bluuebunny/crossref_metadata_2025 huggingface dataset:
Late replication in an X-autosome translocation in the mouse: correlation with genetic inactivation and evidence for selective effects during embryogenesis.
Authors: Disteche C M , Eicher E M , Latt S A
Published: May 2006

A technique involving 5-bromodeoxyuridine, 33258 Hoechst, and fluorescence microscopy has been used to analyze replication kinetics in cells from embryonic and adult mice bearing the Cattanach [T(X;7)ICt] translocation in a balanced or an unbalanced form. In balanced 9- and 13-day female embryos, the translocated X was late replicating in 28 and 22% of the cells, respectively, whereas it was late replicating in only 13% of adult cells. In contrast, in unbalanced females, the translocated X was late replicating in 62 and 70% of 9- and 13-day embryos and in 70% of adult cells. Such divergent late replication frequencies suggest the operation, during development, of selection against cells with extreme genetic imbalance. Within a late-replicating translocated X chromosome, the autosomal segment itself replicated late approximately half of the time, regardless of karyotypic balance. The late replication data are consistent with the measurements of levels of mitochondrial malic enzyme (MOD-2, whose locus is on the autosomal segment) activity in these mice [Eicher E. & Coleman, D. (1977) Genetics 85, 647-658]. The present study also shows a dissociation between the replication timing in X chromatin distal and proximal to the autosomal segment, supporting the hypothesis of at least two inactivation centers in the X chromosome.
RNA ligase reaction products in plasmolyzed Escherichia coli cells infected by T4 bacteriophage.
Authors: David M , Vekstein R , Kaufmann G
Published: May 2006

Searching for a physiological role of T4 RNA ligase [polyribonucleotide synthetase (ATP); poly(ribonucleotide):poly(ribonucleotide) ligase (AMP-forming), EC 6.5.1.3] activity, we developed an acellular system of plasmolyzed Escherichia coli cells infected by T4 bacteriophage. Upon incubation of this system with [gamma-32P]ATP, 32P was transferred into a large number of polyribonucleotides, mostly up to 300-400 residues long. The bulk of 32P in the product polyribonucleotides was found in 5'-terminal phosphate groups, suggesting that they originated by a phosphorylation reaction catalyzed by the endogenous polynucleotide kinase (EC 2.7.1.78). Indeed, these products were not seen in an acellular system from uninfected cells, and their amount and complexity increased with the progress of infection. Analysis of the 32P-labeled polyribonucleotide products by gel electrophoresis, either before or after digestion with alkaline phosphatase (EC 3.1.3.1), revealed that a small fraction of the 32P resided in phosphodiester bonds of several tRNA-sized chains. This specific 32P transfer from [gamma-32P]ATP into phosphodiester bonds was apparently catalyzed by successive polynucleotide kinase and RNA ligase reactions. The possible relationship of the 32P transfer to RNA ligase was investigated next by using a system from cells infected with T4 am M69 (an amber mutant deficient in RNA ligase). Transfer of 32P from [gamma-32P]ATP into phosphodiester bonds was not detected in the am M69 system. However, addition of purified RNA ligase to the am M69 system restored the specific 32P transfer. A system from cells infected with T4 psu-b delta 33 (a deletion mutant lacking the entire tRNA region) sustained the specific 32P transfer into tRNA-sized products, indicating that they were not derived from transcripts of T4 tRNA genes. These data may reflect a role of RNA ligase in posttranscriptional conversion of presumably host polyribonucleotides into novel tRNA species during T4 infection.
Molecular cloning of human epsilon-globin gene.
Authors: Proudfoot N J , Baralle F E
Published: May 2006

Human beta-like globin genes were investigated by use of rabbit beta-globin cDNA plasmid as a cross-species hybridization probe. Normal and beta 0/delta beta 0 thalassemic DNA were compared by filter hybridization procedures. It proved possible to demonstrate that the rabbit probe detected G gamma, A gamma, delta, beta, beta 0, and delta beta 0 human globin genes as well as an additional unidentified beta-like globin gene. By use of an agarose gel elution procedure, fractions of HindIII-digested DNA enriched for beta-like globin genes were purified. One of these fractions, 8.0 kilobases in size, was clonedinto lambda 788, and EK2 lambda HindIII vector. A positive clone was obtained and characterized by restriction mapping and sequence analysis. The sequence data obtained predicted an amino acid sequence that exactly matches a part of human epsilon-globin. The human non-alpha-globin locus is now nearly complete. delta, beta, and gamma human globin genes have already been cloned and analyzed. We describe here the cloning of the remaining non-alpha-globin gene, epsilon.
Inactive mRNA-protein complexes from mouse sarcoma-180 ascites cells.
Authors: Geoghegan T , Cereghini S , Brawerman G
Published: May 2006

Mouse sarcoma ascites cells do not utilize fully their capacity for protein synthesis. A considerable portion of their ribosomes occur as inactive monomers. Also, a substantial amount of the cellular mRNA is in the form of ribonucleoprotein particles that sediment in the 20-70S range. This is indicated both by measurements of poly(A) content and by translation of the RNA in cell-free systems. The population of polypeptides synthesized under the direction of the RNA from these particles is less heterogeneous than that directed by RNA from polysomes. The mRNAs for some polypeptides are present predominantly in the small particles. Others are distributed to varying degrees between particles and polysomes. Incubation of the cells with cycloheximide drives most of the ribosomal monomers and a portion of the untranslated mRNA into polysomes. Some of the mRNAs that were predominantly in the inactive fraction seem to be refractory to this treatment. Particles released from polysomes in cells subjected to starvation are quite effective in promoting polypeptide synthesis in a reticulocyte cell-free system and cause the synthesis of a population of polypeptides similar to that coded by the polysomal RNA. The particles from cells exposed to cycloheximide are inactive but yield active RNA upon deproteinization. It is suggested that some mRNA species are maintained in an inactive state in the cell by a component of the nucleoprotein complex.
Estimation of membrane potentials of individual lymphocytes by flow cytometry.
Authors: Shapiro H M , Natale P J , Kamentsky L A
Published: May 2006

