bluuebunny/crossref_metadata_2025 huggingface dataset:
Scrapie and Creutzfeldt-Jakob disease prion proteins share physical properties and antigenic determinants.
Authors: Bendheim P E , Bockman J M , McKinley M P , Kingsbury D T , Prusiner S B
Published: May 2006

Scrapie of sheep and goats as well as Creutzfeldt-Jakob disease (CJD) of humans are neurologic disorders caused by slow infectious pathogens. The novel molecular properties of the pathogen causing scrapie have prompted introduction of the term "prion" to denote a small proteinaceous infectious particle that resists inactivation by nucleic acid-modifying procedures. Antiserum to the major hamster scrapie prion protein (PrP 27-30) was found to cross-react with murine CJD proteins. The CJD proteins had molecular weights similar to those observed for scrapie prion proteins as determined by NaDodSO4 gel electrophoresis. In addition, the CJD proteins were resistant to digestion by proteinase K and appear to polymerize into rod-shaped particles. The purification procedure developed for scrapie prions was found to be useful in purifying the CJD agent. Purification of the two infectious pathogens by virtually identical procedures provided further evidence for similarities in their molecular structures. We conclude that the molecular and biologic properties of the CJD agent are sufficiently similar to those of the scrapie prion protein that CJD should be classified as a prion disease.
Calcium channel antagonists inhibit the acrosome reaction and bind to plasma membranes of sea urchin sperm.
Authors: Kazazoglou T , Schackmann R W , Fosset M , Shapiro B M
Published: May 2006

As a prerequisite to fertilization, sea urchin sperm undergo an acrosome reaction that is mediated in part by increased permeability to Ca2+, with an attendant rapid, massive intracellular Ca2+ accumulation. The acrosome reaction is inhibited by Ca2+ channel antagonists, including verapamil, D600, and dihydropyridines such as nitrendipine, nimodipine, and nisoldipine. To examine the interaction of Ca2+ antagonists with sperm, a plasma membrane preparation enriched for Na+,K+-ATPase was isolated from sea urchin sperm. These plasma membranes specifically bound [3H]nitrendipine and [3H]verapamil at concentrations similar to those that inhibit the acrosome reaction. The binding of verapamil was sigmoidal and half-maximal at 1 microM. There was a high specificity in the binding interaction, since by competition binding verapamil, (-)-D600, and (+)-D600 had different relative Kd values, 11, 2.5, and 0.5 microM, respectively. These data suggest that sperm mediate the Ca2+ influx required for induction of the acrosome reaction via Ca2+ channels with properties similar, but not identical, to those of other excitable tissues.
Correction of hyperglycemia with phloridzin restores the glucagon response to glucose in insulin-deficient dogs: implications for human diabetes.
Authors: Starke A , Grundy S , McGarry J D , Unger R H
Published: May 2006

In insulin-deprived alloxan-induced diabetic dogs with severe hyperglycemia and marked hyperglucagonemia, glucagon was not suppressed by intravenous infusion of glucose at a progressively increasing rate up to 24 mg/kg of body weight per min. However, when the hyperglycemia was corrected by phloridzin, a blocker of renal tubular glucose reabsorption, the hyperglucagonemia was readily suppressed by as little as 2 mg of glucose per kg/min. Direct perfusion of phloridzin into the isolated pancreas of nondiabetic dogs had no effect on the in vitro glucagon response to increments in glucose. However, in pancreata isolated from dogs whose glucose levels had been lowered by phloridzin pretreatment, in vitro glucagon suppression in response to glucose increments was more than twice that of controls. This enhancing effect of phloridzin treatment was completely abolished by giving an intravenous infusion of glucose for the 5 hr prior to surgery for isolation of the pancreas. It is concluded that (i) alpha cells have a glucose-sensing system that is independent of insulin and beta cells, and (ii) this system is reversibly attenuated by hyperglycemia. Thus, hyperglycemia, a metabolic consequence of islet cell dysfunction, may be a self-exacerbating inducer of further islet cell dysfunction, a possibility with implications for human diabetes.
Radioimmunoassay for 6-D-tryptophan analog of luteinizing hormone-releasing hormone: measurement of serum levels after administration of long-acting microcapsule formulations.
Authors: Mason-Garcia M , Vigh S , Comaru-Schally A M , Redding T W , Somogyvari-Vigh A , Horvath J , Schally A V
Published: May 2006

A sensitive and specific radioimmunoassay for [6-D-tryptophan]luteinizing hormone-releasing hormone [( D-Trp6]LH-RH) was developed and used for following the rate of liberation of [D-Trp6]LH-RH from a long-acting delivery system based on a microcapsule formulation. Rabbit antibodies were generated against [D-Trp6]LH-RH conjugated to bovine serum albumin with glutaraldehyde. Crossreactivity with LH-RH was less than 1%; there was no significant crossreactivity with other peptides. The minimal detectable dose of [D-Trp6]LH-RH was 2 pg per tube. Intra- and interassay coefficients of variation were 8% and 10%, respectively. The radioimmunoassay was suitable for direct determination of [D-Trp6]LH-RH in serum, permitting the study of blood levels of the analog after single injections into normal men and after once-a-month administration of microcapsules to rats. In men, 90 min after subcutaneous injection of 250 micrograms of the peptide, serum [D-Trp6]LH-RH rose to 6-12 ng/ml. Luteinizing hormone was increased 90 min and 24 hr after the administration of the analog. Several batches of microcapsules were tested in rats and the rate of release of [D-Trp6]LH-RH was followed. The improved batch of microcapsules of [D-Trp6]LH-RH increased serum concentrations of the analog for 30 days or longer after intramuscular injection. This was accompanied by suppression of testosterone levels for more than 30 days. This radioimmunoassay should be of value for monitoring [D-Trp6]LH-RH during long-term therapy.
Genomic structure of the cottontail rabbit (Shope) papillomavirus.
Authors: Giri I , Danos O , Yaniv M
Published: May 2006

