bluuebunny/crossref_metadata_2025 huggingface dataset:
Clonal analysis of human cytotoxic T lymphocytes: T4+ and T8+ effector T cells recognize products of different major histocompatibility complex regions.
Authors: Meuer S C , Schlossman S F , Reinherz E L
Published: May 2006

Alloreactive human T lymphocytes were cloned in soft agar or by limiting dilution and subsequently propagated with interleukin 2 and alloantigen for 8 months or more. By indirect immunofluorescence every clone was reactive with anti-Ia antibodies as well as the T cell-specific antibodies anti-T3 and anti-T11 and expressed either T4 or T8 antigens. All 15T8+ clones were highly cytotoxic for the sensitizing alloantigen. In contrast, only two of seven T4+ clones mediated cytotoxic effector function. The specificity of T4+ and T8+ clones and subclones was analyzed on a panel of typing cells and by antibody blocking studies of major histocompatibility complex (MHC) determinants on the stimulating alloantigen. It was found that T8+ clones killed targets that shared class I MHC antigens (HLA-A,B) with the original stimulator cells whereas cytotoxic T4+ clones were directed at class II MHC antigens (Ia-related). Preincubation of the allogeneic target cell with a monoclonal antibody to a nonpolymorphic HLA alpha-chain determinant inhibited killing by the T8+ clones but did not affect T4+ cytotoxic function. In a reciprocal fashion, anti-IA antibodies to common framework structures on the same target cell blocked killing by T4+ but not by T8+ clones. These results indicate that T4+ and T8+ T lymphocytes have receptors for different classes of MHC antigens and suggest tha cytotoxic T4+ subpopulations might be important in human transplantation and autoimmune disorders.
Sequences of the Escherichia coli dnaG primase gene and regulation of its expression.
Authors: Smiley B L , Lupski J R , Svec P S , McMacken R , Godson G N
Published: May 2006

The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the dnaG primase gene [Lupski, J., Smiley, B., Blattner, F. & Godson, G. N. (1982) Mol. Gen, Genet. 185, 120--128] has been determined. The region coding for the dnaG primase has been identified by NH2-terminal and tryptic peptide amino acid analysis of the dnaG protein. The coding region is 1,740 base pairs long (580 amino acids) and is preceded by an unusual ribosome-binding site sequence (G-G-G-G). The dnaG gene is read in the same direction as the adjacent rpoD gene, but no obvious promoter sequences can be found for either gene within several hundred nucleotides upstream. Other unusual features of the dnaG gene that may explain the maintenance of its product at low copy number are the presence of a RNA polymerase terminator 31 nucleotides upstream from the ATG initiator codon and greater use (3--10 times) of certain condons that occur infrequently in other E. coli genes. The nucleotide sequence has also been correlated with data from transposon Tn5 insertional inactivation mapping.
Phospholipids stimulate phosphorylation of vinculin by the tyrosine-specific protein kinase of Rous sarcoma virus.
Authors: Ito S , Richert N , Pastan I
Published: May 2006

The phosphorylation of vinculin by a highly purified tyrosine-specific protein kinase was enhanced more than 10-fold by anionic phospholipids: the phosphorylation of casein, actin, and alpha-actinin was inhibited. The effect of phospholipid was dependent on the divalent cation used. Stimulation was observed by phosphatidylinositol or phosphatidylglycerol in the presence of either 0.5 mM Mn2+ of 5 mM Mg2+; with either phospholipid, more enzyme activity was observed with Mn2+. Maximal stimulation by phosphatidylinositol was observed at about 400 micrograms/ml. In contrast, marked stimulation by phosphatidylserine was observed only with Mn2+ and marked stimulation by phosphatidic acid was observed only with Mg2+. These results raise the possibility that phospholipids modulate vinculin phosphorylation in Rous sarcoma virus-transformed cells.
Synexin facilitates fusion of specific phospholipid membranes at divalent cation concentrations found intracellularly.
Authors: Hong K , Düzgüneş N , Ekerdt R , Papahadjopoulos D
Published: May 2006

The effect of synexin (an adrenal medullary protein) on the kinetics of Ca2+- and Mg2+-mediated membrane fusion was examined. Membrane fusion was studied by monitoring intermixing of the aqueous contents of phospholipid vesicles. Synexin facilitated Ca2+-mediated, but not Mg2+-mediated, fusion of phosphatidate/phosphatidylethanolamine (1:3) and phosphatidate/phosphatidylserine/phosphatidylethanolamine/cholesterol (1:2:3:2) vesicles. The threshold concentration of Ca2+ for fusion was decreased to approximately equal to 10 microM in the presence of synexin at 6 micrograms/ml and 1.5 mM Mg2+ in vesicle suspensions containing 50 microM lipid. This effect of synexin was drastically inhibited by including 25% phosphatidylcholine (mol/mol) in the vesicle membrane. It is proposed that the Ca2+-dependent lipid-specific enhancement of membrane fusion by synexin contributes to an increase in the sensitivity of specific intracellular membranes to Ca2+ with respect to fusion.
Generation of leukotriene C4 from a subclass of mast cells differentiated in vitro from mouse bone marrow.
Authors: Razin E , Mencia-Huerta J M , Lewis R A , Corey E J , Austen K F
Published: May 2006