The membrane potentials of individual cells can be estimated by flow cytometric quantitation of the cells' uptake of the fluorescent lipophilic cationic dye 3,3'-dihexyloxacarbocyanine iodide. Human lymphocytes separated from peripheral blood on Hypaque-Ficoll gradients are uniformly depolarized by gramicidin and hyperpolarized by valinomycin. Concanavalin A and phytohemagglutinin depolarize only a fraction of the lymphocytes. The flow cytometric technique allows precise detection of heterogeneous membrane potential responses to stimuli such as lectins; it could also provide a basis for sorting cells that respond differently to a given stimulus.
Methylation of chloroplast DNAs in the life cycle of
Chlamydomonas
Authors: Royer Hans-Dieter , Sager Ruth
Published: May 2006

Methylation patterns of
Chlamydomonas
chloroplast DNAs (chlDNAs) were examined in the vegetative, gametic, and zygotic stages of the life cycle. Restriction endo-nuclease fragment patterns produced by
Eco
RI,
Bam
HI,
Hpa
II, and
Msp
I were compared; the last two cleave DNA at the sequence C-C-G-G, but
Hpa
II is blocked by prior methylation of the internal cytidine whereas
Msp
I is not. chlDNAs from vegetative cells of both mating types showed no evidence of methylation at C-C-G-G. Gametic
mt
+ chlDNA was heavily methylated at C-C-G-G, whereas the homologous chlDNA from
mt
- gametes showed very slight methylation at C-C-G-G. Methylation of additional sites in chlDNA from
mt
+ gametes but not from
mt
- gametes was shown by blockage of some
Eco
RI and
Bam
HI sites that were cleaved in the chlDNA from vegetative cells. chlDNA from 6-hr zygotes was much more methylated than gametic
mt
+ DNA, as shown by its almost total resistance to cleavage by all four restriction enzymes. These findings support and extend previous evidence that chlDNA of
mt
+ cells is methylated during gametogenesis and that further methylation occurs after gametic fusion in the young zygotes.
Gene expression of an Escherichia coli ribosomal RNA promoter fused to structural genes of the galactose operon.
Authors: Ota Y , Kikuchi A , Cashel M
Published: May 2006

The promoter region of the rrnB ribosomal RNA gene of Escherichia coli has been ligated within the epimerase gene (galE) of the galactose operon in a lambda phage vector. The recombinant lambda phage has been characterized by restriction mapping and assays of both galK (galactokinase) gene activity and galactose messenger RNA hybridization. In such lyosgens, expression of the fused galactose operon occurs as a function of growth rate in a manner characteristic of ribosomal RNA gene expression and is subject to stringent control by amino acid availability for protein synthesis. Galactose messenger RNA arising from the ribosomal promoter is not as metabolically stable as ribosomal RNA.
Crossreactive mixed lymphocyte reaction determinants recognized by cloned alloreactive T cells.
Authors: Fathman C G , Hengartner H
Published: May 2006

We have isolated clones of alloreactive T cells by soft agar cloning of primed responder cells contained in serially restimulated, mixed lymphocyte cultures. These clones recognize unique mixed lymphocyte reaction (MLR) stimulating determinants present on (C57B/6 X A/J)F1 (B6A) spleen cells. By assaying the reactivity patterns of these clones on a panel of stimulator cells, we have identified at least three separate "specificities" of alloreactive T cell clones. Several clones exhibiting one of these three reactivity patterns have been isolated on many occasions from different experiments. Using such clones of alloreactive T cells, we have identified and are attempting to define crossreactive MLR stimulating determinants exhibited by a panel of stimulator cells. Recognition by certain clones of a shared MLR determinant(s) on B6A and DBA/2 and of the lack of this determinant(s) on B6 stimulator cells questions current concepts concerning the nature of MLR stimulating determinants and T cell recognition of alloantigens.
Primary defect of insulin receptors in skin fibroblasts cultured from an infant with leprechaunism and insulin resistance.
Authors: Schilling E E , Rechler M M , Grunfeld C , Rosenberg A M
Published: May 2006

Insulin binding to insulin receptors, on skin fibroblasts established in culture from an infant with insulin resistance and clinical features of leprechaunism was markedly decreased in comparison with cultures from an age-matched control. By contrast, the binding of epidermal growth factor, a polypeptide growth factor chemically unrelated to insulin, to patient's and control fibroblasts was indistinguishable. The selective defect in insulin binding to patient's fibroblasts was reflected in an impaired ability of insulin to stimulate 2-deoxyglucose uptake. These results most likely indicate a primary genetic defect of insulin receptors.
Immunocytochemical localization, in synapses, of protein I, an endogenous substrate for protein kinases in mammalian brain.
Authors: Bloom F E , Ueda T , Battenberg E , Greengard P
Published: May 2006