The nucleotide sequence of the cottontail rabbit papillomavirus (CRPV or Shope papillomavirus) genome has been determined. The overall organization of the genome is similar to that of the other three papillomaviruses already sequenced. The amino acid sequences of the putative viral proteins were compared to the available protein data banks. Of particular interest is the homology found between the COOH terminus of the E2 putative protein of the different papillomaviruses and the viral or cellular mos oncogene product. This analysis also reveals a specific feature of the rabbit virus that may be related to its natural oncogenic potential. One of its open reading frames (E6) shows significant homologies with the beta subunit of a family of ATP synthases from mitochondria, chloroplasts, and bacteria, including the conservation of amino acids residues involved in nucleotide binding. The viral noncoding region includes a highly A+T-rich segment and shows a complex array of repetitions and inverted repeats that may act as control elements for gene expression and genome replication.
Stabilization of a primary loop in myosin subfragment 1 with a fluorescent crosslinker.
Authors: Mornet D , Ue K , Morales M F
Published: May 2006

A bifunctional fluorescent alkylating agent, dibromobimane, has been used to stabilize a preexisting primary loop in myosin subfragment 1 (S-1). The crosslink achieved joins Cys-707 (called sulfhydryl group "SH1") of the 20-kDa domain (formerly called "20K" domain) with a thiol of the 50-kDa domain and seems to place the dibromobimane near the ATP-perturbable tryptophan.
A monoclonal immunotoxin acting on the Na+ channel, with properties similar to those of a scorpion toxin.
Authors: Barhanin J , Meiri H , Romey G , Pauron D , Lazdunski M
Published: May 2006

We describe the properties of a monoclonal antibody against the Na+ channel. The antibody, 72.38, competitively inhibited (Ki = 1.5 X 10(-9) M) the binding of an 125I-labeled toxin from the Brazilian scorpion Tityus serrulatus (125I-TiTX gamma) to Na+ channels of rat brain membranes. No significant inhibition of binding of a number of other Na+ channel toxins was observed. The inhibition of 125I-TiTX gamma binding also was observed with the solubilized Na+ channel from rat brain membranes (Ki = 2 X 10(-9) M). Antibody 72.38 antagonized 125I-TiTX gamma binding to Na+ channels from different animal species (fish, avian, and mammalian) and from different tissues (electroplax, brain, heart, and muscle). Moreover, 72.38 has been used for immunofluorescence labeling of Na+ channels in rat sciatic nodes of Ranvier and cultured dorsal root ganglion cells. Electrophysiological experiments on rat muscle cells fully confirmed the similarity between TiTX gamma and 72.38 seen in binding experiments. Both produce slow oscillations of the membrane potential accompanied by bursts of action potentials which are due to a selective action on the Na+ channel. TiTX gamma and 72.38 are without effect on the ion selectivity of the Na+ channel, but they both drastically change the voltage-dependence of activation and inactivation of the Na+ channel.
Escherichia coli transcription termination factor rho has a two-domain structure in its activated form.
Authors: Bear D G , Andrews C L , Singer J D , Morgan W D , Grant R A , von Hippel P H , Platt T
Published: May 2006

Limited tryptic digestion of Escherichia coli transcription termination factor rho [an RNA-dependent nucleoside triphosphatase (NTPase)] yields predominantly two fragments (f1 and f2) when the protein is bound to both poly(C) and ATP. The apparent molecular masses of the two fragments are 31 kDa for f1 and 15 kDa for f2, adding up to the molecular mass of the intact rho polypeptide chain (46 kDa). Sequence analysis of the amino termini demonstrates that f1 is derived from the amino-terminal portion of rho and that the trypsin cleavage that defines f2 occurs at lysine-283. These results suggest that, in the liganded (activated) form, the native rho protein monomer is organized into two distinct structural domains that are separable by a single proteolytic cleavage. The f1 fragment, purified from NaDodSO4/polyacrylamide gels and renatured, binds poly(C) but the f2 fragment does not; neither regains any ATPase activity. ATP- and polynucleotide-dependent changes in the rate of proteolysis and in the character of the fragments produced suggest that rho undergoes a series of conformational transitions as a consequence of RNA binding, NTP binding and NTP hydrolysis. The rate of loss of rho ATPase activity and of intact rho monomers is slower in the presence of adenosine 5'-[gamma-thio]triphosphate than in the presence of either ATP or ADP, indicating that the hydrolysis of ATP may result in different conformational effects than does the binding of this ligand. These findings are discussed within the context of recent models of rho-dependent transcription termination.
Fast and slow orientational fluctuations in membranes
Authors: Vogel Horst , Jähnig Fritz
Published: May 2006

