bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of apolipoprotein E in the lipolytic conversion of beta-very low density lipoproteins to low density lipoproteins in type III hyperlipoproteinemia.
Authors: Ehnholm C , Mahley R W , Chappell D A , Weisgraber K H , Ludwig E , Witztum J L
Published: May 2006

The beta-very low density lipoproteins (beta-VLDL) that accumulate in type III hyperlipoproteinemic subjects can be divided into two fractions (fraction I and fraction II), which differ in size, lipid composition, and the type of apolipoprotein B (apo-B) present in the particles. The apo-B48-containing particles (fraction I) are of intestinal origin, while apo-B100-containing particles (fraction II) are derived from the liver. Both fractions contain a defective form of apo-E referred to as apo-E2. Intravenous infusion of heparin into two subjects with type III hyperlipoproteinemia resulted in the complete removal of fraction II particles from density less than 1.006 g/ml, while fraction I particles remained at this density. In vitro studies confirmed that fraction I particles did not change density when subjected to hydrolysis with lipoprotein lipase, while fraction II particles shifted to the intermediate density lipoprotein range (approximately equal to 1.02 g/ml). When the beta-VLDL were hydrolyzed by lipoprotein lipase in the presence of density greater than 1.21 g/ml lipoprotein-deficient plasma, the addition of normal apo-E (apo-E3), but not apo-E2, resulted in a shift of fraction II particles to the low density lipoprotein (LDL) range (approximately equal to 1.05 g/ml). Fraction I particles did not undergo a shift to this higher density, supporting previous observations that apo-B48-containing particles are not converted to LDL. The demonstration that apo-B100-containing particles in type III hyperlipoproteinemic subjects could be converted to particles with the density of LDL suggests that apo-E plays a role in the normal conversion of VLDL to LDL. The mutant form of apo-E (apo-E2) found in the beta-VLDL from type III hyperlipoproteinemic subjects appears to impede this conversion, whereas the addition of normal apo-E (apo-E3) allows the processing to occur.
Pertussis toxin differentiates between two mechanisms of attenuation of cyclic AMP accumulation by muscarinic cholinergic receptors.
Authors: Hughes A R , Martin M W , Harden T K
Published: May 2006

It has been proposed elsewhere [Meeker, R.B. & Harden, T. K. (1982) Mol. Pharmacol. 22, 310-319] that muscarinic cholinergic receptor-mediated attenuation of cAMP accumulation occurs through activation of phosphodiesterase in 1321N1 human astrocytoma cells. Pertussis toxin, which ADP-ribosylates the guanine nucleotide regulatory protein involved in receptor-mediated inhibition of adenylate cyclase (Ni), has been utilized to further differentiate between the mechanism of cholinergic regulation of cAMP metabolism in 1321N1 cells and the mechanism involving inhibition of adenylate cyclase in other tissues. Muscarinic receptor-mediated regulation of cAMP accumulation in NG108-15 neuroblastoma-glioma cells occurs through inhibition of adenylate cyclase. Pretreatment of these cells with pertussis toxin completely blocked the capacity of carbachol to attenuate cAMP accumulation. In contrast, concentrations of pertussis toxin two to three orders of magnitude higher than those effective in NG108-15 cells had no effect on muscarinic receptor-mediated attentuation of cAMP accumulation in 1321N1 cells. In addition, no effect of pertussis toxin was observed either on the control rate or the carbachol-stimulated rate of cAMP degradation measured directly in intact 1321N1 cells. A 41,000 Mr protein previously proposed to be the alpha subunit of Ni was labeled during incubation of a plasma membrane fraction from 1321N1 cells with [32P]NAD and pertussis toxin. Pertussis toxin is apparently active in 1321N1 cells, since this protein substrate was not labeled in plasma membrane preparations from cells previously incubated with toxin. Functional activity of Ni was demonstrated by the observation that guanosine 5'-[gamma-thio]triphosphate- and GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity occurred in cell-free preparations from 1321N1 cells. The inhibitory activity of these guanine nucleotides was lost in membrane preparations from pertussis toxin-treated cells. The data suggest that adenylate cyclase is not involved in cholinergic action in 1321N1 cells and, furthermore, Ni is not involved in muscarinic receptor-mediated activation of phosphodiesterase in these cells. Thus, pertussis toxin can be used to differentiate between two mechanisms of cholinergic regulation of cAMP metabolism.
Monoclonal antibody specific for the mutant PiZ alpha 1-antitrypsin and its application in an ELISA procedure for identification of PiZ gene carriers.
Authors: Wallmark A , Alm R , Eriksson S
Published: May 2006