Protein I is a principal endogenous substrate for cyclic AMP- and Ca2+-dependent protein kinases of mammalian brain. Antibodies raised against purified protein I have been used to localize this protein in the rat central nervous system. At the light microscope level, immunoreactivity was detected in punctate deposits in selected zones of synaptic termination. These deposits varied in density among brain regions. At the electron microscope level, immunoreactivity was observed at some but not all synaptic regions and was restricted to the perimeter of synaptic vesicles and to submembranous material in the postsynaptic neuron.
RETRACTED: DNA from recombinogenic lambda bacteriophages generated by arl mutant of Escherichia coli is cleaved by single-strand-specific endonuclease S1.
Authors: Hays J B , Korba B E
Published: May 2006

When propagated on arl strains (a subclass of Escherichia coli hyper-rec mutants), lambda "Red-" duplication phages accumulated an enhanced potential for recombination. The physical properties of the recombinogenic phages thus obtained ("Arl-" phages) were similar to those of phages grown on arl+ bacteria. However, Arl- phage DNA was cleaved by endonuclease S1 under conditions such that the nuclease is specific for single-stranded DNA;DNA from control phages was S1-resistant. The number of S1 sites (defined by the apparent decrease in single-strand molecular weight) reached a maximum (seven to nine sites per strand of lambda DNA) after five or six rounds of growth on arl bacteria. Similarly, the recombinogenicity of Arl- phages reached a limiting value (recombination frequency, 15%) that was 5 times that of Arl+ phages. Recombinogenicity and S1 susceptibility were accumulated concomitantly during growth on arl+ bacteria. If all increased recombination occurred at the S1 sites, then these regions (about 40 bases each) were about 300 times as recombinogenic as normal DNA regions of the same size, and 1.5 times as recombinogenic as UV-induced lesions. Chromosomal DNA and plasmid DNA (pBR322) from arl cells were more susceptible to nuclease S1 than was DNA from arl+ bacteria. Analysis of the cleavage products suggests that the S1 sites on Arl- lambda phage DNA are located randomly.
Effect of colicins Ia and E1 on ion permeability of liposomes.
Authors: Tokuda H , Konisky J
Published: May 2006

Colicins Ia and E1 are shown to inhibit the formation and bring about the collapse of a potassium diffusion potential imposed across the membrane of liposomes prepared from soybean or Escherichia coli phospholipids. Such depolarization results from a colicin-induced increase in membrane ion permeability. Colicins E2 and E3 do not depolarize such membranes. In addition to the colicin Ia-induced rapid efflux of preloaded rubidium, sodium, phosphate, or choline from liposomes, a slower efflux of preloaded sucrose or glucose 6-phosphate occurs. However, treated liposomes do not leak inulin or dextran, demonstrating that the effects of E1 and Ia are not due to a general disruption of membrane structure. The fact that colicin-induced ion efflux is observed in the complete absence of a membrane potential shows that the action of these colicins on liposomes is not voltage dependent. These results provide strong evidence that the depolarization of E. coli cells by colicins Ia and E1 results from a colicin-induced increase in membrane permeability to ions. It is proposed that this is brought about by the direct interaction of the colicin molecules with the bacterial cytoplasmic membrane.
Hormone-induced increase in levels of functional mRNA and α-amylase mRNA in barley aleurones
Authors: Muthukrishnan S. , Chandra G. R. , Maxwell Elizabeth S.
Published: May 2006

Incubation of barley aleurone cells with gibberellic acid produces a progressive increase in the RNA content of the cells. The activity of poly(A)-containing RNA, measured in an
in vitro
wheat germ protein-synthesizing system, reaches a maximum ≈12 hr after hormone addition and declines thereafter. The structurally intact functional mRNA content in these cells, measured as poly(A)-RNA with 5′ “caps,” also shows a maximum at 12 hr and correlates with the translational capacity of poly(A)-RNA. Activation of mRNA by guanylylation or methylation after addition of gibberellic acid is ruled out. Available evidence indicates that gibberellic acid stimulates protein synthesis by increasing the synthesis of mRNA. Studies with cycloheximide suggest that the induction of synthesis of α-amylase mRNA by gibberellic acid requires protein synthesis after hormone addition.
Effect of diethylpyrocarbonate on lactose/proton symport in Escherichia coli membrane vesicles.
Authors: Padan E , Patel L , Kaback H R
Published: May 2006

Exposure of Escherichia coli ML 308-225 membrane vesicles to the histidine-specific reagent diethylpyrocarbonate (DEPC) led to concentration- and time-dependent inactivation of active lactose transport, and the sensitivity of the system to inactivation was enhanced when an electrochemical proton gradient (delta- muH+, interior negative and alkaline) was generated across the vesicle membrane. Although beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside blocked DEPC inactivation, binding of p-nitrophenyl alpha-D-galactopyranoside was not significantly altered, indicating that DEPC does not react at the binding sites of the lac carrier protein. Strikingly, vesicles treated with DEPC exhibited an increased apparent Km for delta- muH+-driven lactose transport and counterflow but no change in the Vmax of these reactions and no change in the apparent Km or Vmax of facilitated diffusion. Moreover, DEPC treatment increased the apparent Km observed for delta- muH+-driven proline and D-lactate transport with no change in Vmax. Finally, the lactose counterflow activity of DEPC-treated vesicles was regenerated by subsequent exposure to hydroxylamine. It is suggested that a histidyl residue(s) in the lac carrier or another protein in the translocation complex is involved either in the binding and translocation of protons or in a conformational change that may occur upon protonation of the lac carrier protein.
Middle repetitive DNA: a fluid component of the Drosophila genome.
Authors: Young M W
Published: May 2006