Fluorescence anisotropy measurements on isotropically distributed membranes yield the well-known orientational order parameter as a measure of orientational fluctuations of the fluorophore that are fast compared to the fluorescence lifetime. Measurements on oriented membranes provide four order parameters, two for fast orientational fluctuations and two for slow, from which their approximate angular distributions can be derived. This is exemplified for the fluctuations of lipids in membranes with and without protein. Steady-state fluorescence anisotropy experiments on oriented membranes are carried out, using diphenylhexatriene as a fluorescence probe for lipid fluctuations and melittin as a membrane protein. Protein molecules in a fluid lipid membrane restrict the fast lipid fluctuations and induce slow lipid fluctuations. The angular distribution of the slow fluctuations indicates that lipid molecules at the protein surface are tilted with respect to the membrane normal. The spatial coherence length of these fluctuations is of the order of 15 Å.
Cells from an immunodeficient patient (46BR) with a defect in DNA ligation are hypomutable but hypersensitive to the induction of sister chromatid exchanges.
Authors: Henderson L M , Arlett C F , Harcourt S A , Lehmann A R , Broughton B C
Published: May 2006

A fibroblast cell strain, 46BR, derived from an immunodeficient patient is hypersensitive to the lethal effects of a wide range of DNA-damaging agents. It is also defective in strand-break rejoining after treatment with dimethyl sulfate and UV light. The present study shows that the cells have a defect in joining Okazaki-type fragments during DNA replication, supporting the interpretation that the basic defect is in ligation of DNA strands. The baseline level of sister chromatid exchange is slightly higher than in normal cells but it does not approach that of Bloom's syndrome or dyskeratosis congenita cells. Sensitivity to the induction of sister chromatid exchange and the hypersensitivity to the lethal effects of a set of DNA-damaging agents are correlated, implying that the basic defect influences both end points in a similar manner. No 6-thioguanine-resistant mutants could be induced by either gamma- or UV-irradiation in these cells, suggesting that error-prone repair pathways for damage induced by these agents may contain a common ligation step in human cells.
Construction of telocentric chromosomes in Saccharomyces cerevisiae.
Authors: Surosky R T , Tye B K
Published: May 2006

We describe a simple method for the construction of large chromosomal deletions in yeast. Diploid yeast cells were transformed with DNA fragments that replace large regions of the chromosomes by homologous recombination. Using this method, we have constructed a telocentric chromosome III in which approximately equal to 100 kilobases (kb) of DNA has been removed from the left arm of the chromosome, so that the centromere is 12 kb from the left telomere. This telocentric chromosome is mitotically stable. Its rate of loss in a diploid strain is 2.5-7.4 X 10(-4) per cell division compared to a rate of loss of 0.36-1.8 X 10(-4) per cell division for a normal chromosome III. It also segregates 2+:2- with fidelity during meiosis. The construction of systematic deletions in a chromosome should be useful in determining the essential features for proper chromosomal segregation and replication.
Production of antibody associated with non-A, non-B hepatitis in a chimpanzee lymphoblastoid cell line established by in vitro transformation with Epstein-Barr virus.
Authors: Shimizu Y K , Oomura M , Abe K , Uno M , Yamada E , Ono Y , Shikata T
Published: May 2006

A continuous cell line of chimpanzee lymphocytes producing an antibody specifically associated with non-A, non-B hepatitis (NANB) was established. Peripheral blood lymphocytes of a chimpanzee convalescent from experimental infection with NANB hepatitis were transformed in vitro by Epstein-Barr virus infection into lymphoblastoid cell lines. Supernatants of the cell cultures were screened by immunofluorescence for antibody activity against the liver tissue of a chimpanzee with NANB hepatitis. Nineteen of the 1402 cultures were found to be positive for the activity. Ten of these 19 gave cytoplasmic reactions and the remaining 9 gave nuclear reactions in hepatocytes. One culture (48-1) stably producing the antibody was further characterized. The antibody produced in 48-1 was IgM and gave granular cytoplasmic reactions in hepatocytes. Cloning of 48-1 was performed by the soft agar method and cloned cell lines stably producing the antibody were obtained. The 48-1 antibody reacted with liver biopsy specimens from 12 chimpanzees obtained during the acute or chronic phase of hepatitis caused by five different NANB strains, but not with biopsy specimens from chimpanzees with hepatitis A or B or from normal chimpanzees. In addition, examinations of serial liver biopsy specimens obtained from 2 chimpanzees experimentally infected with NANB hepatitis demonstrated that the antibody reacted with the biopsies obtained during the preacute, acute, and chronic hepatitis, but not with those obtained before inoculation, early incubation period, or during convalescence. The present results indicate the specific association of the antibody with NANB hepatitis. Immunoelectron microscopy revealed that the antibody reacted with the microtubular aggregates identical to those previously described in a patient and chimpanzees with NANB hepatitis.
Model of cortical organization embodying a basis for a theory of information processing and memory recall.
Authors: Shaw G L , Silverman D J , Pearson J C
Published: May 2006

Motivated by V. B. Mountcastle's organizational principle for neocortical function, and by M. E. Fisher's model of physical spin systems, we introduce a cooperative model of the cortical column incorporating an idealized substructure, the trion, which represents a localized group of neurons. Computer studies reveal that typical networks composed of a small number of trions (with symmetric interactions) exhibit striking behavior--e.g., hundreds to thousands of quasistable, periodic firing patterns, any of which can be selected out and enhanced with only small changes in interaction strengths by using a Hebb-type algorithm.
Specific expression of the human cellular fps/fes-encoded protein NCP92 in normal and leukemic myeloid cells.
Authors: Feldman R A , Gabrilove J L , Tam J P , Moore M A , Hanafusa H
Published: May 2006