Bone marrow-derived mast cells, differentiated in vitro, demonstrate surface IgE, receptors and contain histamine in metachromatic granules, which are composed of chondroitin sulfate E proteoglycan rather than heparin proteoglycan. Activation of this subclass of mast cells with calcium ionophore A23187 resulted in generation of immunoreactive C-6-sulfidopeptide leukotriene in a dose- and time-dependent fashion. Isolation of immunoreactive C-6-sulfldopeptide leukotriene by reverse-phase high-performance liquid chromatography (RP-HPLC) revealed a retention time and a specific biologic activity identical to those of synthetic leukotriene C4 (LTC4). Neither radiolabeled nor immunoreactive conversion products of [3H]LTC4/LTC4 were recognized during RP-HPLC resolution of the supernatants. The failure of fresh bone marrow cultures to generate C-6-sulfidopeptide leukotriene in response to ionophore indicates that leukotriene generation is dependent upon cellular differentiation into a mast cell population. The amount of LTC4 generated during optimal ionophore stimulation, 90.9 +/- 7.5 ng per 10(6) cells, contrasts with the relatively low amounts of C-6-sulfidopeptide leukotriene generated by the conventional heparin-containing rat mast cells or mouse mastocytoma cells.
Ribosomal protein L3 is involved in replication or maintenance of the killer double-stranded RNA genome of Saccharomyces cerevisiae.
Authors: Wickner R B , Ridley S P , Fried H M , Ball S G
Published: May 2006

Ability to secrete the K1 (or K2) toxin protein and immunity to that toxin [the K1 (or K2) killer trait] are determined by a double-stranded (ds) RNA, called M1 (or M2), whose replication and maintenance depend on at least one of the larger (L) ds RNAs and 29 chromosomal genes, called MAK genes (maintenance of killer). The location of the MAK8 gene near TCM1 (trichodermin resistance) on the yeast map suggested the possible identity of these two genes. Of six independently isolated tcm1 mutants, five were clearly mak-, and the sixth was weakly mak-. In each case, the mak- phenotype and the trichodermin-resistant phenotypes cosegregated in meiosis and showed the expected tight linkage to pet17. The mak- mutations in the trichodermin-resistant strains did not complement mak8-1, indicating that MAK8 and TCM1 are the same gene. The mak8-1 mutation does not make strains resistant to trichodermin, and one tcm1 mutation is only slightly mak-. Whereas tcm1 mutants lose M1 or M2 ds RNA, they do not lose L ds RNA. Because TCM1 codes for ribosomal protein L3 [Fried, H. M. & Warner, J. R. (1981) Proc. Natl. Acad. Sci, USA 78, 238--242], we conclude that ribosomal protein L3 is involved in the replication and maintenance of M ds RNA. Mutations in cyh2 or cry1, producing resistance to cycloheximide and crytopleurine due to mutant ribosomal proteins, do not produce a mak- phenotype. In analogy with bacterial ribosome assembly mutants, yeast low-temperature-sensitive (lts) mutants may have defective ribosomes. We thus examined mutants for an effect on the killer system. An lts5 mutant, unable to grow at 5 degrees C, also has a mak- phenotype (at 30 degrees C) that cosegregates in meiosis with the lts- phenotype. Mutations in seven other lts genes do not result in the mak- phenotype.
Transmembrane channel formation by complement: functional analysis of the number of C5b6, C7, C8, and C9 molecules required for a single channel.
Authors: Ramm L E , Whitlow M B , Mayer M M
Published: May 2006

Earlier studies have shown that sequential treatment of resealed erythrocyte ghosts with C5b6, C7, C8, and C9 leads to insertion of hydrophobic peptides from these complement proteins into the membrane and assembly of transmembrane channels. The number of molecules of each of the proteins required for assembly of the membrane-associated channel structure was evaluated by measuring the quantitative relationship between the doses of the individual proteins and the release of two trapped markers, sucrose and inulin, from ghosts after channel formation. The incubation period was sufficient to attain equilibrium of marker distribution between the ghosts and the extracellular fluid. Two markers of different size (sucrose and inulin, 0.9 and 3 nm molecular diameter, respectively) were used in order to develop information on the molecular composition of small and large channels, respectively. We found that participation of C5b6, C7, and C8 in channel formation displayed one-hit characteristics, regardless of marker size. By contrast, the participation of C9 was one-hit with respect to the sucrose marker, whereas with respect to the inulin marker the C9 reaction was multi-hit. Our results are compatible with the view that these markers are released through a channel structure in the membrane that is a monomer of C5b--9 of the composition C5b61 C71C81C9n, in which n = 1 for channels permitting passage of sucrose and n = 2 for channels allowing transit of inulin.
Manipulation of brain DNA synthesis is achieved by using a systemic immunological disease.
Authors: Griffin W S , Crom E N , Head J R
Published: May 2006

Previously, we showed that as early as postnatal day 11 an immunological disease, graft-versus-host disease, induced by grafting allogeneic lymph node cells into an immunoincompetent neonatal rat significantly decreases cerebellar histogenesis--i.e., DNA synthesis and the number of newly formed neurons. Here, we report that, subsequent to successful immunotherapy, there was a reversal of the deleterious graft-versus-host disease-induced alterations in DNA synthesis in individual cerebellar germinal cells. Immunotherapy involved treating the diseased rats on postnatal days 11, 12, and 13 with alloantiserum specifically directed against the grafted lymph node cells injected on the day of birth. On postnatal day 14, diseased, serum-treated, and control littermates were injected with [3H]thymidine and, 15 min later, the cerebella were excised and autoradiographed. A 0.72-mm segment of the external granular layer in the cerebellar fissure between lobules VIB and C was searched for labeled cells. The control group had the greatest number of labeled cells, defined by the presence of six or more autoradiographic grains. (43 +/- 4, mean +/- SEM) and the greatest number of grains per cell (9.5 +/- 0.2). Rats with the disease had few labeled cells (4 +/- 2) and the number of grains per cell was low (6.6 +/- 0.6); however, serum treatment increased both the number of labeled cells (26 +/- 8) and the number of grains per cell (7.4 +/- 0.2). These results show that without mononuclear infiltrates or inflammation in the cerebellum, a systemic immunological disease can dramatically decrease DNA synthesis per germinal cell and, moreover, that halting the disease by alloantiserum therapy can reverse this effect. These findings emphasize the sensitive plastic nature of neuronal cell acquisition in the normally developing brain.
Electronic distributions within protein phenylalanine aromatic rings are reflected by the three-dimensional oxygen atom environments.
Authors: Thomas K A , Smith G M , Thomas T B , Feldmann R J
Published: May 2006

