bluuebunny/crossref_metadata_2025 huggingface dataset:
Ten-nanometer filaments and mitosis: maintenance of structural continuity in dividing endothelial cells.
Authors: Blose S H
Published: May 2006

By indirect immunofluorescence the behavior of the 10-nm filaments was studied at various stages of mitosis in guinea pig vascular endothelial cells. Interphase cells contain a ring of 10-nm filaments that encircles the nucleus and is maintained in a plane parallel to the substrate. During prophase and metaphase the cells round up and the 10-nm filament ring becomes wavy though still a closed structure. As anaphase progresses the ring then elongates into a rectangle that contains the spindle apparatus and chromosomes. In late telophase, cytokinesis cleaves the 10-nm filaments into crescents at the site of the contractile ring. These crescents then close into rings in the daughter cells. If cytokinesis is inhibited with 5 microgram of cytochalasin B per ml, then cleavage of the 10-nm filaments is blocked and the daughter nuclei remain surrounded by the parent ring. At no point during mitosis does the array of 10-nm filaments undergo major disassembly. These results indicate that, in contrast to the other major cytoplasmic structures, ventral microfilament bundles and cytoplasmic microtubules, which disassemble and reassemble during mitosis, 10-nm filaments remain intact throughout this process. The possibility is discussed that these filaments may function in transport of organelles and structural proteins, and provide the daughter cells with topological information about placement and assembly of these elements within the microtrabecular lattice.
Iron—dextran antibody conjugates: General method for simultaneous staining of two components in high-resolution immunoelectron microscopy
Authors: Dutton Anne H. , Tokuyasu K. T. , Singer S. J.
Published: May 2006

We report the preparation and properties of an electron-dense antibody conjugate, made by the covalent bonding of an iron—dextran (Imposil) particle to an antibody molecule. Transmission electron microscopic experiments with the Imposil—antibody conjugates demonstrate their suitability as specific immunostains at high resolution, particularly for simultaneous double staining experiments in conjunction with ferritin—antibody conjugates.
Human T gamma globin chain is a variant of A gamma chain (A gamma Sardinia).
Authors: Saglio G , Ricco G , Mazza U , Camaschella C , Pich P G , Gianni A M , Gianazza E , Righetti P G , Giglioni B , Comi P , Gusmeroli M , Ottolenghi S
Published: May 2006

Isoelectric focusing, cellulose acetate electrophoresis, and carboxymethylcellulose chromatography in the presence of Nonidet P-40 allow the separation of pure gamma chains into two fractions. Amino acid analysis of their cyanogen bromide fragment 3 (gamma CB3) identifies these fractions as the separated G gamma (Gly-136) and A gamma (Ala-136) globin chains. Fingerprint and amino acid analyses of the gamma Tp9 tryptic peptide from the purified A gamma and G gamma fractions from two different patients demonstrate that the commonly occurring gamma Sardinia variant (gamma 75 isoleucine leads to threonine), also known as T gamma chain, has alanine in position 136. From this analysis we suggest that the T gamma gene is an allele of the A gamma locus (A gamma Sardinia) rather than a third gamma locus.
Effects of pseudomonas toxin A, diphtheria toxin, and cholera toxin on electrical characteristics of turtle bladder.
Authors: Brodsky W A , Sadoff J C , Durham J H , Ehrenspeck G , Schachner M , Iglewski B H
Published: May 2006

Rapidly developing changes in the short-circuiting current (Isc), conductance (G), and potential (PD) of turtle bladders in Na-rich or Na-free media are seen after the mucosal addition, at 10 nM, of each of three toxins that contain ADP-ribosylation activity: Pseudomonas aeruginosa toxin A, diphtheria toxin, and cholera toxin. Toxin A irreversibility decreased the Isc, PD, and G of bladders in Na-rich media and the Isc and PD of bladders in Na-free media. Diphtheria or cholera toxin reversibly increased Isc and PD (not G), but only in Na-free media. The effects of toxin A in the turtle bladder, like those in other host cell systems, were eliminated by preexposure of this toxin to heat, specific antitoxin, or dithiothreitol and urea. Because exposure to this last condition increases the ADP-ribosylation activity of toxin A, it is suggested that the proenzyme is the required transport-inhibiting form of toxin A. The effects of all three toxins occurred rapidly, possibly before any of the possible intracellular ADP-ribosylation reactions are initiated. Whereas a recognition binding of toxin of toxin to receptors on the apical membrane completely accounts for the reversible effects of diphtheria or cholera toxin, this and additional toxin-membrane interactions (e.g., translocation) are needed to account for the irreversible effects of toxin A.
Photosensitized electron transport across lipid vesicle walls: quantum yield dependence on sensitizer concentration.
Authors: Ford W E , Otvos J W , Calvin M
Published: May 2006

An amphiphilic tris(2,2'-bipyridine)ruthenium(2+) derivative that is incorporated into the walls of phosphatidylcholine vesicles photosensitizes the irreversible oxidation of ethylenediaminetetraacetate(3-) dissolved in the inner aqueous compartments of the vesicle suspension and the one-electron reduction of heptylviologen(2+) dissolved in the continuous aqueous phase. The quantum yield of viologen radical production depends on the phospholipid-to-ruthenium complex mole ratios. A kinetic model is used to derive an order-of-magnitude estimate for the rate constant of electron transport across the vesicle walls. The results are inconsistent with a diffusional mechanism for electron transport and are interpreted in terms of electron exchange.
The smallest genome RNA segment of influenza virus contains two genes that may overlap.
Authors: Inglis S C , Barrett T , Brown C M , Almond J W
Published: May 2006