Most of the middle repetitive DNA of Drosophila melanogaster appears to be organized into families of 10-100 repeated elements that are found at scattered locations in the chromosome arms and occupy new chromosomal positions as populations of D. melanogaster diverge. These "nomadic" DNA segments can be identified by an analysis of cDm plasmids, hybrids of ColE1 and segments of randomly sheared D. melanogaster DNA. Eighty cDm plasmids were withdrawn, at random, from a library of approximately 17,000 cDm clones. Fifty-seven of these seem to contain either DNA that is not repeated in the D. melanogaster genome or DNA that has a low repetition frequency. The remaining 23 cDm plasmids contain repetitive sequences. Seventeen of these 23 plasmids contain repetitive sequences that are demonstrably scattered to many chromosomal sites that can be mapped in two D. melanogaster strains, g-1 and g-X11. The repeated elements hybridizing with each of the different Dm segments are at quite different chromosomal locations in these two strains. However, the size of each family of repeated sequences remains fairly constant in both strains. It is proposed that the number of elements in each family has been fixed by selection.
Fluctuations in polarized fluorescence: evidence that muscle cross bridges rotate repetitively during contraction.
Authors: Borejdo J , Putnam S , Morales M F
Published: May 2006

Particular thiols of the myosin subfragment 1 moieties of single glycerinated muscle fibers are covalently labeled with rhodamine. By using appropriate solutions such fibers can be relaxed, be in rigor, or develop active isometric tension. The rhodamine is excited by polarized 514.5-nm laser light; the greater than 580-nm fluorescence is resolved into orthogonal components and the intensity of each is measured by a computer-interfaced photon counting system. Fluctuations over-and-above noise appear in steady-state activity but not in relaxation or rigor and not when the fluorophore is actin-attached instead of myosin-attached. Fluctuations also appear in ratios of polarized intensities--quantities sensitive to fluorophore attitude but not to fluorophore number. The fluctuations are dominated by low (approximately 2 Hz) frequencies similar to separately measured ATPase frequencies. The fluctuations are ascribed to repetitive motion of the cross bridges to which the rhodamine is attached.
Partial purification and characterization of DNA from the human X chromosome.
Authors: Schmeckpeper B J , Smith K D , Dorman B P , Ruddle F H , Talbot C C
Published: May 2006

Human X chromosome DNA was partially purified from a mouse-human hybrid cell line containing a single human chromosome, the X. Enrichment of such DNA was accomplished by two sequential reassociations of radiolabeled hybrid cell DNA with large excesses of mouse DNA. Unreassociated hybrid cell DNA was used as a probe for human X chromosome sequences. The human-specific fraction of probe DNA CONTAINED THREE COMPONENTS. Two of these reassociated to human DNAs at rates proportional to the number of X chromosomes present. These two components were thus localized to the X chromosome. One of these X-specific components, representing about 80% of human-specific probe DNA, consisted of single copy or very low order reiterated DNA. The second X-specific component, representing about 10% of human-specific probe DNA, was about 20-30 times more reiterated. The remaining 10% of human-specific probe DNA, although derived from the X chromosome, reassociated to human DNAs at a rate independent of the number of X chromosomes present. This component was thus homologous to autosomal as well as X chromosome DNA. The probe DNA accounts for approximately half of the human X chromosome, suggesting that the remainder may have homology with mouse DNA.
Regulation of mitochondrial malic enzyme synthesis in mouse brain.
Authors: Bernstine E G , Koh C , Lovelace C C
Published: May 2006

In a previous study [Bernstine, E.G. (1979) J. Biol. Chem. 254, 83-87] it was shown that inbred strains of mice fall into two classes based on the specific activity of mitochondrial malic enzyme [L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40] in brain. In this report we demonstrate differences between high- and low-activity strains in the development of enzyme activity levels in adult mice and show that the rate of enzyme synthesis quantitatively accounts for the inherited level of the brain enzyme. Genetic analysis has established that the locus controlling the amount of enzyme in brain (Mdr-1) is located on chromosome 7. Its linkage to Hbb and c places it in the same region of the chromosome as Mod-2, the structural gene for mitochondrial malic enzyme. By making use of deletions and a duplication that include Mod-2, evidence for cis action of Mdr-1 was obtained.
Operon-specific regulation of ribosomal protein synthesis in Escherichia coli.
Authors: Lindahl L , Zengel J M
Published: May 2006

We have cloned a DNA fragment harboring the genes for ribosomal proteins L2, L4, and L23 on a plasmid vector that contains a lac operator and promoter. The cloned ribosomal protein genes are now under the control of lacOP. Addition of a lac inducer to these cells results in a specific 5- to 10-fold increase in the synthesis of the proteins corresponding to the cloned genes. Within 10 min of this induction, the synthesis of ribosomal proteins S3, S19, L3, L16, L22, and L29 stops almost completely. The genes for all these proteins reside in the same chromosomal operon as L2, L4, and L23. We have seen no dramatic effect on the synthesis of any other ribosomal proteins. Thus, the induction of L2, L4, and L23 results in a specific and rapid decrease in the expression of all (or almost all) genes in their own transcription unit.
Absence of heme-localized strain in T state hemoglobin: insensitivity of heme-imidazole resonance Raman frequencies to quaternary structure.
Authors: Kincaid J , Stein P , Spiro T G
Published: May 2006