The atomic environments of 170 phenylalanine-residue aromatic rings from 28 protein crystal structures are transformed into a common orientation and combined to calculate an average three-dimensional environment. The spatial distribution of atom types in this environment reveals a preferred interaction between oxygen atoms and the edge of the planar aromatic rings. From the difference in frequency of interaction of oxygen atoms with the edge and the top of the ring, an apparent net free energy difference of interaction favoring the edge of the ring is estimated to be about -1 kcal/mol (1 cal = 4.184 J). Ab initio quantum mechanical calculations, performed on a model consisting of benzene and formamide, indicate that the observed geometry is stabilized by a favorable enthalpic interaction. Although benzene rings are considered to be nonpolar, the electron distribution is a complex multipole with no net dipole moment. The observed interaction orientation frequencies demonstrate that these multipolar electron distributions, when occurring at the short distances encountered in densely packed protein molecules, are significant determinants of internal packing geometries.
Inhibition of hepatic cholesterol synthesis in mice by sterols with shortened and stereochemically varied side chains.
Authors: Erickson K A , Nes W R
Published: May 2006

Mice were fed cholesterol or various other sterols for 26 hr, after which the amount of hepatic cholesterol synthesis was measured in a cell-free system. The following sterols were as effective as cholesterol itself in depressing the conversion of acetate into sterol: pregn-5-en-3 beta-ol, which lacks an isohexyl group on C-20; (E)-17(20)-dehydrocholesterol, in which the isohexyl group is fixed to the right; (E)-20(22)-dehydrocholesterol, in which C-23 is oriented away from the nucleus; and 20-epicholesterol. Moreover, when the isohexyl group was fixed to the left in (Z)-17(20)-dehydrocholesterol, this dietary sterol, identified in the liver, caused not only a depression in the conversion of both mevalonate and squalene into sterols. The incorporation of acetate into fatty acids was not depressed, nor did the (Z)-sterol appear to have a generalized effect on membranous enzymes, because the activity of glucose-6-phosphatase was unaffected. Thus, feedback inhibition was retained when the stereochemistry of cholesterol's side chain was drastically changed and even after the nearly complete removal of the side chain. This implies that the side chain is only minimally recognized by the mechanisms involved in feedback inhibition.
Requirement for a membrane potential for cellulose synthesis in intact cells of
Acetobacter xylinum
Authors: Delmer Deborah P. , Benziman Moshe , Padan Etana
Published: May 2006

The marked lability in cell-free preparations of the enzyme system involved in cellulose biosynthesis in most organisms studied led us to investigate factors responsible for loss of activity on cellular disruption. Previous studies have led to the suggestion that the existence of a transmembrane electrical potential (ΔΨ) may be one factor responsible for maintaining an active system in intact cells. In this report, we show that dissipation of the ΔΨ in metabolizing cells of
Acetobacter xylinum
results in severe inhibition of cellulose synthesis. The effect can be reversed by restoration of the ΔΨ. Inhibition of cellulose biosynthesis by dissipation of the ΔΨ can be observed under conditions in which no substantial impairment of energy metabolism occurs—i.e., under conditions in which a transmembrane pH gradient is of sufficient magnitude to maintain an adequate overall protonmotive force across the membrane. The inhibition of cellulose biosynthesis is specifically related to changes in the ΔΨ, since the process can proceed normally in the absence of the pH gradient. These results support the suggestion that loss of the ΔΨ on cellular disruption may be one of the factors responsible for the low capacity for cellulose synthesis in isolated membrane preparations and also raise the possibility that modulation of the ΔΨ could be one means of regulating the rate of cellulose synthesis
in vivo
.
Karyophilic proteins: polypeptides synthesized in vitro accumulate in the nucleus on microinjection into the cytoplasm of amphibian oocytes.
Authors: Dabauvalle M C , Franke W W
Published: May 2006

The specific nucleocytoplasmic compartmentalization of proteins has been examined for some major soluble acidic nuclear proteins in oocytes of different amphibia. Proteins synthesized and radioactively labeled by translation in vitro, by using mRNA from ovaries of the frog Xenopus laevis, were injected into the cytoplasm of living oocytes of Xenopus or of the salamander Pleurodeles waltlii. At various times after injection, nucleus and cytoplasm were manually separated and endogenous and injected proteins were analyzed by two-dimensional gel electrophoresis. We show that several major nucleus-specific proteins of different sizes and electrical charges, including the very acidic proteins N1 and N2 (Mr, 110,000 and 100,000) and N4 (Mr, 34,000), are identical in both forms--i.e., as translation products in vitro and as present in the nucleoplasm. We conclude that significantly different cytoplasmic precursor forms to these nuclear proteins do not exist. The experiments indicate that (i) the translation products contain the signal(s) directing the specific sequestration of these proteins within the nucleus, (ii) post-translational processing is not required for the accumulation of these proteins in the nucleoplasm, and (iii) the signals and the mechanisms involved are not species specific.
Modulation of synovial cell products by a factor from a human cell line: T lymphocyte induction of a mononuclear cell factor.
Authors: Amento E P , Kurnick J T , Epstein A , Krane S M
Published: May 2006