The genome of influenza virus consists of eight segments of single-stranded RNA, each of which encodes a different polypeptide. In addition to the eight recognized gene products, the virus specifies a distinct smaller nonstructural polypeptide (NS2), which is translated from a separate species of virus-specific mRNA. The location on the virus genome of the gene encoding this polypeptide was investigated by hybridization of the NS2 mRNA with isolated subgenomic RNA species, and by correlation of the inheritance of a strain-specific NS2 with inheritance of particular genome RNA segments during recombination between two different virus strains. The genetic information for NS2 was found to reside in the smallest genome RNA segment of the virion, which also encodes the NS1 polypeptide. Considering the sizes of the molecules involved, it is likely that the coding sequences for the two polypeptides overlap.
Reconstitution of RNase P activity from inactive RNA and protein.
Authors: Kole R , Altman S
Published: May 2006

RNase P preparations from Escherichia coli can be separated into RNA and protein by chromatography, in buffers containing 7 M urea, on Sephadex G-200, DEAE-Sephadex, or CM-Sephadex columns. Neither RNA nor protein components alone exhibits any RNase activity. RNase P activity can be reconstituted by mixing separated RNA and protein components in buffer containing 7M urea followed by dialysis of this mixture to remove the urea. Of several purified RNAs tried, only M2 RNA, the RNA species found in purified RNase P, is active in the reconstitution experiments.
Trophoblast modulation of maternal allogeneic recognition.
Authors: McIntyre J A , Faulk W P
Published: May 2006

Human syncytiotrophoblast cell membranes prepared by differential ultracentrifugation were extracted with 3 M KCl, solubilized in 1% deoxycholate, and chromatographically separated into two peaks by passage through a column of Bio-Gel P-200. Previous reports from this laboratory have shown that the first peak (PI) is serologically the same as a group of trophoblast membrane antigens tentatively designated as TA1. Microgram amounts of P1 protein were found to completely inhibit the mixed lymphocyte culture (MLC) reaction but had no suppressive effect on lymphocyte responses to the lectins phytohemagglutinin or pokeweed mitogen. Control P1 membrane fractions identically prepared from human erythrocytes and liver powder had no inhibitory effects on either MLC reactions or lymphocyte responses to mitogens. Dose response experiments with P1 from 10 different placentae showed total inhibition of MLC by all preparations when used between 25 and 50 microgram/0.2-ml MLC mixture, but some P1 fractions inhibited significantly at much lower concentrations. Timed experiments revealed that MLC suppression was maximal when P1 was added within 12 hr after culture initiation and that no effect could be found with addition after 48 hr. We have previously shown that TA1 is a lymphocyte product of allogeneic responses, and the present results indicate that P1 proteins are themselves involved in the biology of lymphocyte responses to allogeneic cells. Pregnancy is one of the few natural circumstances in which a mixing of allogeneic cells occurs in vivo, and the presence of P1 proteins at the operational interface in the host-parasite relationship of human pregnancy suggests that this trophoblast membrane constituent is involved in the modulation of maternal allogeneic responses.
Immune response genes have a variable influence on the selection of antigenic foreign and self determinants of insulin.
Authors: Cohen I R , Talmon J , Lev-Ram V , Ben-Nun A
Published: May 2006

We studied the proliferative response of mouse T lymphocytes to determinants on ungulate insulins. The immunopotency of defined determinants on the molecule was found to be regulated by three factors: the immune response genes of the immunized mouse, the mode of presentation of insulin on cells or in adjuvant, and the intramolecular cooperativity between different determinants on the insulin molecule. Autosensitization against self determinants was observed under specific conditions. These findings emphasize the variable expression of immune response genes.
Induction of indoleamine 2,3-dioxygenase in mouse lung during virus infection.
Authors: Yoshida R , Urade Y , Tokuda M , Hayaishi O
Published: May 2006

Indoleamine 2,3-dioxygenase [indoleamine: oxygen 2,3-oxidoreductase (decyclizing)] activity in the supernatant fraction (30,000 X g, 30 min) of mouse lung homogenate increased approximately 120-fold after infection with PR8 influenza virus. Both specific and total enzyme activities started to increase linearly from the 5th day after infection, reached the highest level around the 11th day, and then gradually decreased to normal values in about 3 weeks. Other enzymes in the lung, such as certain lysosomal enzymes and monoamine oxidase, did not change significantly throughout the experiments. The time course of the increase in the enzyme activity was quite different from that of virus replication in the lung (a peak by the 3rd day and persistence until the 9th day) or that of serum antibody content (started to rise on the 9th day). Rather, it appeared to be closely related to the infiltrations of mononuclear and lymphocytic cells. When mice were exposed to a higher dose of virus and did not recuperate, the time course of the increase of the enzyme activity was essentially identical to that seen with a low concentration of virus. A maximum stimulation of the enzyme activity in the lung occurred on the 9th day after infection; the increase was approximately 100-fold. However, serum antibody content was slight and virus titer in the lung remained high.
Formation and involution of Mallory bodies ("alcoholic hyalin") in murine and human liver revealed by immunofluorescence microscopy with antibodies to prekeratin.
Authors: Denk H , Franke W W , Eckerstorfer R , Schmid E , Kerjaschki D
Published: May 2006