The PiZ genetic variant of alpha 1-antitrypsin in its homozygous form is associated with an increased risk for chronic lung and liver disease. About 5% of the population are carriers of one PiZ deficiency gene resulting in intermediate levels of plasma alpha 1-antitrypsin (PiMZ, PiSZ, PiPZ). We report the preparation of a hybridoma cell line, ATZ 11, produced by fusion of spleen cells from BALB/c mice immunized with a purified liver PiZ alpha 1-antitrypsin and Sp 2/0 Ag 14 mouse myeloma cells. ATZ 11 produces monoclonal antibodies (IgG1 kappa) specifically interacting with PiZ alpha 1-antitrypsin. These antibodies were used in an ELISA procedure permitting easy and accurate identification of PiZ gene carriers. The method is especially well-suited for studying large population samples concerning the putative relationship between intermediate alpha 1-antitrypsin deficiency and disease.
Restriction endonucleases can be used to study B-Z junctions in supercoiled DNA.
Authors: Azorin F , Hahn R , Rich A
Published: May 2006

Plasmids containing (C-G)n inserts have been used to study the inhibition of cleavage by restriction endonucleases due to Z-DNA formation in negatively supercoiled plasmids. The enzyme BssHII, which recognizes G-C-G-C-G-C, is strongly inhibited when the insert forms Z-DNA. The BamHI recognition sequence (G-G-A-T-C-C) was placed in four different positions near the B-Z junction and the inhibition of BamHI cleavage was determined as a function of negative superhelical density. Formation of Z-DNA in the (C-G)n insert inhibited cleavage by BamHI when its recognition sequence was located immediately adjacent to the insert or four base pairs away from it. However, no inhibition was found when the BamHI recognition site was eight base pairs away. These experiments help to define the limits of the structural perturbation associated with the B-Z junction.
Lumen formation and redistribution of inframembranous proteins during differentiation of ducts in the rat mammary gland.
Authors: Dulbecco R , Allen W R , Bowman M
Published: May 2006

During the development of the rat mammary gland, ducts are formed from end-buds, which contain the stem cells. In this process a lumen is formed in the semisolid mass of the end-bud, and the cells acquire polarity. We have studied this process by following the localization of three inframembranous proteins present in the cells of both end-buds and ducts: microvillin, the microvillar protein p80, and the desmosomal plaque protein p205. We find that the development of ducts is accompanied by a redistribution of these proteins, which in immature parts of the end-buds are found together in the cell. Microvillin and p80 go together to the apical pole of the cells, in contact with the lumen, whereas p205 goes to the basal surface, in contact with cells of the myoepithelial lineage. The acquisition of polarity occurs at the same time as a lumen begins to form by local gaps between cells. It seems likely that the redistribution of the inframembraneous proteins is the consequence of the localization of surface glycoproteins that affect in opposite ways the adhesion between the cells.
Suppressor sufJ: a novel type of tRNA mutant that induces translational frameshifting.
Authors: Bossi L , Smith D M
Published: May 2006