A human cell line (U937) can be stimulated to produce a soluble factor (MCF) by either lectin-activated T lymphocytes or their soluble products. In prior studies, we showed that MCF produced by peripheral blood mononuclear cells can increase production of collagenase and prostaglandin E2 by adherent synovial cells obtained from enzymatically dispersed rheumatoid synovium. We show here that peripheral blood T lymphocytes or cloned human T lymphocyte lines are capable of inducing MCF production by the monocyte-like U937 cells. MCF can be demonstrated in the supernatant fluid from cocultures of U937 cells and T lymphocytes that have been stimulated with phytohemagglutinin or concanavalin A for 24-48 hr. In addition, the supernatant fluid from 24-hr lectin-stimulated T lymphocytes can be transferred onto the U937 cells and subsequently, MCF activity can be recovered from the U937 culture medium. The activity of the soluble T cell product on the U937 cells is both time- and dose-dependent. A human cell line capable of MCF production in continuous culture has not been previously available. The use of a monocyte-like cell line (U937) and cloned T lymphocytes now makes it possible to demonstrate the role of discrete cell populations in the production of MCF and other mediators.
Magnetic characterization of the primary state of bacterial photosynthesis
Authors: Norris J. R. , Bowman M. K. , Budil D. E. , Tang J. , Wraight C. A. , Closs G. L.
Published: May 2006

The results of reaction yield-detected magnetic resonance (RYDMR) experiments carried out on modified bacterial photosynthetic reaction centers (RCs) are interpreted in terms of a model that assigns the initial charge-separated radical ion-pair state,
P
F
, as the carrier of the spectrum. The radical pair theory, which has been invoked to explain magnetic field effects in RCs, was significantly expanded to take into consideration the electron dipole-dipole interaction. It is shown that this is the largest interaction between the components of the radical ion pair. Quantum statistical calculations are described simulating the RYDMR spectra and low-field effects in quinone-depleted RCs. The experimental data on which the simulations are based are (
i
) the magnitude of the field effect at 3,000 G, (
ii
) the field at which 0.5 of the maximal field effect is observed, (
iii
) the
P
F
population as a function of time at zero magnetic field, (
iv
) the RYDMR linewidth for low microwave field strength, (
v
) the RYDMR intensity and width as a function of microwave field, and (
vi
) the maximum RYDMR intensity at
H
I
≃ 2ǀ
J
ǀ. With this information it was found possible to characterize
P
F
in terms of four parameters, two containing structural information and two with kinetic implications. These are the dipole-dipole interaction,
D
= -47 ± 10 × 10
-4
cm
-1
; the exchange interaction,
J
= -7.5 ± 1.9 × 10
-4
cm
-1
; and the inverse rate constants of the decay of the radical pair states with singlet and triplet spin functions, respectively,
k
S
-1
= 15 ± 4 nsec and
k
T
-1
= 1.8 ± 0.2 nsec. The structural and dynamic implications of these parameters are discussed.
The pathogenic antigen of Heymann nephritis is a membrane glycoprotein of the renal proximal tubule brush border.
Authors: Kerjaschki D , Farquhar M G
Published: May 2006

Purified brush border fractions prepared from rat kidneys were solubilized in detergent, iodinated, and subjected to immunoprecipitation to identify the pathogenic antigen present in brush border membranes that is responsible for the production of Heymann nephritis (HN). Purified IgG prepared from the sera of rabbits or rats immunized with a crude cortical preparation, known as Fx1A, precipitated multiple peptides, whereas IgG eluted from glomeruli of rats with active or passive HN specifically immunoprecipitated a single large glycoprotein (Mr = 330,000). This protein (gp330) was subsequently purified by gel filtration and lentil lectin affinity chromatography from detergent-solubilized brush border membranes. When rats were immunized with purified gp330, they developed anti-brush border antibodies and active HN. IgG prepared from the serum of rats with active HN caused passive HN when injected into normal recipients. Rats immunized against brush border membrane proteins depleted of gp330 developed anti-brush border antibodies but did not develop HN. Further analysis of gp330 indicated that it is solubilized by detergent treatment of isolated brush border microvilli, and its antigenic component is released from intact microvilli by trypsin. By immunoperoxidase staining it was localized to the luminal side of the brush border membranes. These results indicate that (i) gp330 is the pathogenic antigen of HN; (ii) the antigen is a glycoprotein of the brush border membrane; and (iii) it is disposed with its pathogenic domain(s) facing the tubule lumen.
IgE immune complexes stimulate arachidonic acid release by mouse peritoneal macrophages.
Authors: Rouzer C A , Scott W A , Hamill A L , Liu F T , Katz D H , Cohn Z A
Published: May 2006