Antibodies raised against prekeratin intensely and specifically stain, in immunofluorescence microscopy, Mallory bodies ("alcoholic hyalin") present in livers of human alcoholics and griseofulyin-treated mice. The high sensitivity of this method allows the identification of small distinct cytoplasmic structures that are observed during early stages of Mallory body formation, especially frequent in the perinuclear cytoplasm, as well as during stages of Mallory body disintegration and disappearance, such as after withdrawal of the drug. In the latter situation, the prekeratin-containing small particles exhibit a characteristic pattern of arrangement in the hepatocyte periphery. Electron microscopy illustrates that such small bodies are heap-like aggregates of typical Mallory body filaments. Immunofluorescence studies with antibodies to isolated prekeratin polypeptides from bovine hoof or muzzle epidermis show that Mallory body filaments, in particular those in human liver, are immunologically more closely related to prekeratin of tonofilaments from living epidermal cells (stratum spinosum). The data indicate that Mallory bodies contain a pathologic form of prekeratin-like material. They also suggest that disorders of cytoskeletal structures of the intermediate-sized filament class are associated with specific diseases and can be visualized and characterized by immunofluorescence microscopy by using antibodies to constitutive proteins of such filaments.
Decussion geometrics in the goldfish nervous system: correlation with probability of survival.
Authors: Roth R L
Published: May 2006

In the goldfish, the optic nerve decussation occurs without intermingling of fibers from the two eyes. In two-thirds of juvenile and adult specimens, the left optic nerve is dorsal at the midline. In about 60% of the specimens, the decussation of Mauthner's neuron also has a left-dorsal-to-right (L/R) configuration. Concordance for decussation geometry is greater than 80%, with smaller specimens accounting for a disproportionate number of discordant cases. In embryos and very young larvae, the L/R configuration occurs in slightly less than 50% of optic chiasmata and in slightly more than 50% of Mauthner's cell chiasmata, and there is no significant tendency toward concordance. However, larval specimens that survive 1 month after hatching are markedly skewed toward adult decussation patterns and somewhat skewed toward concordance. These observations, together with results of cannibalization and predation experiments, suggest that the adult population pattern can be achieved through selection pressures against discordant individuals and, perhaps separately, against individuals with right-dorsal-to-left (R/L) chiasmatic configurations. Decussation patterns correlate with embryonic postures, larval orientation preferences, and growth rates after hatching, which may themselves serve as a basis for selection.
Identification of mannose 6-phosphate in glycoproteins that inhibit the assimilation of β-galactosidase by fibroblasts
Authors: Distler Jack , Hieber Virginia , Sahagian Gary , Schmickel Roy , Jourdian George W.
Published: May 2006

Bovine testicular β-galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23) is rapidly and selectively assimilated by human skin fibroblasts. The assimilation of the enzyme is strongly inhibited by mannose 6-phosphate and by a glycoprotein fraction isolated from bovine testes (glycoprotein inhibitors). These results suggest that β-galactosidase and the glycoprotein inhibitors have a common recognition marker that contains mannose 6-phosphate. The presence of mannose phosphate in the glycoprotein inhibitors was demonstrated by acid hydrolysis of the glycoproteins to liberate mannose phosphate followed by reduction with NaB
3
H
4
to give [
3
H]mannitol phosphate. The
3
H-labeled compound was identified by paper electrophoresis and by the release of [
3
H]mannitol on treatment with phosphatase. The [
3
H]mannitol phosphate was oxidized with periodate and the resulting phosphorylated fragment, on reduction with NaB
3
H
4
, yielded [
3
H]ethylene glycol phosphate, indicating substitution of phosphate on carbon 6 of mannitol. Mannose 6-phosphate was also found in a major carbohydrate-containing fraction of peptides produced from the glycoprotein inhibitors by tryspin digestion. It was estimated that about 2% of the mannose residues were present as mannose 6-phosphate. Phosphorylated oligosaccharides were also identified in hydrolysates of the glycoprotein inhibitors. One, a disaccharide, was identified as α-(mannosyl-6-phosphate)-(1 → 2)-mannose. These observations suggest that the recognition marker of β-galactosidase contains α1,2-linked mannose 6-phosphate; terminal α1,2-linked mannose residues are known to occur in the high-mannose type oligosaccharides present on β-galactosidase.
Synthesis of fluorescent nucleotide analogues: 5'-mono-, di-, and triphosphates of linear-benzoguanosine, linear-benzoinosine, and linear-benzoxanthosine.
Authors: Leonard N J , Keyser G E
Published: May 2006

The fluorescent nucleotide analogues (the 5'-mono-, di-, and triphosphates of lin-benzoguanosine, lin-benzoxanthosine, and lin-benzoinosine) have been prepared for use as dimensional probes of enzyme binding sites. They have quantum yields in aqueous solution of 0.39, 0.55, and 0.04 and fluorescent lifetimes of 6, 9, and approximately equal to 1.5 nsec, respectively. lin-Benzoinosine 5'-monophosphate is a substrate for xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2), providing lin-benzoxanthosine 5'-monophosphate, and lin-benzoinosine 5'-diphosphate is a substrate for polynucleotide phosphorylase (polyribonucleotide:orthophosphate nucleotidyltransferase. EC 2.7.7.8), giving poly(lin-benzoinosinic acid). The benzologues of the purine diphosphates are substrates for pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40), which is used to prepare the triphosphates.
Leukotriene C: a slow-reacting substance from murine mastocytoma cells.
Authors: Murphy R C , Hammarström S , Samuelsson B
Published: May 2006