We have found that both an antibody directed against a synthetic peptide representing an amino acid sequence of the conserved kinase domain of transforming protein P140 of Fujinami sarcoma virus and a regressing tumor antiserum recognized the products of the c-fps/fes genes of both avian and mammalian cells. The anti-peptide antibody also recognized a 94-kilodalton protein that was related to but distinct from the c-fps/fes product in structure and in tissue distribution. A 92-kilodalton protein, NCP92, was found to be the mammalian counterpart of the previously identified avian c-fps/fes protein NCP98 by its structural similarity to NCP98, its associated tyrosine kinase activity, and its similar tissue distribution. The highest levels of NCP92 were found in tissue macrophages and in bone marrow. In bone marrow NCP92 expression was restricted to cells of the monocyte/macrophage and granulocyte lineages. That the expression of NCP92 is limited to these cell types was confirmed by the analysis of murine and human hematopoietic tumors representing different cell lineages: NCP92 was positive in leukemic cells of granulocytic and monocytic origin but not in B-lymphocytic, T-lymphocytic, or erythroid tumor cells. The expression of NCP92 seems to be related to the capacity of myeloid cells to differentiate and to respond to certain colony-stimulating factors.
cDNA clones encoding IgE-binding factors from a rat-mouse T-cell hybridoma.
Authors: Martens C L , Huff T F , Jardieu P , Trounstine M L , Coffman R L , Ishizaka K , Moore K W
Published: May 2006

cDNA clones encoding rodent IgE-binding factors (IgE-BF) were isolated from cDNA libraries of a rat-mouse T hybridoma that secretes IgE-suppressive factor (IgE-SF) upon incubation with rat IgE. COS7 cells transfected with two of the cDNAs expressed IgE-BF, which selectively potentiate an in vitro IgE response. IgE-BF expressed in COS7 cells are glycoproteins of approximately equal to 60 and approximately equal to 11 kDa. DNA sequence analysis of an IgE-BF cDNA revealed a 556-amino acid (62 kDa) protein coding region. The results suggest that IgE-potentiating and IgE-suppressive factors share common precursor polypeptides and that the 11-kDa IgE-BF is derived from a 60-kDa precursor.
Pertussis toxin inhibition of chemotaxis and the ADP-ribosylation of a membrane protein in a human-mouse hybrid cell line.
Authors: Backlund P S , Meade B D , Manclark C R , Cantoni G L , Aksamit R R
Published: May 2006

When WBC264-9C cells are preincubated with pertussis toxin, chemotaxis is inhibited and ADP-ribosylation of a membrane protein with a subunit Mr 41,000 is observed. Both the inhibition of chemotaxis and the ADP-ribosylation by pertussis toxin display a similar time lag, temperature dependence, and pertussis toxin-concentration dependence. Although the inhibition of chemotaxis and the ADP-ribosylation of the membrane protein are qualitatively correlated, nearly complete inhibition of chemotaxis occurs when there is only partial ADP-ribosylation of the membrane protein. Pertussis toxin-catalyzed ADP-ribosylation of the Mr 41,000 protein in WBC264-9C membranes is stimulated by GDP, GTP, and to a lesser extent by GMP; the nonhydrolyzable GTP analog guanosine 5'-[beta, gamma-imido]triphosphate has no effect. WBC264-9C membranes have a high-affinity GTPase activity, which is partially inhibited in membranes from pertussis toxin-treated cells. Neither GTPase activity nor adenylate cyclase activity in membranes from WBC264-9C cells is affected by fMet-Leu-Phe, an attractant for these cells. Our results suggest that a guanine nucleotide binding protein may be involved in chemotaxis, but they do not indicate an involvement of adenylate cyclase.
Differential scanning calorimetry of nuclei reveals the loss of major structural features in chromatin by brief nuclease treatment.
Authors: Touchette N A , Cole R D
Published: May 2006

Differential scanning calorimetry revealed that chromatin melts in four distinct transitions in intact HeLa nuclei at 60 degrees C, 76 degrees C, 88 degrees C, and 105 degrees C. Calorimetry of whole cells was characterized by the same four transitions along with another at 65 degrees C, which was probably RNA. Isolated chromatin, however, melted in only two transitions at 72 degrees C and 85 degrees C. Very brief digestion of HeLa nuclei with either micrococcal nuclease or DNase I resulted in the conversion of a structure that melted at 105 degrees C to one that melted at 88 degrees C. Further digestion with micrococcal nuclease to the level of the mononucleosome did not result in any further substantial changes in either enthalpy or melting temperatures. In contrast, further DNase I digestion that resulted in cleavage within the nucleosome produced a pronounced shift in melting temperatures to broad transitions at 62 degrees C and 78 degrees C.
The 140-kDa adenovirus DNA polymerase is recognized by antibodies to Escherichia coli-synthesized determinants predicted from an open reading frame on the adenovirus genome.
Authors: Friefeld B R , Korn R , de Jong P J , Sninsky J J , Horwitz M S
Published: May 2006

Sequence studies of the adenovirus 2 genome have revealed the presence of a large open reading frame (ORF) from 22.9 to 14.2 map units that is believed to encode most of the adenovirus DNA polymerase (Ad Pol). An 838-base-pair fragment (19.6-17.3 map units) containing approximately 25% of this ORF has been cloned and expressed in a beta-galactosidase-chloramphenicol acetyltransferase (lacZ-CAT) expression vector under the control of the trp-lac hybrid promoter. This recombinant vector directed the synthesis of a 58-kDa lacZ-Ad Pol-CAT fusion protein that has CAT activity. This fusion protein was easily purified by affinity chromatography in which chloramphenicol, the substrate for CAT, was covalently bound to a matrix. Antisera were prepared against the purified 58-kDa lacZ-Ad Pol-CAT fusion protein and were found to react specifically with the 140-kDa Ad Pol by ELISA and immunoblot analysis. In addition, these antisera recognized 120- and 29-kDa polypeptides in immunoblot analysis of partially purified terminal protein precursor (pTP)-Ad Pol complex. The exact nature of the 120- and 29-kDa polypeptides is not known, but they may be breakdown products of Ad Pol. Although the lacZ-Ad Pol-CAT fusion protein is not active in any of the Ad Pol enzymatic reactions, antibody against the prokaryotic fusion protein should be useful for screening bacteria harboring plasmids that have been constructed to express the entire Ad Pol ORF.
Purification of the platelet-derived growth factor receptor by using an anti-phosphotyrosine antibody.
Authors: Daniel T O , Tremble P M , Frackelton A R , Williams L T
Published: May 2006