Resident mouse peritoneal macrophages were labeled with [3H]arachidonic acid and challenged with Sephadex beads coated with immune complexes of IgE and antigen. Arachidonic acid release by the cells was assessed by the quantity or radiolabel recovered from the culture medium. Freshly isolated macrophages responded to IgE immune complexes with a release of [3H]arachidonic acid that was linear for 1-2 hr. The magnitude of the response was dependent on both the number of immune complex-coated beads and on the degree of opsonization of the beads. Under conditions of maximal stimulation, macrophages challenged with IgE immune complex-coated Sephadex released 23 +/- 4.5% of their incorporated radiolabel. This is compared to values of 34.2 +/- 0.5% and 38.1 +/- 3.3% for cultures that received IgG immune complex-coated Sephadex or zymosan, respectively. Macrophages did not release arachidonic acid upon exposure to soluble IgE and antigen given sequentially or simultaneously, and soluble IgE did not inhibit the cells' response to IgE immune complexes. Incubation of macrophages for longer than 3 hr prior to challenge resulted in a selective loss in the cells' ability to respond to IgE immune complexes. After 16 hr of culture, macrophages released only 3.9 +/- 0.3% of their incorporated 3H on exposure to IgE immune complexes; however radiolabel release in response to zymosan (42.0 +/- 0.8%) was identical to that of freshly isolated cells. These data indicate that macrophages are capable of releasing arachidonic acid in response to preformed particulate immune complexes of IgE and antigen. Because Sephadex beads are too large to be interiorized by the cells, this response results from the interaction of the immune complexes with the macrophage plasma membrane.
Synthesis and secretion of alpha 2-plasmin inhibitor by established human liver cell lines.
Authors: Saito H , Goodnough L T , Knowles B B , Aden D P
Published: May 2006

The site of synthesis of alpha 2-plasmin inhibitor (alpha 2-PI), a physiologic inhibitor of plasmin, is not known with certainty. We have studied the production and secretion of alpha 2-PI by three established human liver cell lines derived from hepatocellular carcinoma and hepatoblastoma (Hep G2, Hep 3B, and PLC/PRF/5). As measured by a specific radioimmunoassay, the titer of alpha 2-PI increased in the medium of Hep G2 and Hep 3B cells with time, but no significant amount of alpha 2-PI was found in the medium of PLC/PRF/5. There was no evidence for a significant intracellular pool of this protein. On immunodiffusion against anti-alpha 2-PI serum, alpha 2-PI secreted by Hep G2 (G2 alpha 2-PI) formed a simple precipitin line of complete identity with the alpha 2-PI present in plasma (plasma alpha 2-PI). G2 alpha 2-PI behaved similarly to plasma alpha 2-PI in Sephadex G-150 gel filtration, sucrose density-gradient centrifugation, and crossed immunoelectrophoresis. G2 alpha 2-PI inhibited plasmin activity instantaneously in a functional assay and formed a complex with plasmin demonstrable by crossed immunoelectrophoresis. De novo synthesis of alpha 2-PI was shown by the presence of specific immunoprecipitable radioactivity in the medium after 5 hr of labeling of the cells with [35S]methionine. Analysis of the immunoprecipitates by NaDodSO4/polyacrylamide gel electrophoresis showed a single peak of radioactivity corresponding to Mr 68,000. These results indicate that the liver is a site of alpha 2-PI production.
Biosynthesis of aromatic compounds: 13C NMR spectroscopy of whole Escherichia coli cells.
Authors: Ogino T , Garner C , Markley J L , Herrmann K M
Published: May 2006

13C and 31P NMR spectra of wild-type Escherichia coli showed resonances from metabolic intermediates of glycolysis and ATP formation but no detectable signals from aromatic amino acids. However, tyrosine biosynthesis from D-[l-13C]glucose was observed in cells harboring a feedback-resistant allele of aroF, the gene encoding tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC4.1.2.15], one of the isoenzymes that control carbon flow through the common aromatic biosynthetic pathway. A similar accumulation of tyrosine and phenylalanine is seen in cells carrying a multiple-copy plasmid that carries a wild-type aroF allele in addition to pheA and tyrA, the structural genes for controlling enzymes of the terminal pathways to phenylalanine and tyrosine biosynthesis. These in vivo measurements by a noninvasive probe suggest feedback inhibition as the quantitatively major mechanism controlling carbon flow in the common aromatic compound biosynthetic pathway. In strains accumulating aromatic amino acids, a transient accumulation of trehalose was detected, indicating that previously unknown changes in Escherichia coli metabolism accompany overproduction of aromatic compounds.
Tissue-specific regulation of gene function: Presetting and erasure
Authors: Schwartz Drew
Published: May 2006

Evidence is presented for presetting and erasure at the
P
locus of maize. The presence of an
Mp
transposable controlling element in the

P
vv

allele affects the presetting and thus the regulation of gene function in different tissues.
The genome of frog virus 3, an animal DNA virus, is circularly permuted and terminally redundant.
Authors: Goorha R , Murti K G
Published: May 2006

We examined the structure of the frog virus 3 (FV 3) genome by using electron microscopic and biochemical techniques. The linear FV 3 DNA molecules (Mr approximately 100 x 10(6) formed circles when partially degraded with bacteriophage lambda 5'-exonuclease and annealed, but not when the annealing was done without prior exonuclease digestion. The results suggest that the DNA molecules contain direct terminal repeats. The repeated region composed about 4% of the genome. Complete denaturation of native FV 3 DNA molecules followed by renaturation produced duplex circles each bearing two single-stranded tails at different points along the circumference. The tails presumably represent the terminal repeats. The formation of duplex circles suggests that the FV 3 genome is circularly permuted. This is further borne out by (i) failure to identify a specific restriction endonuclease fragment containing the label when the molecular ends were radiolabeled by using the polynucleotide kinase procedure, and (ii) similarity in the restriction patterns of virion DNA and large concatemeric replicating viral DNA as revealed by endonucleolytic cleavage of both DNAs with HindIII. From the above data, we conclude that the FV3 genome is both circularly permuted and terminally redundant--unique features for an animal virus.
Phosphorylation of synthetic peptides by a tyrosine protein kinase from the particulate fraction of a lymphoma cell line.
Authors: Casnellie J E , Harrison M L , Pike L J , Hellström K E , Krebs E G
Published: May 2006