Murine mastocytoma cells treated with calcium ionophore A23187 produced a slow-reacting substance (SRS) that caused guinea pig ileum to contract. The response was reversed by the SRS antagonist FPL 55712. On the basis of isotope incorporation experiments, spectroscopy, and chemical degradations, the SRS was identified as a cysteine-containing derivative of 5-hydroxy-7,9,11,14-icosatetraenoic acid. This amino acid was attached in thioether linkage at C-6. The SRS is structurally related to previously identified epoxy and dihydroxy metabolites of arachidonic acid in leukocytes. A common feature is the presence of a conjugated triene, and the name "leukotriene" has been introduced to designate these compounds. Leukotriene A (5,6-epoxy-7,9,11,14-icosatetraenoic acid) is an intermediate in the formation of leukotriene B (5,12-dihydroxy-6,8,10,14-icosatetraenoic acid) and is proposed to be a precursor also of leukotriene C, which is the SRS identified here.
Energy-dependent processing of cytoplasmically made precursors to mitochondrial proteins.
Authors: Nelson N , Schatz G
Published: May 2006

Earlier work has shown that mitochondrial proteins synthesized in the cytosol are initially made as larger precursors which are then transferred into the organelles and processed to their mature size in the absence of protein synthesis. It is now demonstrated that depletion of the mitochondrial matrix ATP in intact yeast spheroplasts by various combinations of inhibitors and mutations prevents the processing of precursors to the three largest subunits of the mitochondrial F1-ATPase and two subunits of the cytochrome bc1 complex. These polypeptides are all synthesized outside the mitochondria and transported to the mitochondrial matrix or inserted into the mitochondrial inner membrane. In contrast, depletion of the matrix ATP does not inhibit processing of the precursor to cytochrome c peroxidase; this enzyme is located in the mitochondrial intermembrane space which is freely accessible to ATP made in the cytosol. The processing of extramitochondrially made precursors or the transfer of these precursors across the mitochondrial inner membrane is thus dependent on ATP.
Comparison of amino acid sequences of two human histocompatibility antigens, HLA-A2 and HLA-B7: location of putative alloantigenic sites.
Authors: Orr H T , Lopez de Castro J A , Parham P , Ploegh H L , Strominger J L
Published: May 2006

The complete amino acid sequence for papain-solubilized HLA-B7 heavy chain is compared with the partial sequences of HLA-A2 and H-2Kb heavy chains. Although these molecules are highly conserved (i.e., 80% homology in comparing HLA-B7 with HLA-A2; 72% and 74% homology in comparing H-2Kb with HLA-A2 and HLA-B7, respectively), two stretches of greater variability are observed. These clusters of variability are discussed in terms of their possible involvement in the alloantigenic determinant(s) characteristic of these highly polymorphic membrane antigens.
Phospholipid methylation stimulates lactogenic binding in mouse mammary gland membranes.
Authors: Bhattacharya A , Vonderhaar B K
Published: May 2006

Addition of the methyl donor S-adenosyl-L-methionine to membranes prepared from mammary glands of lactating mice results in increased binding of 25I-labeled human growth hormone to the lactogenic receptors. This stimulation is dose dependent and specific for S-adenosyl-L-methionine and is partially inhibited by simultaneous addition of S-adenosyl-homocysteine to the reaction. Pretreatment of the membranes with S-adenosyl-L-methionine for 30 min at 37 degrees C is sufficient to cause enhanced binding. Scatchard analysis shows that treatment with S-adenosyl-L-methionine results in an increase in the number of lactogenic binding sites without changing the apparent affinity constant for 125I-labeled human growth hormone. The increase in the number of binding sites is believed to be due to alteration in the phospholipid composition of the membrane because methylation of phospholipids is observed under these conditions.
Isolation of glycosaminoglycans (heparan sulfate) from glomerular basement membranes
Authors: Kanwar Yashpal S. , Farquhar Marilyn Gist
Published: May 2006

Glycosaminoglycans were isolated from purified fractions of glomerular basement membranes and partially characterized by chemical analysis and cellulose acetate electrophoresis. Basement membranes were prepared by detergent treatment of rat glomeruli and subjected to digestion with papain and Pronase. Glycosaminoglycans were isolated from the digests by precipitation with cetyl pyridinium chloride and ethanol. Results of cellulose acetate electrophoresis of the isolated glycosaminoglycan fraction revealed the presence of one major and one minor spot. The major spot was identified as heparan sulfate because it comigrated with the heparan sulfate standard and was sensitive to heparinase and to nitrous acid oxidation but insensitive to chondroitinase ABC and to testicular or leech hyaluronidase. The minor spot was tentatively identified as hyaluronic acid based on its migratory behavior and sensitivity to leech and testicular hyaluronidase. The chemical composition of the isolated glycosaminoglycan was typical of that of heparan sulfate (high carbazole/orcinol ratio, high sulfate content, absence of galactosamine). The data support and confirm the cytochemical data obtained previously [Kanwar, Y. S. & Farquhar, M. G. (1979)
Proc. Natl. Acad. Sci. USA
76, 1303-1307] demonstrating that heparan sulfate is the only sulfated glycosaminoglycan detectable in the glomerular basement membrane. The present results suggest that in addition to sulfated glycosaminoglycan some nonsulfated glycosaminoglycan (hyaluronic acid) may also be present in the glomerular basement membrane.
On the evolution of accuracy and cost of proofreading tRNA aminoacylation.
Authors: Savageau M A , Freter R R
Published: May 2006