The platelet-derived growth factor (PDGF) receptor is a 180-kDa membrane glycoprotein. A protein of identical size, lectin affinity, and isoelectric point has been identified as a major substrate for PDGF-activated tyrosine kinase in stimulated 3T3 cells. We have purified this tyrosine-phosphorylated protein to homogeneity by using anti-phosphotyrosine immunoaffinity and lectin affinity steps. Demonstration that this purified tyrosine phosphoprotein is the PDGF receptor necessitated development of an assay capable of identifying specific 125I-labeled PDGF binding activity in soluble receptor preparations. PDGF receptor solubilized from 3T3 cell membranes with the detergent octyl beta-D-glucoside was precipitated on an artificial liposome matrix after receptor aggregation with concanavalin A. Precipitated binding sites display affinity and kinetic characteristics of PDGF receptors in cells and membranes. Preparations of the 180-kDa phosphoprotein that are greater than 90% homogeneous by silver stain and by [35S]methionine protein autoradiography have specific high affinity 125I-labeled PDGF binding sites (equilibrium dissociation constant, 0.1 X 10(-9) M). Binding activity enrichment in this preparation reflects an 11,000-fold purification of binding activity in intact cells. These data demonstrate that the 180-kDa substrate of the PDGF-stimulated tyrosine kinase is the PDGF receptor. Furthermore, these methods provide a means of purifying this and other tyrosine kinase substrates from growth factor-stimulated cells.
Cloning of Physarum actin sequences in an exonuclease-deficient bacterial host.
Authors: Nader W F , Edlind T D , Huettermann A , Sauer H W
Published: May 2006

A genomic library of Physarum was constructed in the replacement vector EMBL3. Efficient propagation of the recombinant phages occurred only on the recBC-sbcB- host Escherichia coli CES200, which is deficient in the exonucleases I and V. Thirteen different recombinants with actin-related sequences were detected and 10 were purified from 90,000 plaques (the equivalent of 6 Physarum genomes) on strain CES200. Comparison of the plating efficiencies of the library and the actin-related isolates suggests that palindromic DNA sequences are responsible for the instability of Physarum DNA in E. coli. In one of these isolates, lambda PpA10, and in a 2.81-kilobase subclone of that isolate in plasmid pBR322, a deletion of 360 base pairs was detected that led to stable propagation of the recombinant DNA molecules in Rec+ E. coli. Electron microscopic analysis of the 2.81-kilobase fragment, after denaturation and self-hybridization, revealed secondary structures consistent with "foldback" structures. Restriction and DNA blot analysis of lambda PpA10 suggest that the unstable DNA segment is in close proximity to, if not part of, the previously defined actin-gene locus ardA.
Generation of leukotrienes by human monocytes upon stimulation of their beta-glucan receptor during phagocytosis.
Authors: Czop J K , Austen K F
Published: May 2006

Human monocytes possess a receptor for ingestion of particulate activators of the human alternative complement pathway that functions in the absence of plasma proteins and is distinct from the receptors for Fc-IgG and the major cleavage fragment of the third component of complement (C3b). Incubation of monolayers of monocytes with 1.1 X 10(6) to 2.2 X 10(7) glucan particles per ml initiated a phagocytic response comparable to that obtained with zymosan particles, of which beta-glucan is a constituent along with mannan. Maximal quantities of 4.93 +/- 3.43 ng of leukotriene B4 (LTB4) and 0.43 +/- 0.23 ng of leukotriene C4 (LTC4) (mean +/- SD, n = 3) were released by 10(6) monocytes stimulated with 1.1 X 10(7) glucan particles per ml. Preincubation of monocytes with 50 micrograms of soluble beta-glucan per ml reduced subsequent monocyte ingestion of 5 X 10(6) zymosan particles per ml and 2.2 X 10(6) glucan particles per ml by 52% and 55%, respectively, and diminished release of LTB4 by monocytes stimulated with 2 X 10(8) zymosan particles per ml and 8.6 X 10(6) glucan particles per ml by 73% and 61%, respectively. Preincubation with 1 mg of soluble mannan per ml had little effect on monocyte phagocytosis or LTB4 generation in response to either zymosan or glucan particles, and neither soluble beta-glucan nor mannan stimulated generation of LTB4 or LTC4. The effect of pretreatment of monocytes with soluble beta-glucan was time dependent, with the maximal effect being evident within 20 min of pretreatment, and was specific for zymosan or glucan particles in that the LTB4 and LTC4 release induced by 2.5 microM calcium ionophore A23187 was unaffected. That both phagocytosis and leukotriene generation are inhibited by soluble beta-glucan but not by mannan at a rate compatible with the phagocytic process of monocyte monolayers indicates ligand specificity for a beta-glucan receptor. As the beta-glucan receptor recognizes particulate activators of the alternative complement pathway, the nonimmune response to a single stimulus induces complement activation, phagocytosis, and leukotriene generation.
Platelet-derived collagenase inhibitor: characterization and subcellular localization.
Authors: Cooper T W , Eisen A Z , Stricklin G P , Welgus H G
Published: May 2006