The particulate fraction from a lymphoma cell line, LSTRA, was found to contain an apparent high level of tyrosine protein kinase activity. When this fraction was incubated with [gamma-32P]ATP in the presence of 10 mM MnCl2, hydrolyzed, and assayed, 70--80% of the radioactivity recovered in phosphoamino acids was in phosphotyrosine. Gel electrophoresis of the proteins showed that a large portion of the 32P was in a single protein with a molecular weight of approximately 58,000. The phosphorylated residue in this protein was identified as phosphotyrosine. Detergent extracts of the particulate fraction from LSTRA cells contained both the Mr 58,000 protein and the enzyme responsible for its phosphorylation. These extracts were found to catalyze the phosphorylation of the tyrosine residue in the synthetic peptide, Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly, corresponding to the sequence around the tyrosine that is phosphorylated in pp60src; the Km for the peptide in this reaction was 5 mM. High-performance liquid chromatography was used to assay for this phosphorylation. A second peptide was synthesized that contained two additional arginine residues whose presence permitted the phosphorylation of the peptide to be measured by a simple assay using phosphocellulose paper. The Km for this peptide was 3--4 mM, indicating that the presence of the additional arginine residues did not alter the apparent affinity of the kinase for the peptide.
Regulation of actin-activated ATP hydrolysis by arterial myosin.
Authors: Chacko S , Rosenfeld A
Published: May 2006

Myosin was isolated from the main pulmonary artery of swine and was phosphorylated or dephosphorylated by utilizing the endogenous kinase or phosphatase, respectively. The myosins, phosphorylated to various degrees, were purified free of kinase and phosphatase activities by gel filtration on Sepharose CL-4B agarose columns. The level of actin-activated ATPase activity was dependent upon the degree of myosin light chain phosphorylation. Fully phosphorylated myosin reconstituted with actin and tropomyosin (actin/tropomyosin = 61:1) had the highest ATPase activity (0.1 mumol of Pi/mg . min). The actin-activated ATPase activity showed maximal (60--65%) Ca2+ sensitivity at 2 mol of Ca2+ bound per mol of myosin. The actin-activated ATPase activity, Ca2+ binding, and Ca2+ sensitivity of arterial myosin were also dependent upon Mg2+ concentration. The ATPase activity was maximal at 2--3 mM Mg2+ and, at low (0.5 mM) Mg2+ concentration, the activity was only one-third of the maximal activity. Increasing the Mg2+ above 3 mM was not associated with a further increase in ATPase activity, but the Ca2+ binding and Ca2+ sensitivity decreased with increasing Mg2+ concentration. The maximal Ca2+ sensitivity was observed at 2--3 mM Mg2+, a concentration at which the myosin bound 2 mol of Ca2+/mol. Both the ATPase activity and the Ca2+ sensitivity were more remarkable when actin that contained tropomyosin was used to activate the ATPase activity. The data indicate that calcium regulates the actin-activated ATP hydrolysis not only by its effects on the phosphorylation system but also by direct binding to the myosin.
Regulation of 6-phosphofructo-2-kinase activity by cyclic AMP-dependent phosphorylation.
Authors: el-Maghrabi M R , Claus T H , Pilkis J , Pilkis S J
Published: May 2006

Addition of glucagon to isolated rat hepatocytes resulted in inhibition of 6-phosphofructo-2-kinase (ATP:D-fructose-6-phosphate-2-phosphotransferase) activity in extracts of the cells and in a decrease in the intracellular level of fructose 2,6-bisphosphate. The effect on 6-phosphofructo-2-kinase was characterized by a decrease in the affinity of the enzyme for fructose 6-phosphate. To investigate the mechanism of action of glucagon, 6-phosphofructo-2-kinase from rat liver was partially purified by polyethylene glycol precipitation, DEAE-cellulose chromatography, (NH4)2SO4 fractionation, Sephacryl S-200 gel filtration, DEAE-Sephadex chromatography, and Sephadex G-100 gel filtration. Incubation of the purified enzyme with the catalytic subunit of the cyclic AMP-dependent protein kinase from rat liver and [gamma-32P]ATP resulted in 32P incorporation into a protein with a subunit Mr of 49,000 as determined by NaDodSO4 disc gel electrophoresis. Associated with this phosphorylation was an inhibition of 6-phosphofructo-2-kinase activity that was also characterized by a decrease in the affinity of the enzyme for fructose-6-phosphate. Both the phosphorylation and the inhibition of the purified 6-phosphofructo-2-kinase were blocked by addition of the heat-stable protein kinase inhibitor. It is concluded that the glucagon-induced decrease in fructose 2,6-bisphosphate levels observed in isolated hepatocytes is due, at least in part, to cyclic AMP-dependent phosphorylation and inhibition of 6-phosphofructo-2-kinase.
Isolation of an altered form of DNA polymerase I from Escherichia coli cells induced for recA/lexA functions.
Authors: Lackey D , Krauss S W , Linn S
Published: May 2006