Aminoacylation of tRNA occurs with a high degree of accuracy in many cases because misacylated molecules are effectively proofread on the aminoacyl-tRNA synthetase by preferential hydrolysis. This hydrolysis releases an ATP equivalent of energy. An explicit relationship between cost of proofreading and the resulting degree of accuracy is presented. Experimental data from Escherichia coli for isoleucyl-tRNA synthetase proofreading valyl-tRNAIle are examined by means of this relationship, and a conjecture concerning the natural selection of accuracy and proofreading costs is put forth and tested. We have found the energy cost of accurate proofreading to be high. The minimum error, derived in previous theoretical studies, is never actually reached. Instead, higher values, determined by the balance between energy wasted in the cell as a consequence of error and the energy cost of proofreading, appear to be selected. The total cost of proofreading all types of tRNA aminoacylations is estimated to be approximately 2% of the energy required to synthesize a bacterial cell.
Activation of mating type genes by transposition in Saccharomyces cerevisiae.
Authors: Klar A J , Fogel S
Published: May 2006

Yeast Saccharomyces cerevisiae may express an a or alpha mating type. These cells types may be interconverted as a consequence of heritable genetic alteractions at the mating type locus (MAT). According to the more general controlling element model [Oshima, U. & Takano, I. (1971) Genetics 67, 327--335] and the specific cassette model [Hicks, J., Strathern, J. & Herskowitz, I. (1977) in DNA Insertion Elements, Plasmids and Episomes, eds. Bukhari, A. I., Shapiro, J.A. & Adhya, S. L.(Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 457--462], the regulatory information required for switching the MAT locus exists at two other loosely linked loci, HMa and HMalpha. Specifically, the HMa and HMalpha loci are proposed to carry silent alpha and silent a genes, respectively. According to these models, switching occurs when a replica of a silent gene replaces the resident information at the mating type locus and is thereby expressed. These models predict that mutations at the silent ("storage") loci would generate defective MAT loci subsequent to the switching process. Therefore, the behavior of HMalpha mutants during the mating type interconversion was investigated. The results demonstrate that defective MATa alleles are generated by switching the MATalpha locus in HMalpha mutants. Thus, the genetic information from HMalpha is transposed to the mating type locus. These results provide genetic evidence in support of these models.
Identification of DNA fragments carrying ecotropic proviruses of AKR mice.
Authors: Steffen D , Bird S , Rowe W P , Weinberg R A
Published: May 2006

The proviruses of the N-tropic, ecotropic virus (AKV) of AKR mice (Akv-1, Akv-2) have been studied by the Southern gel--filter transfer technique. These proviruses can be detected by cleavage of cell DNA by BamHI endonuclease, which yields characteristic subgenomic DNA fragments upon cleavage of this type of provirus. Proviruses integrated into different sites in the mouse genome can be resolved with EcoRI endonuclease, which does not cleave the AKV proviruses. Use of congenic and backcrossed mice and a radioactive DNA probe enriched for AKV sequences has allowed identification of the EcoRI fragments carrying the proviruses of the genetically defined Akv-1 and Akv-2 loci. Novel proviruses introduced by superinfection of cultured AKR cells with AKV and present in leukemic cells from AKR mice have also been identified. Comparison of substrains of AKR mice indicates some heterogeneity in their spectra of proviruses.
Monoclonal antibody directed to a B-cell antigen present in rats, mice, and humans.
Authors: Gasser D L , Winters B A , Haas J B , McKearn T J , Kennett R H
Published: May 2006

Spleen cells from a LEW.AVN rat immunized with cells from an MNR rat were fused with mouse myeloma cells to produce hybrid cell lines. One of these hybridomas produced a monoclonal antibody that was cytotoxic for bone marrow-derived (B) but not thymus-derived (T) cells. The antigen defined by this antibody is determined by a gene linked to the major histocompatibility complex (MHC). The antigen is also present on B cells of most mouse strains and is determined by an MHC-linked gene in this species as well. In both rats and mice, the gene determining the antigen maps within the immune response region of the MHC. All human B-cell lines, but not T-cell lines, and B but not T cells of all human donors tested so far are also positive for this antigen. Among human-mouse somatic cell lines that have lost various human chromosomes, this B-cell antigen is present on all lines that are positive for HLA antigen but is absent from all lines that have lost HLA.
Sodium transport inhibitor from bovine hypothalamus.
Authors: Haupert G T , Sancho J M
Published: May 2006

A low molecular weight, basic, nonpeptidic factor that possesses sodium transport inhibitory properties has been prepared from bovine hypothalamus by acid/acetone extraction and gel filtration. Concentration and partial purification was achieved by ion-exchange chromatography. This substance inhibits active Na+ transport across anuran membranes, inhibits ouabain binding to frog urinary bladder, and directly inhibits renal Na+,K+-ATPase. This substance thus possesses the putative characteristics of a natriuretic factor of hypothalamic origin.
Clonal hybrid cell lines expressing cholinergic and adrenergic properties.
Authors: Heumann R , Ocalan M , Kachel V , Hamprecht B
Published: May 2006

Different cholinergic cell lines were fused with an adrenergic neuroblastoma cell line (N115-BU-8). Its fusion with a cholinergic neuroblastoma-glioma hybrid produced a "hybrid-hybrid" line containing cholinergic and adrenergic enzyme activities. Both activities were also present in subclones of this line. The presence of catecholamines in single cells was confirmed by microspectrofluorimetry. These results are discussed with respect to the possibility of a simultaneous synthesis of noradrenaline and acetylcholine in single cells. The cholinergic and adrenergic enzyme activities are influenced by cell density, by dexamethasone, and by conditioned medium.
Properties of a copper-containing cytochrome c1aa3 complex: a terminal oxidase of the extreme thermophile Thermus thermophilus HB8.
Authors: Fee J A , Choc M G , Findling K L , Lorence R , Yoshida T
Published: May 2006