We describe the DNA sequence change responsible for the creation of a frameshift-suppressor gene in Salmonella typhimurium. The suppressor, sufJ, results from a base-pair insertion in the gene coding for a threonine transfer RNA (tRNA3Thr). Unlike previously studied frameshift suppressor mutations, the sufJ insertion does not fall within the sequence corresponding to the tRNA anticodon. The insertion (a G.C base pair) occurs within a run of three G.C base pairs in that region of the gene coding for one strand of the anticodon stem. In the secondary structure of the mature tRNA, the net result is that an extra, unpaired cytidine residue is pushed into the anticodon loop, thus increasing the size of the loop to eight nucleotides. These findings are discussed in connection with the peculiar "three-out-of-four" method of reading by the sufJ suppressor. A unifying model is presented accounting for the contrasting decoding behaviors of tRNAs with eight-nucleotide-long anticodon loops.
Adenosine release from stimulated mast cells.
Authors: Marquardt D L , Gruber H E , Wasserman S I
Published: May 2006

Adenosine release has been documented in lung tissue exposed to hypoxic conditions or antigen challenge. Exogenous adenosine potentiates mediator release from stimulated rat serosal and mouse bone marrow-derived mast cells. To investigate the production and release of adenosine from stimulated mast cells, rat serosal mast cells were purified on metrizamide gradients, sensitized with anti-dinitrophenol IgE for 30 min at 37 degrees C, and challenged in the presence of 1 microM deoxycoformycin with either dinitrophenol-conjugated bovine serum albumin antigen, the calcium ionophore A23187, or compound 48/80. Reactions were terminated by centrifugation, and the supernatants and pellets were assayed for adenosine and ATP content, respectively, by high performance liquid chromatography. The adenosine concentration of the supernatants increased from 0.036 +/- 0.003 nmol per 10(6) cells to 0.049, 0.056, and 0.129 nmol per 10(6) cells 60 sec after challenge with antigen, 48/80, or A23187, respectively. After ionophore stimulation, increased extracellular adenosine was evident by 15 sec, peaked by 60 sec, and remained constant for at least 5 min. A significant decline in stimulated ATP levels was observed within 30 sec, suggesting that the enhanced adenosine concentrations may result from the breakdown of ATP. Cultured mouse bone marrow-derived mast cells under similar conditions also displayed augmented extracellular adenosine levels with A23187 challenge. This endogenous source of adenosine may act locally through a positive feedback mechanism to potentiate immediate hypersensitivity reactions.
Frequent generation of nonrescuable reorganized Moloney murine sarcoma viral genomes.
Authors: Shtivelman E , Zakut R , Canaani E
Published: May 2006

Nonproducer transformants infected with wild-type Moloney murine sarcoma virus were screened for the generation of variants with reorganized genomes. Seven of 20 lines contained such viral genomes, 5 of which were found to be nonrescuable. Blotting analysis indicated that viral RNA molecules transcribed from these variant genomes could not be encapsidated into virions. The nonrescuable genomes were molecularly cloned and all were found to have suffered deletions or deletions/inversions involving the 5' long terminal repeat as well as some adjacent sequences. Nucleotide sequence analysis suggested that the long terminal repeat or the tetranucleotides G-G-T-C and G-A-C-C (or both) were involved in the generation of these mutants. Transfection studies showed that the cloned DNAs of the 5 mutants transformed NIH/3T3 monolayers. Removal of the 3' long terminal repeat from the genomes that lacked the 5' long terminal repeat or carried it in an inverted orientation abolished or considerably reduced the transforming activity.
Specific covalent binding of platelet-derived growth factor to human plasma alpha 2-macroglobulin.
Authors: Huang J S , Huang S S , Deuel T F
Published: May 2006