A novel form of DNA polymerase I (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, DNA nucleotidyltransferase, EC 2.7.7.7) activity has been isolated from Escherichia coli cells that had been activated for expression of the DNA damage-inducible genes. Induction was by treatment of normal cells or cells carrying the spr-51 and tif-1 mutations with nalidixic acid. This activity, DNA polymerase I, seems to be a form of DNA polymerase I because it is insensitive to N-ethylmaleimide, is inhibited by antibody to DNA polymerase I, and does not appear in a polA1 strain. DNA polymerase I activity sediments through sucrose gradients as a broad peak with s20.w = 6.6--10.5, compared with an s20,w = 4.8--5.5 for DNA polymerase I. The fidelity during polymerization reactions of DNA polymerase I is relatively low with a variety of synthetic templates and deoxynucleoside triphosphates, although the enzyme appears to have a normal level of 3' greater than 5' exonuclease. Polymerase I has properties that might implicate it in some form of mutagenic DNA repair.
Internal pH of isolated newly formed and aged neurohypophysial granules.
Authors: Scherman D , Nordmann J J
Published: May 2006

The pH gradient (delta pH) across the membrane of bovine neurohypophysial granules was estimated by using the [14C]methylamine partition technique. The granule membrane showed high permeability to sugars such as sucrose or sorbitol and to cations. Granules suspended in sucrose medium showed an acidic internal delta pH that decreased with external pH from 1.0 pH unit at pH 7.5 to 0 at pH 4.5. High ionic strength of the external medium destroyed the delta pH, indicating that it originated from a Donnan equilibrium. In a medium constituted to simulate cytoplasm, the delta pH was approximately 0.5 pH unit. Aged neurosecretory granules were more acidic inside than were newly formed granules. The results are discussed in relationship to the nature and the duration of maturation and degradation process of the granule matrix.
Isolation of transforming DNA by cosmid rescue.
Authors: Lund T , Grosveld F G , Flavell R A
Published: May 2006

A procedure has been developed to allow the recovery of an integrated plasmid genome from a transformed cell, together with large areas of the flanking DNA sequences. DNA from Saccharomyces cerevisiae BAS2, in which the pBR322--ura 3 plasmid (Y1p5) is integrated at the yeast histone H2A and H2B locus, was used to generate a cosmid library, using a new cosmid vector (pTL5) that is ampicillin sensitive and tetracycline resistant. Colonies were selected for ampicillin resistance, which was conferred by the incorporation of the integrated pBR322 beta-lactamase gene into the recombinant cosmid. Restriction enzyme and blot hybridization analyses show that the rescued clones contain the yeast histone genes in addition to the Y1p5 sequences; a total of approximately 50 kilobase pairs of DNA sequences flanking the plasmid was recovered as a series of overlapping cosmids. This approach should allow the recovery of most genes that can be linked to a marker pBR322 sequence and for which a specific phenotype can be selected in a recipient eukaryotic cell.
Role of DNA repair in mutagenesis of Chinese hamster ovary cells by 7-bromomethylbenz[a]anthracene.
Authors: Thompson L H , Brookman K W , Carrano A V , Dillehay L E
Published: May 2006

The role of DNA repair in mutagenesis was studied in normal, repair-proficient Chinese hamster ovary cells and in two mutant strains that are deficient in excision repair. By using the mutagen 7-bromomethylbenz[a]anthracene (7-BrMeBA) and the technique of alkaline elution of DNA, the mutants were found to be defective at or before the incision step of excision repair. Dose--responses were determined for cell killing, mutation induction at three loci, and sister chromatid exchanges over a survival range of 1.0--0.1 after 7-BrMeBA treatment. The mutants were 5-fold more sensitive to killing than were the normal cells, but the degree of hypersensitivity to mutation induction varied depending on the mutant strain, the genetic marker, and the dose of mutagen. In each instance, the dose--response curve for mutations was essentially linear in the repair-deficient cells. In the normal cells, however, the curves for induced resistance to thioguanine and azaadenine were complex and were curvilinear with increasing slope at low doses. This behavior may be attributable to saturation of the excision repair system. No difference was seen in the efficiency of inducing ouabain-resistant mutations in the repair-deficient cells compared to the normal cells, indicating a qualitatively different behavior of this marker. These results are consistent with excision repair of 7-BrMeBA damage being error-free in Chinese hamster ovary cells. Sister chromatid exchange, another manifestation of DNA damage, also was induced with greater efficiency in the repair-deficient cells.
Receptor-mediated endocytosis of transferrin and the uptake of fe in K562 cells: identification of a nonlysosomal acidic compartment.
Authors: van Renswoude J , Bridges K R , Harford J B , Klausner R D
Published: May 2006

At physiological temperature, the Fe-carrier transferrin is taken up by K562 human erythroleukemia cells through receptor-mediated endocytosis. Both ligand (now minus Fe) and receptor recycle back to the cell surface where the receptor is rapidly reutilized. After endocytosis, transferrin becomes transiently lodged within an acidic compartment inside the cell, as judged by the changed spectral characteristics and quantum yield of fluorescein isothiocyanate-labeled transferrin that is cell-associated at 37 degrees C. Upon binding to transferrin, anti-fluorescein antibody strongly quenches the emission of the fluorescein-labeled residues on the protein and is used to assess whether the transferrin is at the cell surface (incubation at 0 degrees C) or mainly internalized into the cell (incubation at 37 degrees C). Using Percoll gradient fractionation of postnuclear supernatants, we show that the acidic compartment is not the lysosomal compartment.
Specific interactions of Saccharomyces cerevisiae proteins with a promoter region of eukaryotic tRNA genes.
Authors: Klemenz R , Stillman D J , Geiduschek E P
Published: May 2006

