channels.
Selective expression of endogenous lactose-binding lectins and lactoseries glycoconjugates in subsets of rat sensory neurons.
Authors: Regan L J , Dodd J , Barondes S H , Jessell T M
Published: May 2006

Cell-surface glycoconjugates and endogenous lectins have been implicated in cellular interactions that contribute to embryonic development. Functional subsets of primary sensory neurons in mammalian dorsal root ganglia (DRG) have been shown recently to express specific cell-surface oligosaccharide structures. We report here that endogenous lectins with affinity for sensory neuron glycoconjugates are also synthesized by subsets of DRG neurons and are present in the dorsal horn of the developing spinal cord. The distribution of two endogenous lactose-binding lectins, RL-14.5 and RL-29 (subunit Mrs of 14,500 and 29,000, respectively), was examined by immunoblotting and by immunocytochemistry in embryonic and postnatal rat DRG and spinal cord. The two lectins appear soon after the formation of the DRG and are present in the cell bodies and terminals of subsets of DRG neurons that also express cytoplasmic and cell-surface lactoseries glycoconjugates. RL-14.5 and RL-29 are present in overlapping, but not coincident, subsets of DRG neurons that project to the superficial dorsal horn of the spinal cord. In addition, RL-14.5, but not RL-29, is expressed in spinal motoneurons from embryonic day 14. The preferential localization of lactoseries glycoconjugates and lactose-binding lectins in the DRG and the dorsal horn of the spinal cord suggests that these complementary molecules contribute to the development and function of primary sensory neurons.
Sporulation of the yeast Saccharomyces cerevisiae is accompanied by synthesis of adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate.
Authors: Jakubowski H
Published: May 2006

Two-dimensional TLC analysis of 32P-labeled nucleotides extracted from the yeast Saccharomyces cerevisiae reveals that two highly phosphorylated nucleotides are synthesized during sporulation. These nucleotides have been identified as adenosine 5'-tetraphosphate (ppppA) and adenosine 5'-pentaphosphate (pppppA). The synthesis of ppppA and pppppA commences late in sporulation and follows formation of ascospores. The maximum concentration of ppppA and pppppA in sporulating yeast cultures was 2% and 1.5%, respectively, that of ATP. Adenosine 5'-tetraphosphate and 5'-pentaphosphate are unique to this stage of yeast development and are absent in vegetative yeast cells. Since these nucleotides are also absent in asporogenous a/a and alpha/alpha cells, it is reasonable to propose that they are signal nucleotides marking one of the stages of yeast development--i.e., ascospore formation.
Fc receptor triggering induces expression of surface activation antigens and release of platelet-activating factor in large granular lymphocytes.
Authors: Malavasi F , Tetta C , Funaro A , Bellone G , Ferrero E , Franzone A C , Dellabona P , Rusci R , Matera L , Camussi G
Published: May 2006

Triggering of large granular lymphocyte (LGL) Fc receptor with a specific monoclonal antibody (AB8.28) linked to an insoluble matrix induces cell activation, as witnessed by expression of HLA class II (DR and DQ) molecules and interleukin 2 receptor. Moreover, this event is accompanied by a concomitant release of platelet-activating factor by LGL. We conclude that the Fc receptor molecule identified by mAb AB8.28 represents a trigger for LGL activation.