Purified human platelets were found to contain a collagenase inhibitor that is immunologically, functionally, and chromatographically identical to that produced by human skin fibroblasts. None of the other formed elements of the blood (erythrocytes, granulocytes, mononuclear cells) possessed detectable quantities of this protein. Virtually all the collagenase inhibitor contained within platelets was released following platelet activation with thrombin. Similarly, platelet activation accompanying blood clotting also resulted in the release of this protein, the ratio of plasma to serum inhibitor levels being approximately equal to 0.5. When platelets were subjected to subcellular fractionation, essentially all of the platelet-associated collagenase inhibitor was found to be located in the alpha-granule. Studies with radiolabeled inhibitor failed to detect uptake of inhibitor by platelets. Furthermore, immunologically reactive protein of similar quantity to that found in platelets was identified in human megakaryocyte lysates. Thus, the data suggest that the collagenase inhibitor is endogenously produced and stored within platelet alpha-granules. The platelet-derived collagenase inhibitor was antigenically identical to the collagenase inhibitor from human skin fibroblasts in double immunodiffusion and, like its fibroblast counterpart, inhibited collagenase on a 1:1 stoichiometric basis. When subjected to several of the chromatographic procedures utilized to purify the fibroblast protein, the platelet inhibitor behaved in an indistinguishable manner. Platelet factor 4, previously reported to be a collagenase inhibitor, was found to be immunologically unrelated to the platelet-derived collagenase inhibitor. Furthermore, platelet factor 4 displayed no collagenase inhibitory activity. Although the function of platelet-derived collagenase inhibitor is unknown, such a protein released by activated platelets may serve to regulate collagen turnover during the early stages of the inflammatory process.
Nucleotide sequence of a yeast Ty element: evidence for an unusual mechanism of gene expression.
Authors: Clare J , Farabaugh P
Published: May 2006

We have determined the DNA sequence of the transposable element Ty912 of yeast. The 5918-base-pair element encodes two genes, tya912 and tyb912, which specify proteins similar to sequence-specific DNA-binding proteins of Escherichia coli and retroviral reverse transcriptases, respectively. The tyb912 gene is atypical of eukaryotic genes since (i) it begins 1336 nucleotides into the Ty912 mRNA (i.e., downstream of the tya912 gene) and (ii) the first in-frame AUG is 921 nucleotides into the coding frame. Protein blot analysis of Ty-lacZ fusions shows that the tyb912 gene is translated starting at the 5' end of the tya912 gene and that the primary translational product is a tya912::tyb912 fusion protein. We have shown that synthesis of this fusion protein probably does not occur by RNA splicing. The data are consistent with a mechanism of translational frameshifting occurring within the region of overlap between the 3' end of tya912 and the 5' end of tyb912.
Structure of a human histone cDNA: evidence that basally expressed histone genes have intervening sequences and encode polyadenylylated mRNAs.
Authors: Wells D , Kedes L
Published: May 2006

We have isolated and sequenced full-length cDNA clones encoding the human basally expressed H3.3 histone from a human fibroblast cDNA library. Several features of this atypical cDNA distinguish it and its gene from the well-characterized cell-cycle regulated histone genes and their RNA transcripts. The H3.3 mRNA is approximately equal to 1200 bases long, contains unusually long 5' and 3' untranslated regions, and has a 3' polyadenylylated terminus. In addition, we have isolated and characterized a cDNA clone that is a precursor to the H3.3 mRNA and contains an intervening sequence interrupting its 5' untranslated region. Hybridization of subsegments of the cDNA to human genomic DNA reveals a complex multigene family. The differences in the structures of basal and cell-cycle histone genes suggest a model to explain the differences in their expression.
A drug-resistant mutation in the ribosomal DNA of Tetrahymena.
Authors: Bruns P J , Katzen A L , Martin L , Blackburn E H
Published: May 2006

A mutation that confers resistance to the drug paromomycin is shown to be in the structural gene that codes for the ribosomal RNA in Tetrahymena. This observation was made by exploiting a variant of the ribosomal DNA that distorts amplification of this locus when a new somatic nucleus develops during conjugation. Because the allelic forms of this locus have a restriction endonuclease site polymorphism, it was possible to correlate drug resistance with presence of a specific allele. The genetic results have been confirmed by sequence analysis (presented elsewhere). Thus, the crosses presented here provide a unique opportunity to identify mutations in the ribosomal DNA.
Insertion of an unstable element in an intervening sequence of maize Adh1 affects transcription but not processing.
Authors: Rowland L J , Strommer J N
Published: May 2006

Three independently isolated and unstable mutants of the maize alcohol dehydrogenase 1 gene (Adh1) have arisen by insertion of the Mu transposing element into the first intervening sequence of the progenitor Adh1 allele. The mutants have been selected for their decreased levels of alcohol dehydrogenase 1 activity. The original mutants were unstable, giving rise to both revertant alleles and altered mutant alleles. From one of the original mutants, two derivative mutants have been recovered and described. We analyzed the effect of Mu insertion in all five of these mutants by measuring relative levels of run-off transcripts from the progenitor and mutant alleles and by comparing levels of run-off transcripts corresponding to regions lying 5' and 3' to the insertion sites. In this paper we present evidence that early transcriptional events are affected, but that, in spite of the inclusion of a 1.4-kilobase transposing DNA element, processing of transcripts occurs normally.
Rearrangement of T-cell receptor beta-chain genes during T-cell development.
Authors: Born W , Yagüe J , Palmer E , Kappler J , Marrack P
Published: May 2006