Attempts to measure the platelet-derived growth factor (PDGF) in human plasma resulted in the discovery of a specific plasma binding protein. The 125I-labeled PDGF (125I-PDGF)-plasma binding protein complex retained mitogenic activity but lost reactivity against rabbit anti-PDGF antiserum. Copurification of the plasma binding protein and alpha 2-macroglobulin (alpha 2M) in human plasma, the formation of a complex between 125I-PDGF and purified alpha 2M, and the comigration of the 125I-PDGF-plasma binding protein complex and the 125I-PDGF-alpha 2M complex in NaDodSO4/polyacrylamide gel electrophoresis and in pore-limiting polyacrylamide gel electrophoresis strongly suggested that alpha 2M is the plasma binding protein for 125I-PDGF. Immunoprecipitation of 125I-PDGF-alpha 2M and 125I-PDGF-plasma binding protein complexes by anti-human alpha 2M antiserum further established that alpha 2M and the plasma binding protein are the same molecule. Approximately 20% of 125I-PDGF is complexed by alpha 2M; further 125I-PDGF is complexed if the remaining 125I-PDGF is incubated with additional alpha 2M. Complex formation of 125I-PDGF with plasma or with alpha 2M was completely inhibited by 0.2 mM p-chloromercuric benzoate or 0.2 mM N-ethylmaleimide. The 125I-PDGF-alpha 2M complex or 125I-PDGF-plasma binding protein complex was not dissociated by 8 M urea, 1 M acetic acid, 0.1 M NaOH, or 1% NaDodSO4 but was dissociated by 2-mercaptoethanol, suggesting that the covalent binding of 125I-PDGF to alpha 2M occurs through a disulfide/sulfhydryl exchange reaction. The 125I-PDGF-alpha 2M complex (780,000 daltons) appears to contain two molecules of 125I-PDGF and two dimers of alpha 2M. The precise physiological role of the 125I-PDGF-alpha 2M interaction is unknown. alpha 2M may serve to limit PDGF released locally at sites of blood vessel injury. Alternatively, because of the nearly complete homology between the partial amino acid sequence of PDGF and the predicted amino acid sequence of the transforming protein of the simian sarcoma virus, p28sis, alpha 2M may play an important role in limiting the activity of a PDGF-like activity expressed by virus-transformed cells.
Regulated expression of a human interferon gene in yeast: control by phosphate concentration or temperature.
Authors: Kramer R A , DeChiara T M , Schaber M D , Hilliker S
Published: May 2006

The promoter/regulator region from the yeast repressible acid phosphatase gene was used to construct a vector for the regulated expression of cloned genes in yeast. The gene for human leukocyte interferon was inserted into this vector. Yeast cells transformed with the resulting plasmid produced significant amounts of interferon only when grown in medium lacking inorganic phosphate. Mutants in two acid phosphatase regulatory genes (coding for a defective repressor and a temperature-sensitive positive regulator) were used to develop a yeast strain that grew well at a high temperature (35 degrees C) but produced interferon only at a low temperature (23 degrees C), independent of phosphate concentration.
Cell cycle regulation of human histone H1 mRNA.
Authors: Plumb M , Marashi F , Green L , Zimmerman A , Zimmerman S , Stein J , Stein G
Published: May 2006

A cloned genomic DNA fragment containing a human histone H1 gene has been used to analyze histone H1 gene expression in two human cell lines (HeLa S3 and WI-38). The cellular abundance of histone H1 mRNA was compared with that of core (H2A, H2B, H3, and H4) histone mRNAs as a function of the cell cycle: core and H1 histone mRNA levels are related both to each other and to the apparent rate of DNA synthesis and are rapidly destabilized after DNA synthesis inhibition. The use of three synchronization protocols, and of transformed and normal diploid cells in culture, suggests that the detected core and H1 histone mRNA levels are regulated by similar mechanisms in continuously dividing human cell lines and nondividing cells stimulated to proliferate.
Dynamics of enzymatic reactions.
Authors: Warshel A
Published: May 2006

The detailed molecular dynamics of an actual bond-breaking event in a fluctuating enzyme substrate complex is simulated. The method developed allows one to explore what type of fluctuations are involved in enzymatic reactions and to evaluate entropic contributions to enzyme catalysis. The fluctuations of the enzyme electrostatic potential are found to be a key dynamical factor in reactions that involve a large change in the polarity of the reacting bonds.
Plasma membrane reorganization induced by tumor promoters in an epithelial cell line.
Authors: Packard B S , Saxton M J , Bissell M J , Klein M P
Published: May 2006