From the plasma membrane of Thermus thermophilus HB8 we have partially purified a detergent-solubilized complex of cytochromes a and c1 that actively catalyzes the transfer of electrons from ascorbate via a redox dye to oxygen. The complex is composed of two types of polypeptides, with molecular weights of approximately 55,000 and 33,000. Quantitative analysis revealed the presence of heme a, heme c, and copper in a ratio of 2:1:2, with the heme a being present at 10 +/- 1.3 nmol/mg of protein. The heme c was shown to be associated with the molecular weight 33,000 peptide and is suggested to be of the c1 type. The optical and electron paramagnetic resonance properties of this complex were found to be similar to those of eukaryotic cytochrome oxidase, suggesting the following arrangement of chromophores: a magnetically isolated cytochrome c1 and an oxygen-reducing functional unit consisting of two heme a groups and two copper ions associated with one or more larger peptides.
Inhibition of receptor-mediated clearance of lysine and arginine modified lipoproteins from the plasma of rats and monkeys
Authors: Mahley Robert W. , Weisgraber Karl H. , Melchior George W. , Innerarity Thomas L. , Holcombe Kathleen S.
Published: May 2006

Reductive methylation of at least 30% of the lysine residues or 1,2-cyclohexanedione modification of 45% of the arginine residues prevented low density lipoproteins (LDL) from binding to cell surface receptors of fibroblasts
in vitro
, without significantly altering other physical or chemical properties of the LDL. When rat or human LDL with more than 30% of the lysine residues methylated were injected intravenously into rats, the clearance of these lipoproteins from the plasma was slowed considerably. The half-life of the reductively methylated LDL was approximately twice that obtained for control (unmodified) LDL, and the value for the fractional catabolic rate was approximately half that of the control. Furthermore, when human LDL modified by reductive methylation were injected into rhesus monkeys, the rate of clearance was similarly retarded, and the value for the fractional catabolic rate was reduced by approximately 50% as compared with the value for control LDL. A dual isotope labeling technique (
125
I and
131
I) was used to compare the disappearance of the control and modified LDL in the same animal. It was demonstrated that not only modification of lysine residues but also modification of the arginine residues with 1,2-cyclohexanedione retarded the plasma clearance of the rat LDL. However, the cyclohexanedione modification was spontaneously reversible at 37°C, whereas reductive methylation of the lysine residues was stable. It is concluded that the selective chemical modification of lysine or arginine residues of LDL interferes with the normal uptake of these lipoproteins
in vivo
as well as by fibroblasts
in vitro
. These data provide an estimation of the level of receptor-mediated clearance of LDL from the plasma, a value that may be as high as 50% in rats and monkeys.
Phospholipids as the molecular instruments of ion and solute transport in biological membranes.
Authors: Green D E , Fry M , Blondin G A
Published: May 2006

Partition studies have established that phospholipids generally have the capabilities to mediate the transmembrane transport of the full range of ions and solutes that physiologically cross biological membranes. The list of transportable species includes cations, anions, amino acids, citric acid cycle intermediates, nucleotides, and sugars. Phospholipid-mediated transport can be readily modulated by altering the phospholipid mixture or by addition of detergents, nucleotides, divalent metals, proteins, peptides, or ring compounds. Containment of phospholipid within channels in protein appears to be the precondition for the formation of the micellar structure requisite for solute transport. Phospholipid-mediated transport is postulated to be a central feature of energy coupling, membrane-spanning systems, and membrane-bound, phospholipid-requiring enzymes.
Cell-cell recognition in yeast: Purification of
Hansenula wingei
21-cell sexual agglutination factor and comparison of the factors from three genera
Authors: Burke David , Mendonça-Previato Lucia , Ballou Clinton E.
Published: May 2006

Trypsin digestion of
Hansenula wingei
21-cells releases a protein (21-factor-T) that inhibits the agglutination of 21-cells by purified 5-agglutinin obtained from 5-cells by subtilisin digestion [Crandall, M. A. & Brock, T. D. (1968)
Bacteriol. Rev.
32, 139-163]. We have purified this inhibitor 415-fold by ion-exchange chromatography, affinity adsorption to 5-cells, and gel permeation chromatography. The material shows a diffuse band, on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, with an apparent
M
r
of 27,000. It has a pI of 3.8, is rich in acidic amino acids, contains 5% mannose and a trace of glucosamine, and is stable to reducing agents but is inactivated by heat. Zymolyase (β1→3-glucanase) digestion of 21-cells releases a similar inhibitor that, after purification, has a larger size than 21-factor-T. This 21-factor-Z appears to contain an additional portion that may serve to anchor 21-factor in the cell wall. Haploid cells of the yeasts
Pichia amethionina
and
Saccharomyces kluyveri
also show a constitutive sexual agglutination, and little or no crossreactivity is observed in heterologous mixtures. The agglutination factors in all three genera, however, have parallel properties; one cell type of each pair is heat stable and is inactivated by reducing agents (
H. wingei
5-cells,
P. amethionina
α-cells, and
S. kluyveri
16-cells), and the other is heat labile and is unaffected by reducing agents
H. wingei
21-cells,
P. amethionina a
-cells, and
S. kluyveri
17-cells). Because
S. kluyveri
16-cells respond to
Saccharomyces cerevisiae
α-factor with the typical morphogenetic change of a mating half-reaction, the heat-stable agglutinin appears related to the
S. cerevisiae a
mating type and the heat-labile factor to the
S. cerevisiae
α mating type.
Meiotic synthesis of testis histones in the rat.
Authors: Brock W A , Trostle P K , Meistrich M L
Published: May 2006