Substitution of pentadeuterated 2-methylimidazole in (2-methylimidazole)-Fe(II)-protoporphyrin IX, a model complex for deoxyHb, shifts three bands in the low-frequency resonance Raman spectrum 380 leads to 373 cm-1, 348 leads to 345 cm-1, and 220 leads to 218 cm-1. The first of these is assigned primarily to Fe-imidazole stretching, and the other two are assigned to porphyrin deformation modes with substantial Fe-pyrrole stretching contributions. The three bands are observed in deoxyHb and Mb. The Fe-pyrrole modes are at essentially the same frequencies in the two proteins, but the Fe-imidazole mode is 6 cm-1 lower in deoxyHb than Mb, implying a slight alteration in the heme-imidazole linkage. No change greater than 2 cm-1 is observed when Hb Kempsey is switched from the R to the T state. This observation places an upper limit on the energy stored in the Fe-imidazole bond of T state deoxyHb, which is estimated to be less than 0.2 kcal/mol (less than 836.8 J/mol).
Do cleavages of amides by serine proteases occur through a stepwise pathway involving tetrahedral intermediates?
Authors: Komiyama M , Bender M L
Published: May 2006

The mechanism of the serine protease-catalyzed cleavage of amides (acylation) was examined in terms of the basicity of the functional groups participating in the catalysis. It is proposed that the reaction does not proceed through a stepwise pathway, as opposed to the cleavage of esters and anilides, which start with general base-catalyzed formation of the tetrahedral intermediate followed by its general acid-catalyzed breakdown. Instead, the proton abstracted from the hydroxyl group of the serine by the imidazolyl group of the histidine is donated to the nitrogen atom of the leaving group of the amide before the bond between the carbonyl carbon atom of the amide and the attacking serine oxygen atom is completed. Reactions proceed by a SN2-like reaction through the cooperation of acid catalysis by the imidazolyl cation and nucleophilic attack by the serine. The mechanisms of the enzymatic hydrolyses of anilides and esters proceed through a discrete tetrahedral intermediate, but the enzymatic hydrolyses of amides probably do not.
Amino-acid sequence of Tetrahymena histone H4 differs from that of higher eukaryotes.
Authors: Glover C V , Gorovsky M A
Published: May 2006

A partial amino-acid sequence of Tetrahymena histone H4 has been determined and differs significantly from the sequence of calf or pea H4. The amino terminus of Tetrahymena H4, unlike that of other H4s so far examined, is not acetylated. Of 66 residues determined, one is a single-residue insertion, one a single-residue deletion, and thirteen are amino-acid replacements with respect to the calf thymus H4 sequence. Most of the amino-acid replacements are nonconservative and are distributed nonrandomly, with a strong concentration in the amino-terminal arm. The first four lysines are partially acetylated. All but two of the replacements can be explained by single nucleotide substitutions at the level of the gene. The similarity in sequence of calf and pea H4 coupled with the substantial differences displayed by Tetrahymena H4 suggest that the divergence of protozoa and higher eukaryotes substantially antedates the divergence of plants and animals. Furthermore, quantitative analysis of the data requires either that the rate of H4 evolution be considerably more rapid than previously thought or that the rate be different for different periods of evolution or for different lines of descent.
Opiates and enkephalins inhibit synthesis of gangliosides and membrane glycoproteins in mouse neuroblastoma cell line N4TG1.
Authors: Dawson G , McLawhon R , Miller R J
Published: May 2006

When mouse neuroblastoma clonal cell line N4TG1 cells were cultured in the presence of opiates or enkephalins, in the range 10(-6)-10(-10) M for 24 hr, a dose-dependent inhibition of the incorporation of [3H]glucosamine and [14C]-galactose into sialoglycosphingolipids and glycoproteins was observed. The gangliosides most affected comigrated in thinlayer chromatographic systems with GM2 (GalNAc[AcNeu]-Gal-Glc-ceramide), GM1 (Gal-GalNAc[AcNeu]Gal-Glc-ceramide), and GDla (AcNeu-Gal-GalNAc[AcNeu]Gal-Glc-ceramide). The effects were stereospecific and naloxone-reversible. Polyacrylamide gel electrophoresis revealed that the synthesis of a large number of membrane glycoproteins was also stereospecifically inhibited. Synthesis of other proteins and glycoproteins, proteoglycans, DNA, and membrane phospholipids and the rate of cell division were not altered in any specific or stereospecific manner. Moreover, clonal cell lines (neuroblastomas and oligodendroglioma) and human skin fibroblasts, which do not possess opiate receptors, did not respond to opiates or enkephalins in a stereospecific manner.
Parvovirus RNA transcripts containing sequences not present in mature mRNA: a method for isolation of putative mRNA precursor sequences.
Authors: Jay F T , de la Maza L M , Carter B J
Published: May 2006

We report here a method of RNA preparation that may enrich for precursor RNA sequences and the results of an investigation of adeno-associated virus (AAV) RNA transcription that used this method. Whole cells were lysed with detergent and high salt and separated into supernatant and pellet (crude chromatin) fractions. These fractions were then separately deproteinized by proteolytic digestion and phenol extractions. DNA was removed from the preparation by two cycles of pancreatic DNase digestion and phenol extraction. Hybridization analyses of the RNA obtained from AAV/adenovirus-infected KB (human) cells revealed some AAV-specific RNA sequences that were not present in the mature 20S mRNA. These additional sequences were contained in AAV RNA molecules present in the pellet fraction, whereas the 20S AAV mRNA accumulated in the supernatant. A species of AAV-specific RNA (about 22S), which was associated only with the pellet fraction and was labeled only after a short pulse, appeared to have a kinetic relationship with the more stable cytoplasmic 20S mRNA. These putative AAV mRNA "precursors" and precursor sequences were not observed previously when conventional methods were used to obtain RNA from either whole cells or isolated nuclei.
Integration and transcription of mouse mammary tumor virus DNA in rat hepatoma cells.
Authors: Ringold G M , Shank P R , Varmus H E , Ring J , Yamamoto K R
Published: May 2006