The effects of phorbol ester tumor promoters on the lateral diffusion in plasma membrane lipid environments were examined by the technique of fluorescence recovery after photobleaching. To this end, the probe collarein, a fluorescent lipid analog that has the property of exclusive localization in the plasma membrane, was synthesized. Measured decreases in three parameters [percentage of fluorescence bleached (30%), percentage of recovery (52%), and half-time for recovery (52%)] connoted the appearance of an immobile fraction upon exposure to tumor promoters. These data are consistent with lipid reorganization in response to a reorganization of the intra- and perimembranous macromolecular scaffolding upon the interaction of cells with tumor promoters. The idea of induced reorganization is supported by experiments in which cell shape change, brought about by either exposure to cytochalasin B or growth on matrices of collagen, fibronectin, or laminin, resulted in values in the fluorescence recovery after photobleaching technique similar to those with active phorbol esters.
Structural intermediates of deletion mutagenesis: a role for palindromic DNA.
Authors: Glickman B W , Ripley L S
Published: May 2006

A model is presented for deletion mutations whose formation is mediated by palindromic and quasipalindromic DNA sequences. It proposes that the self-complementarity of palindromes allows the formation of DNA secondary structures that serve as deletion intermediates. The structures juxtapose the end points of the deletion and thus direct deletion specificity. While misaligned DNA intermediates that explain deletion termini occurring in repeated DNA sequences have been described, no explanations have been offered for deletion termini occurring in other sequences. The DNA secondary structures whose formation is mediated by palindromic sequences appear to explain many of these. In this paper, secondary-structure intermediates are described for a series of spontaneous deletions of known sequence in the lacl gene of Escherichia coli. The model is supported by its failure to predict structures that can juxtapose simulated deletion termini in the lacl gene. We have found a strong association between palindromic sequences and repeated sequences at lacl deletion termini that suggests the joint participation of repeated and palindromic DNA sequences in the formation of some deletions. Sequences of deletions in other organisms also suggest the participation of palindromic DNA sequences in the formation of deletions.
Drift and mutation with a finite number of allelic states
Authors: Cockerham C. Clark
Published: May 2006

The frequencies with which alleles are alike within (
Q
) and between (
q
) populations are formulated for monoecious populations undergoing drift and mutation with unequal mutation rates among alleles for a finite number (
k
) of allelic states. The effective number of alleles and an application to Nei's measure of genetic distance [Nei, M. (1972)
Am. Nat.
106, 283-292] are also considered for this model. The equilibrium values of
Q
and
q
increase as
k
decreases. Unequal mutation rates further increase the equilibrium values and reduce the rates of approach of
Q
and
q
to these values. The transitional values of
Q
and
q
are very dependent on the initial population frequency composition when mutation rates are unequal. Reducing
k
, of course, reduces the effective number of alleles, which is further reduced by unequal mutation rates. Complications introduced by initial population composition, unequal mutation rates, and number of allelic states, coupled with data limitations for long-term measures of genetic distance or population differentiation, with mutation as the main driving force, are discussed.
Immunoglobulin gene rearrangement as a diagnostic criterion of B-cell lymphoma.
Authors: Cleary M L , Chao J , Warnke R , Sklar J
Published: May 2006