The specific binding of one or several Saccharomyces cerevisiae proteins to a segment of genes that code for different yeast tRNAs has been demonstrated with the use of the DNase I-protection "footprint" assay of Galas and Schmitz. The analyzed binding occurs near the 3' ends of the genes and is centered on an 11-base-pair DNA sequence that has been well conserved among eukaryotic tRNA genes. Others have shown the involvement of this sequence in initiating the transcription of tRNA genes by RNA polymerase III. The adenovirus gene that codes for VAI RNA also contains this conserved sequence element, and we detect binding of yeast protein(s) to this gene. Competition experiments show that a common set of proteins binds to different tRNA genes. The DNA-protein complex is quite stable at 20 degrees C and low ionic strength.
Purification and characterization of epidermal growth factor receptor/protein kinase from normal mouse liver.
Authors: Cohen S , Fava R A , Sawyer S T
Published: May 2006

We have purified the epidermal growth factor (EGF) receptor/protein kinase from the livers of normal mice by affinity chromatography. The biochemical properties of the liver receptor are very similar to those of the EGF receptor previously prepared from the human tumor cell line A-431 [Cohen, S., Ushiro, H., Stoscheck, C. & Chinkers, M. (1982) J. Biol. Chem. 257, 1523-1531]. The liver receptor for EGF is a glycoprotein of Mr 170,000. It binds 125I-labeled EGF and possesses an EGF-stimulable protein kinase activity specific for tyrosine residues. Both autophosphorylation and kinase activity toward exogenous substrates are demonstrable. The EGF receptor purified from normal mouse liver is antigenically related to the receptor purified from human A-431 cells.
Distance from cohesive end site cos determines the replication requirement for recombination in phage lambda.
Authors: Stahl F W , Kobayashi I , Stahl M M
Published: May 2006

Previous work showed that crossing-over in the middle of the chromosome of phage lambda requires more DNA replication than does crossing-over near the termini. Relocation of cos, the sequence that determines the lambda termini, alters the requirements for replication in a given marked interval, demonstrating that distance from cos determines the amount of DNA replication that is required for genetic exchange. This result supports a break and copy mechanism for recombination mediated by the red system of phage lambda.
Possible mechanism for donor DNA binding and transport in Haemophilus.
Authors: Kahn M E , Maul G , Goodgal S H
Published: May 2006

Morphological differences were observed in competent and noncompetent Haemophilus parainfluenzae and Haemophilus influenzae when thin sections of these cells were examined by electron microscopy. The membranous extensions present on the surface of competent H. parainfluenzae cells disappeared on treatment with transforming DNA, while vacuole-like structures appeared in the periplasm. Noncompetent cells had 1/5th as many extensions on their surface as competent cells, and no vacuoles were observed after treatment with homologous DNA. Competent cells treated with radiolabeled DNA were disrupted and the clarified lysate was centrifuged on CsCl density gradients. Material having a density of 1.34 g/ml was found to contain the majority of the DNase-resistant radioactive DNA recovered from the bacteria and was shown by electron microscopy to be composed of membrane vesicles. The polypeptide composition of this dense membrane fraction was similar to that of H. parainfluenzae outer membrane.
Appropriate constraints for variational optimization of electronic density matrices and electron densities
Authors: Payne Philip W.
Published: May 2006

Euler equations characteristic of the electronic reduced density matrix have previously been obtained by minimizing an energy functional subject to trace constraints 〈

P
K

〉 =
N
, in which
N
is the number of particles. It is shown that these constraints are inadequate to exclude variations that violate
N
-representability conditions. However, a first-order reduced density matrix that does satisfy
N
-representability conditions is formulated as a functional of the orbital densities through use of angular momentum recoupling. Modified variational constraints are proposed.
Expression of abbreviated mouse dihydrofolate reductase genes in cultured hamster cells.
Authors: Gasser C S , Simonsen C C , Schilling J W , Schimke R T
Published: May 2006

We have constructed a number of abbreviated dihydrofolate reductase (DHFR) genes by using cloned mouse genomic and cDNA sequences. These genes contain 1.0 kilobase of 5' flanking genomic sequence and varying portions of the 3' non-coding region. Two of the genes contain the first two introns of the DHFR gene; the other three lack introns. Transfection of DHFR-deficient Chinese hamster ovary cells with any of these constructed genes results in cells with the DHFR+ phenotype. Treatment of the transfectants with methotrexate, a folate antagonist, leads to the emergence of methotrexate-resistant colonies which have amplified the transfected genes.
Ectopic germ cells: natural model for the study of germ cell sexual differentiation.
Authors: Upadhyay S , Zamboni L
Published: May 2006

In the course of a study on the morphogenesis of the adrenal gland in random-bred Swiss albino mice, we noted the presence of ectopic germ cells in the adrenal cortexes and medullas in animals of both sexes, from day 12 1/2 of fetal development to postnatal day 12. Up to day 15 of fetal development, the cells exhibited characteristics of primordial germ cells. At day 17, and irrespective of the sex of the fetus, they all entered meiosis in synchrony with those in the ovary. Postnatally, in females as well as males, all ectopic germ cells displayed morphologic characteristics identical to those of young oocytes in unilaminar ovarian follicles. No germinal elements were seen in the adrenal glands past day 12 of life. Our study shows that mammalian germ cells are capable of undergoing sustained differentiation outside the gonads and that, in ectopic sites, they all differentiate into oocytes as they normally would in the ovary, even in males.