Rat hepatoma cells infected with mouse mammary tumor virus (MMTV) acquire viral DNA that becomes covalently linked to the cell DNA. Using restriction endonucleases and the DNA transfer procedure of Southern [Southern , E.M. (1975) J. Mol. Biol. 98, 503--517], we have studied the sites in cellular DNA into which MMTV DNA inserts. These experiments indicate that: (i) there are many sites in cell DNA into which MMTV DNA integrates; (ii) the junctions between viral and cellular DNA occur within a limited portion of the viral genome, (iii) clones that contain MMTV DNA do not necessarily produce viral RNA; and (iv) the extent of transcription and glucocorticoid responsiveness of MMTV proviruses may be dependent on the site(s) in cell DNA in which the viral DNA resides.
Structural bases of the inhibitory effects of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S.
Authors: Nagel R L , Bookchin R M , Johnson J , Labie D , Wajcman H , Isaac-Sodeye W A , Honig G R , Schilirò G , Crookston J H , Matsutomo K
Published: May 2006

We have previously found that the inhibitory effect of hemoglobin F (Hb F) on the polymerization of Hb S proceeds via the formation of asymmetrical hybrid tetramers of the type alpha2betasgamma. Examination of the gelling properties of binary mixtures of Hb S and several Hb variants now shows that, among the gamma chain amino acid residues that differ from those of the beta chain, residues gamma80 (EF4) and gamma87 (F3) are at least partly responsible for this inhibition. Furthermore, we find that mixing Hb A2(alpha2delta2) with Hb S strongly inhibits gelling to an extent similar to that seen with Hb S/Hb F mixtures; this inhibition is attributable to amino acid differences between the delta and beta chain sequences at positions delta22 (B4) and delta87 (F3). Therefore, residues 22, 80, and 87 of the beta chain appear to be involved in intermolecular contact sites that stabilize the deoxy Hb S polymers.
Degradation of abnormal proteins in intact mouse reticulocytes: accumulation of intermediates in the presence of bestatin.
Authors: Botbol V , Scornik O A
Published: May 2006

Incubation of intact mouse reticulocytes with bestatin (a competitive inhibitor of aminopeptidases) produced the accumulation of low molecular weight intermediates in the degradation of puromycinyl-peptides or analog-containing proteins that had been pulse labeled with L-[1-14C]leucine. A large fraction of the radioactive protein was degraded to trichloroacetic acid-soluble products within 10 min. In the presence of bestatin (0.5 mg/ml), one-fourth of these products appeared to be dipeptides, tripeptides, or both: they were resistant to ninhydrin at acid pH (a treatment that decarboxylates only free amino acids) except after intensive acid hydrolysis, and they eluted from a Sephadex G-10 column with an apparent average size of 300 daltons. These radioactive products did not appear if incorporation of the tracer was prevented by prior treatment with cycloheximide, demonstrating that they originated from polypeptide precursors. Thus, a peptidase inhibitor has been successfully used in the production of low molecular weight intermediates in the in vivo degradation of cellular proteins.
Steady-state phase or cooperative transitions between biochemical cycles
Authors: Hill Terrell L.
Published: May 2006

In a steady-state lattice of interacting enzyme molecules that have a multicycle kinetic diagram, a cooperative or phase transition may involve not only the conventional sudden change in the relative importance of the different states of a molecule but also a sudden change in the dominant cycles of the diagram. The latter effect implies a sudden switch in the dominant biochemistry (e.g., a sudden onset of active transport). An explicit example is discussed.
Labeling of complex III, with [35S]diazobenzenesulfonate: orientation of this electron transfer segment in the mitochondrial inner membrane.
Authors: Bell R L , Sweetland J , Ludwig B , Capaldi R A
Published: May 2006

[34S]Diazobenzenesulfonate has been used to tag the surface-exposed polypeptides of isolated complex III. All nine different component polypeptides were labeled, indicating that each is at least partially exposed on the surface of the isolated, detergent-dispersed complex. Labeling studies were also conducted on the membrane-bound complex. Preparations of intact mitochondria and submitochondrial particles were separately labeled with [35S]diazobenzenesulfonate in order to determine the distribution of the polypeptides of complex III between the outer (cytoplasmic) and inner (matrix) surfaces of the mitochondrial inner membrane, respectively. Polypeptides II and III were the only components labeled in a significant amount in submitochondrial particles (i.e., from the matrix side). Polypeptides III, IV, and VI were heavily labeled in mitochondria (i.e., from the cytoplasmic side). Polypeptides I,II, V, and VII were also labeled in mitochondria but to a much lesser extent. Polypeptides VIII and IX were not significantly labeled from either side of the membrane. The labeling data and information obtained from previous crosslinking studies [Smith, R.J. & Capaldi, R.A. (1977) Biochemistry 16, 2629-2633] are used to derive a picture of the arrangement of complex III in the mitochondrial inner membrane.
Self-catalyzed destruction of cytochrome P-450: covalent binding of ethynyl sterols to prosthetic heme.
Authors: Ortiz de Montellano P R , Kunze K L , Yost G S , Mico B A
Published: May 2006