We describe the use of the Southern blot hybridization technique to diagnose B-cell lymphoma by detecting clonal immunoglobulin gene rearrangements in lymph node and other biopsy tissues. DNA was isolated from a wide variety of neoplastic and non-neoplastic specimens and analyzed for the presence of rearranged immunoglobulin genes using radiolabeled DNA probes specific for the heavy- and light-chain immunoglobulin constant region genes. Among the specimens examined, clonal immunoglobulin gene rearrangements were found only in biopsy samples of B-cell lymphoma and not in samples containing reactive lymphoid processes or non-B-cell cancers. In lymphomas, the presence of rearrangements for either the kappa or lambda light-chain gene correlated with expression of one or the other of these chains when cellular immunoglobulins could be detected by frozen-section immunophenotyping techniques. The analysis of immunoglobulin gene rearrangements offers several advantages over conventional diagnostic methods for lymphomas, including improved sensitivity in detecting minor populations of neoplastic lymphocytes composing as little as 1% of the total cell population. In addition, clonal immunoglobulin gene rearrangements are demonstrable in a subset of lymphomas that lack detectable surface or cytoplasmic immunoglobulin, thus offering positive evidence for both malignancy and the B-cell origin of these tumors. Our studies indicate that detection of immunoglobulin gene rearrangements is a valuable method for diagnosis and classification of various lymphoproliferative disorders that are difficult to evaluate histologically or that lack distinctive antigenic markers.
Regression of myelocytic leukemia in rats after hypophysectomy.
Authors: Huggins C B , Ueda N
Published: May 2006

A standard series of five intravenous injections of N-nitroso-N-methylurea (NMU) elicited selective patterns of neoplasms in male rats of the Sprague-Dawley strain; the neoplastic pattern was related to the age of the recipients. When the injections of NMU were started in weanling male rats, a specific triad of neoplasms developed that consisted of mammary carcinoma, primary myelocytic leukemia, and ear duct cancer. When the injections of NMU were started in males at age 100 days, a specific dyad of neoplastic growths was evoked that consisted of primary myelocytic leukemia and ear duct cancer, whereas mammary cancer and other neoplasms were not evident. Secondary myelocytic leukemia and myelocytic sarcoma were produced when blood from the foregoing rats with advanced primary myelocytic leukemia was injected in a subcutaneous site in newborn allogeneic rats. In a proportion of animals, primary and secondary myelocytic leukemia and myelocytic sarcoma underwent dramatic and continued regression after hypophysectomy.
Free cytoplasmic Ca2+ and neurotransmitter release: studies on PC12 cells and synaptosomes exposed to alpha-latrotoxin.
Authors: Meldolesi J , Huttner W B , Tsien R Y , Pozzan T
Published: May 2006

The relationship between the free cytoplasmic Ca2+ concentration, [Ca2+]i, and neurotransmitter release was investigated in guinea pig brain synaptosomes and the neurosecretory cell line PC12. Release was induced by alpha-latrotoxin, which acts in both Ca2+ -containing and Ca2+ -free incubation media, or by the classical depolarizing agents high K+ and veratridine, which require extracellular Ca2+. Two complementary approaches were used to reveal changes of [Ca2+]i: (i) direct measurement by a fluorescent Ca2+ indicator (quin2) and (ii) study of the Ca2+ -dependent phosphorylation of a protein, synapsin I, located at the cytoplasmic surface of synaptic vesicles. Depolarizing agents, when applied in Ca2+ -containing medium, induced the [Ca2+]i to increase promptly 3- to 6-fold, drastically increased synapsin I phosphorylation, and caused stimulation of transmitter release. With alpha-latrotoxin, the [Ca2+]i increase was delayed and occurred at a slower rate, the increase of synapsin I phosphorylation was less drastic, and the release response was much more pronounced. In Ca2+ -free medium, depolarizing agents released no transmitter and had no effect on [Ca2+]i or synapsin I phosphorylation, whereas with alpha-latrotoxin these processes were dissociated: considerable stimulation of the release without apparent change of [Ca2+]i and synapsin I phosphorylation. We conclude that the relationship between average [Ca2+]i and transmitter release is not straightforward and, in particular, that the release evoked by alpha-latrotoxin in Ca2+ -free medium is mediated by a factor(s) other than bulk redistribution of Ca2+ from intracellular stores.
Characterization of the Bacillus subtilis tryptophan promoter region.
Authors: Shimotsu H , Henner D J
Published: May 2006