The distribution and synthesis of the testis-specific variants of histones H1 and H2B, TH1 and TH2B, respectively, and of the somatic histones were studied in rat testis cells. Rat testis cells were labeled in vivo with intratesticular injections of [3H]lysine. The cells and nuclei were then separated into different developmental classes by velocity sedimentation and the histones were analyzed. TH1 and TH2B, as well as the somatic histones, were present in spermatocytes and round spermatids, but none of them were detectable in elongated spermatids. The synthesis and nuclear accumulation of TH1 and TH2B took place throughout pachytene, as well as in earlier stages, but not in the round spermatids. In addition, there was synthesis during pachytene of a histone that migrates electrophoretically with H2A. However, somatic histone synthesis, with the possible exception of H2A and H2B, was not detectable at the pachytene stage. In vivo treatment of rats with hydroxyurea reduced DNA synthesis in the testis to 1% of control values and significantly reduced the synthesis of H3, H2B, H2A, and H4, with the greatest effect being on H3 and H4. However, the hydroxyurea treatment did not significantly decrease the synthesis of TH1, H1, or TH2B. These results prove that the synthesis of several histones during the meiotic prophase is not dependent upon concurrent S-phase DNA synthesis.
Mutant and recombinant avian retroviruses with extended host range.
Authors: Tsichlis P N , Conklin K F , Coffin J M
Published: May 2006

Avian retroviruses of subgroups B and D efficiently infect chicken (C/E) but not turkey (T/BD) cells. We describe here three variants of subgroup B and D viruses that infect both cell types equally well. One of these viruses, NTRE-4, was a recombinant between transformation-defective Prague (Pr) strain Rous sarcoma virus (RSV) subgroup B and the endogenous virus RAV-0; the second, SR-DE-1, was a recombinant between Schmidt-Ruppin RSV subgroup D and defective endogenous virus information. T1 oligonucleotide fingerprint analysis of the genomes of these two viruses showed only a small alteration in the portion of the env gene responsible for subgroup specificity, as indicated by the presence of a single subgroup E oligonucleotide in an otherwise purely subgroup B or D gene. The third virus, hrBO1Pr-B, was a variant of Pr-RSV-B that did not appear to be a recombinant and whose altered host range we attribute to mutation. Analysis of the host range of all three viruses by infection of selectively resistant cells and by interference testing indicates that all use the subgroup B receptor on chicken cells and the subgroup E receptor on turkey cells. These viruses may be analogous to the polytropic recombinant viruses recently found to be associated with leukemia in some strains of mice.
Specific binding of phorbol ester tumor promoters
Authors: Driedger Paul E. , Blumberg Peter M.
Published: May 2006

[20-
3
H]Phorbol 12,13-dibutyrate bound to particulate preparations from chicken embryo fibroblasts in a specific, saturable, reversible fashion. Equilibrium binding occurred with a
K
d
of 25 nM; this value is very close to the 50% effective dose (ED
50
), 50 nM, previously determined for the biological response (induction of fibronectin loss) in growing chicken embryo fibroblasts. At saturation, 1.4 pmol of [20-
3
H]phorbol 12,13-dibutyrate was bound per mg of protein (approximately 7 × 10
4
molecules per cell). Binding was inhibited by phorbol 12-myristate 13-acetate (
K
i
= 2 nM), mezerein (
K
i
= 180 nM), phorbol 12,13-dibenzoate (
K
i
= 180 nM), phorbol 12,13-diacetate (
K
i
= 1.7 μM), phorbol 12,13,20-triacetate (
K
i
= 39 μM), and phorbol 13-acetate (
K
i
= 120 μM). The measured
K
i
values are all within a factor of 3.5 of the ED
50
values of these derivatives for inducing loss of fibronectin in intact cells. Binding was not inhibited by the inactive compounds phorbol (10 μg/ml) and 4α-phorbol 12,13-didecanoate (10 μg/ml) or by the inflammatory but nonpromoting phorbol-related diterpene esters resiniferatoxin (100 ng/ml) and 12-deoxyphorbol 13-isobutyrate 20-acetate (100 ng/ml). These data suggest that biological responses to the phorbol esters in chicken embryo fibroblasts are mediated by this binding activity and that the binding activity corresponds to the phorbol ester target in mouse skin involved in tumor promotion. Binding was not inhibited by the nonphorbol promoters anthralin (1 μM), phenol (1 mM), iodoacetic acid (1.7 μM), and cantharidin (75 μM), or by epidermal growth factor (100 ng/ml), dexamethasone acetate (2 μM), retinoic acid (10 μM), or prostaglandin E
2
(1 μM). These agents thus appear to act at a target distinct from that of the phorbol esters.
Covalent labeling of protein components of the sodium channel with a photoactivable derivative of scorpion toxin.
Authors: Beneski D A , Catterall W A
Published: May 2006

Azidonitrobenzoyl mono[125I]iodo scorpion toxin can be covalently attached to its receptor site in electrically excitable neuroblastoma cells or synaptosomes by photolysis. A polypeptide of Mr approximately 250,000 is specifically labeled in neuroblastoma cells. Labeling is blocked by unlabeled scorpion toxin and by depolarization. This polypeptide is not labeled in a variant neuroblastoma clone lacking voltage-sensitive sodium channels. Polypeptides of Mr approximately 250,000 and Mr approximately 32,000 are specifically labeled in synaptosomes. This labeling is also blocked by unlabeled toxin and by depolarization. These results identify specific polypeptides of Mr approximately 250,000 and Mr approximately 32,000 that are components of the sodium channel.
Autoradiographic localization of gamma-aminobutyric acid (GABA) receptors in the rat cerebellum.
Authors: Palacios J M , Young W S , Kuhar M J
Published: May 2006