The hepatic pigment accumulated as a consequence of the self-catalyzed destruction of cytochrome P-450 by norethisterone (17-hydroxy-19-nor-17 alpha-pregn-4-en-20-yn-3-one), after acidic methylation and purification, consists of two virtually identical, probably isomeric, porphyrins. Radiolabeled norethisterone is incorporated into both porphyrin products. The major of the two porphyrins exhibits a mass spectrometric molecular ion exactly equivalent to the sum of norethisterone and dimethylprotoporphyrin IX, less two hydrogen atoms: unequivocably demonstrating covalent association of the sterol with this porphyrin in a 1:1 ratio. Cytochrome P-450 is therefore destroyed by self-catalyzed addition of norethisterone to its heme prosthetic group. Cytochrome P-450 is also destroyed by norgestrel (13-ethyl-17-hydroxyl-18, 19-dinor-17 alpha-pregn-4-en-20-yn-3-one) and by 1-ethynylcyclohexanol but not by 17-hydroxy-19-nor-17alpha-pregn-4,20-dien-3-one. The destructive potential is thus clearly a property of the propargylic alcohol function. A mechanism involving enzymatic oxidation of the triple bond is postulated.
Evidence for role of glycoprotein carbohydrates in membrane transport: specific inhibition by tunicamycin.
Authors: Olden K , Pratt R M , Jaworski C , Yamada K M
Published: May 2006

Using tunicamycin, we have investigated the role of glycoproteins in membrane transport. Tunicamycin is a glucosamine-containing antibiotic that specifically inhibits dolichol pyrophosphate-mediated glycosylation of asparaginyl residues of glycoproteins. Inhibition of protein glycosylation in chick embryo fibroblasts by tunicamycin or other inhibitors of glycosylation resulted in defective transport of glucose, uridine, and amino acid analogs (alpha-aminoisobutyrate and cycloleucine). The defect in glucose transport is accompanied by decreased glucose metabolism, as determined by rates of CO2 and lactate production. In contrast, tunicamycin treatment did not affect other membrane-associated processes, such as secretion of fibronectin and procollagen, uptake of glucose by passive diffusion, Na+/K+ ATPase and adenylate cyclase activities, or stimulation of adenylate cyclase by prostaglandin and cholera toxin. Two glucose/glycosylation-regulated membrane proteins with apparent subunit molecular weights of 95,000 and 75,000 were induced by tunicamycin treatment. Our results indicate that glycoprotein glycosylation is required for membrane transport.
Xenogeneic gene expression in chimeric mice derived from rat--mouse hybrid cells.
Authors: Illmensee K , Croce C M
Published: May 2006

Thymidine kinase-deficient OTT6050 mouse teratocarcinoma cells were fused with hypoxanthine phosphoribosyltransferase-deficient Fu5AH rat hepatoma cells by means of inactivated Sendai virus. The resulting hybrid cells, which were selected in hypoxanthine/aminopterin/thymidine medium, retained almost all of the mouse chromosomes and various numbers of rat chromosomes, and showed many chromosomal rearrangements. The hybrid cells, as well as both parental lines, formed tumors after subcutaneous injection into athymic nude mice. Single rat--mouse hybrid cells from a clonally established subline were transplanted into C57BL6/J mouse blastocysts carrying many genetic markers suitable for the detection of hybrid cell-derived tissue contributions. From 144 blastocysts, each of which was injected with a hybrid cell and then surgically transferred to the uterus of a pseudopregnant foster mother, 62 adult mice developed without any visible coat mosaicism. However, three of these mice showed internal hybrid-cell participation in their livers and a limited number of organs of endomesodermal origin. A tumor classifiable as hemangio endothelioma was found in the liver, the only mosaic tissue, of one of the chimeric mice. Nine different rat-specific enzyme variants were detected in the mosaic organs. A considerable number of variations concerning the presence and quantitative activity of the foreign gene products probably resulted from chromosomal segregation, tissue-specific gene activity, or dosage compensation during differentiation in vivo. Our results demonstrate that cultured malignant rat--mouse hybrid cells differentiate normally and become functionally integrated during development. The appearacne in vivo of certain rat-specific gene products that are not found in the hybrid cells under conditions in vitro indicates differential gene expression of the introduced xenogeneic chromosomes.
NADPH cytochrome P-450 reductase activation of quinone anticancer agents to free radicals.
Authors: Bachur N R , Gordon S L , Gee M V , Kon H
Published: May 2006

With NADPH as the electron donor, rat liver NADPH cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) catalyzes the single-electron reduction of several quinone antibiotics to a semiquinone or free radical state. The benzanthraquinones adriamycin, daunorubicin, carminomycin, 7-O-methylnogalarol, and aclacinomycin A and the N-heterocyclic quinones streptonigrin and mitomycin C are activated to free radical intermediates which can transfer their single electron to molecular oxygen to form superoxide. The overall Km range for this electron transfer is 0.4 to 42.1 X 10(-4) M. We postulate that the formation of the "site-specific free radical/ intermediate is central to the cytotoxic action of these antibiotics.
Interactions among Hageman factor, plasma prekallikrein, high molecular weight kininogen, and plasma thromboplastin antecedent.
Authors: Ratnoff O D , Saito H
Published: May 2006