The nucleotide sequence of the control region of the trp operon of Bacillus subtilis has been determined. The region was shown to contain the trp promoter by deletion analysis and by determination of the transcription start site. The trp promoter shows similarity to the consensus sequence for Escherichia coli and B. subtilis promoters. The presence of the trp control region on a high-copy-number plasmid confers resistance to the tryptophan analogue 5-methyltryptophan. It appears that an approximately 120-base-pair region comprising not only the trp promoter but also adjacent direct repeat sequences is necessary to confer 5-methyltryptophan resistance. We postulate that this region is involved in tryptophan regulation and confers 5-methyltryptophan resistance by titration of a trp regulatory protein. Removal of either the trp promoter or the adjacent direct repeat sequences abolished the 5-methyltryptophan-resistance phenotype. Placement of unrelated promoters adjacent to the direct repeat sequences restored 5-methyltryptophan resistance. This suggests that promoter activity is necessary for the regulatory function.
Homonuclear 1H double-resonance difference spectroscopy of the rat brain in vivo.
Authors: Rothman D L , Behar K L , Hetherington H P , Shulman R G
Published: May 2006

We have used 1H homonuclear double-resonance difference spectroscopy at 360.13 MHz to resolve specific metabolite resonances in the brains of intact rats. Metabolite resonances resolved include previously obscured proton resonances of alanine, gamma-aminobutyric acid (GABA), glutamate, and taurine. The gamma-aminobutyric acid alpha- and gamma-CH2 proton resonances were observed in the living rat in the difference spectrum obtained upon irradiation of the beta-CH2 proton resonance at 1.91 ppm. A 3-fold increase in the intensity of the alpha- and gamma-CH2 resonances of gamma-aminobutyric acid was observed 30 min after death. The alpha-CH and gamma-CH2 resonances of glutamate were also resolved in vivo by selective irradiation of the beta-CH2 protons to which they are spin-coupled. In addition, this technique was used to observe the beta-CH3 protons of lactate through the intact scalp of a rat. Large lipid signals arising from scalp tissue were eliminated in the difference spectrum, revealing the lactate beta-CH3 resonance.
Phorbol ester, serum, and rous sarcoma virus transforming gene product induce similar phosphorylations of ribosomal protein S6.
Authors: Blenis J , Spivack J G , Erikson R L
Published: May 2006

The addition of phorbol 12-myristate 13-acetate (PMA), a potent tumor promoter, to serum-starved quiescent chicken embryo fibroblasts (CEF) or C127 murine cells resulted in increased phosphorylation of 40S ribosomal protein S6. The effect of PMA on S6 phosphorylation in quiescent CEF was half-maximal at approximately equal to 100 nM and was readily observed at 16 nM. In addition, S6 phosphorylation was increased in serum-starved CEF incubated with the diacylglycerol derivative, 1-oleoyl-2-acetylglycerol. S6 phosphorylation in PMA-stimulated, serum-stimulated, and serum-starved Rous sarcoma virus-transformed CEF was analyzed by phospho amino acid analysis, two-dimensional polyacrylamide gel electrophoresis, limited proteolysis with V8 protease, and two-dimensional thin-layer electrophoresis of chymotryptic digests. Comparison of S6 phosphorylation by these methods suggests that phosphorylation of S6 stimulated by PMA, serum, or oncogenic transformation with Rous sarcoma virus occurs through common pathways. This is further supported by the observation that the simultaneous addition of PMA and serum to CEF or of either PMA or serum to Rous sarcoma virus-transformed CEF did not significantly further increase the incorporation of phosphate into S6.
Weak HLA and beta 2-microglobulin expression of neuronal cell lines can be modulated by interferon.
Authors: Lampson L A , Fisher C A
Published: May 2006