The kinetics and order of rearrangements in the gene complex encoding T-cell-receptor beta chains were studied by Southern blot hybridization in a collection of hybridomas derived from fetal thymocytes at various stages of ontogeny (day 14 to day 17). Our results show a steady increase in the frequency of rearranged beta complexes during this period and suggest that these rearrangements occur within the thymus. beta-chain diversity region (D beta) to beta-chain joining region (J beta) joining preceded other types of rearrangements. More complex hybridization patterns consistent with fully rearranged functional beta-chain genes did not begin to accumulate until day 16, 1 day prior to significant surface expression of the receptor protein.
A mechanism for memory storage insensitive to molecular turnover: a bistable autophosphorylating kinase.
Authors: Lisman J E
Published: May 2006

A mechanism is proposed for a molecular switch that can store information indefinitely, despite the complete turnover of the molecules that make up the switch. The design of the switch is based on known types of biochemical reactions. Central to the mechanism is a kinase that is activated by phosphorylation and capable of intermolecular autophosphorylation. It is shown that such a kinase and an associated phosphatase form a bistable chemical switch that can be turned on by an external stimulus and that is not reset by protein turnover.
Kin discrimination by worker honey bees in genetically mixed groups
Authors: Breed Michael D. , Butler Linda , Stiller Tammy M.
Published: May 2006

We tested the hypothesis that in a genetically mixed assemblage of worker honey bees, individual workers would behave differently toward unfamiliar sisters than toward unfamiliar nonsisters. Groups of worker honey bees of mixed genetic composition were assembled by collecting pupae from separate colonies and placing the worker bees together on eclosion. A total of 10 workers, 5 from each of two kin groups, were used to form each group. When the workers were 5 days old, a worker of one of the two kin groups was introduced into the mixed group. This worker had previously been held in a group of its sisters, without contact with queen or nonsister bees. The interactions with the introduced bee indicate that in a mixed kin group, individual workers learn the composite identity of the group and do not attack unfamiliar bees differentially on the basis of kinship. However, kinship does influence the total number of interactions in which an introduced bee engages when placed in a genetically mixed group; bees interacted significantly more often with sisters than with nonsisters. There was a trend for bees to be involved in more feeding interactions with sisters. This finding indicates an ability of a bee to learn and use its own cues. In mixed groups, each bee maintains its genotypically correlated identity; the bees' odors do not comingle into a “group” or “gestalt” odor. The significance of these results is discussed in light of the genetic structure of natural colonies of honey bees.
Origin and evolution of the transcribed repeated sequences of the Y chromosome lampbrush loops of
Drosophila hydei
Authors: Hareven Dana , Zuckerman Mathi , Lifschytz Eliezer
Published: May 2006

The molecular evolution and patterns of conservation of clones from four Y chromosome lampbrush loops of
Drosophila hydei
were investigated. Each loop contains a discrete family of transcribed repeats that are only slightly conserved even in the
hydei
subgroup species. Sequencing of clones from the four
D. hydei
loops indicates that all transcribed repeats evolved from A+T-rich elements of the genome. Evidence is presented that suggests a Y-specific family evolved as a result of the transposition of repeated sequences from an autosomal site to the Y chromosome with the concomitant acquisition of transcriptional activity and loss of non-Y sequences. The results support a structural role for the loops in shaping a spermatocyte-specific nuclear organization. Transcribed heterochomatic sequences could play a similar role in nuclear organization in many cell types.
Incorporation of calcium channels from cardiac sarcolemmal membrane vesicles into planar lipid bilayers.
Authors: Ehrlich B E , Schen C R , Garcia M L , Kaczorowski G J
Published: May 2006

When purified porcine cardiac sarcolemmal membrane vesicles are incorporated into planar lipid bilayers formed at the tip of patch electrode pipettes, individual divalent cation channels can be monitored. Channel activity is increased in the presence of the Ca2+ channel agonist Bay K 8644, is voltage dependent, and selects for divalent cations over anions. The activity does not inactivate because it is maintained during prolonged depolarizations. Determination of divalent cation selectivity from the reversal potential of single-channel currents indicates a relative permeability ratio for Ba/Ca/Mg of 1:0.45:0.08. Mean channel conductance in 0.1 M Ba2+/0.01 M Mg2+ is 8 pS. Channels are reversibly blocked by the Ca2+ channel inhibitor nitrendipine, and inhibition can be competitively antagonized by Bay K 8644. Binding studies with 3H-labeled D-600 demonstrate the presence of high-affinity receptors for D-600 in sarcolemmal membranes (Kd = 6.4 X 10(-9) M; Bmax = 3 pmol per mg of protein). In addition, experiments with resolved D-600 stereoisomers indicate that (-)D-600 is at least 25-fold more potent than (+)D-600 in competing for this aralkyl amine receptor. Consistent with this, (-)D-600 is much more effective than the (+) isomer in inhibiting bilayer-incorporated channels. These results demonstrate that the divalent cation channel that has been reconstituted in planar lipid bilayers possesses many of the characteristics of voltage-regulated Ca2+ channels in heart and suggest that receptors for Ca2+ entry blockers are functionally associated with this channel.
Small heat shock proteins of Drosophila associate with the cytoskeleton.
Authors: Leicht B G , Biessmann H , Palter K B , Bonner J J
Published: May 2006