To investigate the earliest steps of the intrinsic clotting pathway, Hageman factor (Factor XII) was exposed to Sephadex gels to which ellagic acid had been adsorbed; Hageman factor was then separated from the gels and studied in the fluid phase. Sephadex-ellagic acid-exposed Hageman factor, whether purified or in plasma, activated plasma thromboplastin antecedent, but only when high molecular weight kininogen was presnet. In the absence of plasma prekallikrein, maximal activation of plasma thromboplastin antecedent was slightly delayed in plasma, a delay not observed with similarly treated purified Hageman factor. Thus, high molecular weight kininogen was needed for expression of Hageman factor's clot-promoting properties and plasma prekallikrein played a minor role in the interaction of ellagic acid-treated Hageman factor and plasma thromboplastin antecedent.
Effect of estrogen on gene expression in chicken oviduct: Evidence for transcriptional control of ovalbumin gene
Authors: Swaneck George E. , Nordstrom Jeffrey L. , Kreuzaler Fritz , Tsai Ming-Jer , O'Malley Bert W.
Published: May 2006

The transcription of structural and intervening sequences of the chicken ovalbumin gene was studied in nuclei isolated from the oviduct, liver, and spleen of chickens in different states of estrogen simulation. The concentration of transcripts of structural and intervening DNA sequences was determined by hybridizing the newly synthesized [
3
H]RNA to filters containing cloned ovalbumin cDNA (pOV230) or fragments of the natural ovalbumin gene (pOV2.4 and pOV1.8). Of the RNA synthesized by oviduct nuclei from chickens chronically stimulated with diethylstilbestrol, 0.23% corresponded to ovalbumin mRNA and 0.17% were transcripts of intervening sequences. No detectable ovalbumin mRNA sequences were synthesized by nuclei from spleen and liver. After 60 hr of hormone withdrawal, synthesis of ovalbumin mRNA by oviduct nuclei could not be detected. After readministration of estrogen, a gradual increase in ovalbumin mRNA synthesis was observed which began at 1 hr and reached a plateau by 8 hr. For the intervening sequences, similar kinetics were observed for the initial 4 hr. Previously we had identified multiple species of putative precursors of ovalbumin mRNA in oviduct nuclei from chickens chronically stimulated with diethylstilbestrol. We demonstrate here that withdrawal of diethylstilbestrol resulted in a depletion of high-molecular-weight ovalbumin RNA and of mature ovalbumin mRNA and that readministration of the estrogen induced the nuclear accumulation of both forms of ovalbumin RNA. These findings indicate that: (
i
) a method exists to assay synthesis of hormone-inducible specific eukaryotic [
3
H]mRNA
in vitro;
(
ii
) the estrogen-mediated preferential expression of the ovalbumin gene is maintained in isolated oviduct nuclei; (
iii
) after hormone withdrawal, a single injection of diethylstilbestrol induces transcription of ovalbumin structural and intervening sequences, with nuclear accumulation of high-molecular-weight ovalbumin RNA and mature ovalbumin mRNA; and (
iv
) these results are consistent with regulation of ovalbumin mRNA at the level of ovalbumin gene transcription.
Cross-sectional views of hemoglobin S fibers by electron microscopy and computer modeling.
Authors: Garrell R L , Crepeau R H , Edelstein S J
Published: May 2006

Fibers of deoxyHb S have been investigated by thin-section electron microscopy, utilizing a tannic acid embedding procedure. On the basis of numerous measurements of cross-sectional center-to-center distances for adjacent fibers in pairs or arrays, fiber diameters (mean +/- SD) of 205 +/- 5 A in embedded cells and 212 +/- 8 A in embedded hemolysates were obtained. This is an agreement with values obtained by conventional embedding procedures [Crepeau, R. H., Dykes, G., Garrell, R. L. & Edelstein, S. J. (1978) Nature (London) 274, 616--617]. The use of tannic acid has resulted in improved resolution of fiber cross sections, revealing individual strands of Hb S molecules. Because the section thickness corresponds to approximately one-fifth of the fiber helical repeat distance, the strands in projection superimpose to form characteristic image patterns. Additional superposition patterns arise in sections taken at small deviations from perpendicularity to the longitudinal fiber axis. These patterns are consistent with the 14-strand structure for hemoglobin S fibers [Dykes, G., Crepeau, R. H. & Edelstein, S. J. (1978) Nature (London) 272, 506--510], as indicated by computer models of cross-sectional patterns for various thicknesses and angular deviations of sections.
Transfer of glucagon receptor from liver membranes to a foreign adenylate cyclase by a membrane fusion procedure
Authors: Schramm Michael
Published: May 2006

Previous work had demonstrated the coupling of a β-adrenergic receptor on an erythrocyte with the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of a tissue culture cell when the two cells were fused by Sendai virus. The validity of this finding for animal tissues in general, for membrane preparations, and for peptide hormone receptors could hitherto not be assessed. Available fusion procedures worked efficiently only with certain intact cells from tissue culture and with erythrocytes. In the present work a membrane fusion method was developed that causes the transfer of the glucagon receptor from purified rat liver membranes to Friend erythroleukemia cells; even direct transfer to a membrane fraction prepared from Friend cells became feasible. It can therefore be concluded that a peptide hormone receptor in a normal tissue membrane has properties similar to those demonstrated for a β-adrenergic receptor in an erythrocyte: it exists in the membrane as a dissociable independent unit that can readily couple with the adenylate cyclase of a foreign cell. The efficiency of the membrane fusion procedure is due to the combined action of polyethylene glycol, phospholipids, stearylamine, and ATP in a salt medium. The method promises to be applicable to membranes of various cells and tissues, and it can probably be used to analyze hormone receptors and adenylate cyclase systems in states of malfunction by transfer to their respective counterpart in a normal cell membrane. Studies in biochemical hybridization of membrane components need not be limited to hormone activation of adenylate cyclase. With the aid of the membrane fusion method, this approach could be applied to any dissociable multicomponent system in biological membranes.