[3H]Muscimol was used in the light microscopic autoradiographic localization of gamma-aminobutyric acid (GABA) receptors in the rat cerebellum. The binding of [3H]-muscimol to the slide-mounted tissue sections had all of the characteristics associated with a GABA receptor. It was saturable with appropriate kinetic constants and was blocked only by other GABAergic drugs. Autoradiographic studies revealed a very high density of receptors over the granule cell layer, a low level over the molecular layer, and negligible receptor binding over white matter. This suggested a high localization of GABA receptors to granule cells, which is supported by other neurochemical studies. The autoradiographic procedure used here should be a useful method in further studies of receptors requiring high sensitivity and increased anatomical resolution.
Photophysical properties of zinc and magnesium tris(pyrochlorophyllide
a
) 1,1,1-tris(hydroxymethyl)ethane triesters
Authors: Yuen M. J. , Closs G. L. , Katz J. J. , Roper J. A. , Wasielewski M. R. , Hindman J. C.
Published: May 2006

The zinc and magnesium tris(pyrochlorophyllide
a
) 1,1,1-tris(hydroxymethyl)ethane triesters have properties that are very similar to those exhibited by pairs of pyrochlorophyll
a
or chlorophyll
a
linked by ethylene glycol through their propionic acid side chains. In the open configuration, the fluorescence lifetimes and emission spectra of the triesters resemble those of chlorophyll
a
monomers. However, the triesters have a mechanism for energy dissipation not present in monomeric chlorophyll
a
, which prevents the build-up of significant concentrations of molecules in S
1
states. Optical pumping of these compounds in their folded configuration results in the formation of high concentrations of molecules in S
1
states and in laser light emission, which originates from the two folded macrocycles. The optical data indicate that efficient energy transfer occurs from the open to the folded configurations.
Ambient air concentration of sulfur dioxide affects flight activity in bees
Authors: Ginevan M. E. , Lane D. D. , Greenberg L.
Published: May 2006

Three long-term (16-29 days) low-level (0.14-0.28 ppm) sulfur dioxide fumigations showed that exposure to this gas has deleterious effects on male sweat bees (
Lasioglossum zephrum
). Although effects on mortality were equivocal, flight activity was definitely reduced. Because flight is necessary for successful mating behavior, the results suggest that sulfur dioxide air pollution could adversely affect this and doubtless other terrestrial insects.
Regulation of
Rhizobium
nitrogen fixation by the unadenylylated glutamine synthetase I system
Authors: Ludwig Robert A.
Published: May 2006

Rhizobium
sp. 32H1 glutamine auxotrophs have a complex phenotype: a highly adenylylated glutamine synthetase [GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] I and an undetectable GS II (GS II
-
). They are defective in the adenylylation cascade system for GS I. Prototrophic revertants are of two classes: those (3204 type) which retain the adenylylated GS I phenotype but become GS II
+
, and those (3205 type) which acquire a constitutive unadenylylated GS I but remain GS II
-
. Like the parent auxotroph, 3204 remains incapable of nitrogen fixation both in culture and in root nodules of
Macroptileum atropurpureum
. In contrast, 3205 is nitrogenase constitutive. This implies that GS I or associated adenylylation proteins are involved in the control of
Rhizobium
32H1
nif
gene expression and that GS II is not so involved. Normally, rhizobia fix atmospheric N
2
only during symbiosis and, in so doing, only transiently synthesize nitrogenase. Moreover, whereas wild-type strains export ammonium, constitutive strains can assimilate ammonium produced by nitrogenase. This phenotype allows direct selection of nitrogen fixation-defective mutants in
Rhizobium
.
Platelet-derived growth factor stimulates low density lipoprotein receptor activity in cultured human fibroblasts.
Authors: Witte L D , Cornicelli J A
Published: May 2006

Human platelets contain a mitogen, the platelet-derived growth factor (PDGF), that stimulates the proliferation of a variety of cell types in culture and that may play a role in atherogenesis. Studies were conducted to explore the effects of PDGF on low density lipoprotein (LDL) receptor activity of cultured human fibroblasts. The PDGF utilized in these studies was partially purified from human platelet-rich plama by ion exchange chromatography and gel filtration. LDL receptor activity was assessed by both specific binding of 125I-labeled LDL to the fibroblast's surface at 4 degrees C, and the incorporation of [14C]oleate into cholesteryl esters. Exposure of normal human fibroblasts to increasing amounts of PDGF (0.1-10 microgram/ml) for 48 hr caused a dose-related increase in 125I-labeled LDL binding to a maximum of approximately 300%. In the presence of added LDL, this increase in LDL binding was not seen. Cholesterol esterifiction was also stimulated following a 48-hr exposure to PDGF. Following a conditioning period in LDL- and PDGF-depleted medium, cholesterol esterification was greatly increased during a 48-hr exposure to LDL alone; a smaller but significant increase occurred with PDGF alone. However, both PDGF and LDL were required to return the level of esterification to that observed with whole human serum. Fibroblasts from a patient with homozygous familial hypercholesterolemia, which lack the LDL receptor, also showed a significant increase in cholesteol esterification with PDGF alone, whereas LDL had no effect. These studies demonstrate that PDGF can stimulate the LDL receptor activity in cultured human fibroblasts. The effect on other related activities of the LDL receptor system and the mechanism involved remain to be defined.