Fractionation of heat-shocked Drosophila melanogaster Kc cells reveals that both the small heat shock proteins (hsp28, -26, -23, and -22) and vimentin-like intermediate filament proteins (IFPs) are abundantly represented in the nuclear fraction. Cofractionation of the IFPs with nuclei is due to the collapse of the IFP network against the nucleus upon heat shock, raising the possibility that cofractionation of the small hsps is by a similar mechanism. Indirect immunofluorescence supports this possibility. In salivary glands, both the hsps and the IFPs are cytoplasmic after mild-to-moderate heat shocks and only enter the nucleus upon severe--indeed, lethal--shocks. Double-label experiments with Schneider line 2 cells show that the IFPs and small hsps colocalize to the same perinuclear aggregates in 70% of the cells examined. Thus, the small hsps are associated with the cytoskeleton rather than with nuclear structures.
Genomic cloning, characterization, and multilineage growth-promoting activity of human granulocyte-macrophage colony-stimulating factor.
Authors: Kaushansky K , O'Hara P J , Berkner K , Segal G M , Hagen F S , Adamson J W
Published: May 2006

Through the use of long single-sequence oligonucleotide probes, the complete gene for human granulocyte-macrophage colony-stimulating factor (hGM-CSF) has been cloned from a human genomic library. The gene is 2.5 kilobases in length, contains three introns, and is present as a single copy in the human genome. When subcloned into the mammalian expression vector pD3, the gene directs the synthesis of authentic hGM-CSF. In addition to its stimulation of in vitro granulopoiesis and monopoiesis, recombinant hGM-CSF stimulates in vitro erythropoiesis and megakaryopoiesis.
Cloning, sequence, and expression of bovine interleukin 2.
Authors: Cerretti D P , McKereghan K , Larsen A , Cantrell M A , Anderson D , Gillis S , Cosman D , Baker P E
Published: May 2006

Interleukin 2 (IL-2) cDNA clones have been isolated from both human and murine sources. We report here the isolation of a cDNA clone encoding bovine IL-2. This was accomplished by screening a cDNA library constructed from lectin-stimulated bovine lymph node cells, using a human IL-2 probe. Bovine IL-2 is composed of 155 amino acids and has a predicted molecular weight of 19,555. Alignment of the amino acid sequence with human IL-2 indicates that mature bovine IL-2 is composed of 135 amino acids and has a predicted molecular weight of 15,452. It has an amino acid homology of 65% with human IL-2 and 50% with murine IL-2. Bovine IL-2 is unique among IL-2 homologs in that it has a single N-linked glycosylation site. Biologically active bovine IL-2 was synthesized in an Escherichia coli expression system.
Molecular cloning of a functional bovine interleukin 2 cDNA.
Authors: Reeves R , Spies A G , Nissen M S , Buck C D , Weinberg A D , Barr P J , Magnuson N S , Magnuson J A
Published: May 2006

A cDNA clone of the bovine interleukin 2 (IL-2) gene has been isolated and demonstrated to be functional in the production of secreted bovine IL-2 protein when transfected into monkey cells. The bovine IL-2 clone is 791 base pairs in length and contains an open reading frame of 474 base pairs coding for a bovine IL-2 precursor polypeptide of 158 amino acids with an estimated molecular weight of 17,884. The putative hydrophobic leader or signal sequence of the precursor protein is 23 amino acid residues long, suggesting that, after removal by processing, the mature secreted bovine IL-2 protein contains 135 amino acids and has a molecular weight of 15,464. Comparisons of both the nucleotide sequence and the predicted amino acid sequence of bovine IL-2 with those of the human and mouse IL-2 show extensive regions of sequence conservation between the species, interspersed with other regions of less similarity. The 3' untranslated region of the bovine IL-2 gene shares as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes as do the transcribed coding regions of these genes, suggesting an involvement of this region in regulation. In particular, a tandemly repeated sequence, (TATT)n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of the other known interleukin and interferon genes, as well as in similar regions of many other inducible genes of the lymphoid and immune response systems, suggesting a cell or tissue-specific regulatory function for these evolutionarily conserved sequences.
Retroviral mutants efficiently expressed in embryonal carcinoma cells.
Authors: Franz T , Hilberg F , Seliger B , Stocking C , Ostertag W
Published: May 2006

The myeloproliferative sarcoma virus (MPSV) is a unique member of the Moloney murine sarcoma virus family. Due to mutations in the U3 region of its long terminal repeat, MPSV has an expanded host range that includes cells of the hematopoietic compartment. Using a MPSV recombinant containing the gene for neomycin-resistance (NeoR-MPSV), we demonstrate that the host range of MPSV also includes undifferentiated F9 embryonal carcinoma cells. Transfer of G418-resistance with NeoR-MPSV to F9 cells is almost as efficient as G418-resistance transfer to fibroblasts, in contrast to G418-resistance transfer to PCC4 embryonal carcinoma cells, which is at least 3 orders of magnitude lower. To isolate NeoR-MPSV mutants that are efficiently expressed in PCC4 cells, G418-resistant PCC4 cell lines were induced to differentiate, and the provirus was rescued by superinfection with murine leukemia virus. Viral isolates (PCMV-5 and -6; PCMV = PCC4 cell-passaged NeoR-MPSV) were obtained and assayed for expression in embryonal carcinoma cells. The efficiency of NeoR transfer was equally as high in both F9 and PCC4 as in fibroblasts. mos oncogene expression was unaltered as judged by transformation capability. No gross alteration in the coding region and in the long terminal repeat was detectable by restriction enzyme analysis. NeoR-MPSV and its mutants PCMV-5 and -6 can thus be utilized as vectors for the efficient transduction of genes into embryonic cells.