Previous work showed that each of four human neuronal cell lines expresses less than or equal to 0.5% of the HLA-A,B,C and beta 2-microglobulin seen in glial, lymphoid, and other cell types, and there is a corresponding weak expression in neuroblastoma tumor and adult brain. Here, we probe the genetic basis of this weak expression. For each of three neuroblastoma cell lines, we show that HLA-A,B,C and beta 2-microglobulin can be induced by interferon and that the induction occurs within every cell of the population. Class II (Ia) molecules are not detected. Microscopic assay and radioimmunoassay of intact cells suggest that the induced antigen appears at the cell surface as well as within each cell. Immunoblot analysis confirms that the induced proteins have the structure of class I molecules. Thus, the normal weak HLA and beta 2-microglobulin expression of these cell lines appears to reflect a regulatory control rather than a primary genetic lesion. According to current theory, lack of HLA-A,B,C should protect transformed, infected, or damaged neurons--but also neurons in neural transplants--from T-cell-mediated immunosurveillance. The possibility that neuronal HLA-A,B,C expression may be under regulatory control is of importance in this context.
Partial characterization of the neurotrophic factor for maintenance of acetylcholinesterase and butyrylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat in vivo.
Authors: Koelle G B , Sanville U J , Rickard K K , Williams J E
Published: May 2006

In continuation of previous reports, it was found that the neurotrophic factor (NF) of the central nervous system of the cat for the maintenance of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7; AcChoEase) in the denervated cat superior cervical ganglion is a heat-stable compound of low molecular weight (less than 1,000) and that it is probably a peptide. Acetylcholine and nerve growth factor were eliminated as the NF; cyclic AMP produced an effect similar to that of the NF. The NF is probably not present in significant amounts in liver or skeletal muscle; it appears to be present in small intestine. It does not modify the AcChoEase content of the nondenervated cat superior cervical ganglion. Possible mechanisms of action of the NF are discussed.
Functional alteration of the beta-adrenergic receptor during desensitization of mammalian adenylate cyclase by beta-agonists.
Authors: Kassis S , Fishman P H
Published: May 2006

Exposure of several mammalian cell lines to isoproterenol resulted in a desensitization of the beta-adrenergic receptor-adenylate cyclase system in membranes isolated from the cells. Under the experimental conditions chosen, desensitization was accompanied by a minimal loss of beta-receptors. The cells tested included HeLa, S49 cyc- lymphoma, and rat glioma C6. The functional activity of the beta-receptors was determined by coupling them to a foreign adenylate cyclase by membrane fusion. The donor membranes were treated to inactivate the regulatory and catalytic components of adenylate cyclase. The acceptor membranes were from Friend erythroleukemic cells (Fr cells), which lack beta-receptors, and HeLa cells treated overnight with isoproterenol to eliminate their receptors. The fused membranes were assayed for agonist-stimulated activity, which was always reduced when the donor beta-receptors were from the desensitized membranes. The desensitization appeared to be specific for beta-receptors, as the activity of other receptors and cyclase components was not altered. By fusing HeLa membranes with intact Fr cells, we directly measure the intrinsic activity of native and desensitized beta-receptors. For an equal amount of transferred beta-receptors, the activity was 40%-50% lower when the donor membranes were from desensitized cells. Our results clearly indicate that desensitization mediated by a beta-agonist in mammalian cells results in a functional alteration of the beta-receptor.
The translational mobility of substances within the cytoplasmic matrix.
Authors: Jacobson K , Wojcieszyn J
Published: May 2006

The translational mobility of fluorescent-labeled molecules injected into the cytoplasm of living cells can be measured by the fluorescence recovery after photobleaching (FRAP) technique. In the fibroblast cytoplasm, the diffusion coefficients, D, of test macromolecules ranging in molecular weight from 12,000 to 440,000 are about 10(-8) cm2/sec and exhibit almost no dependence on molecular weight. FRAP experiments also showed that macromolecular diffusion within Sepharose beads having an effective pore size smaller than the "microtrabecular lattice" is only slightly retarded compared to buffer values--in contrast to the marked retardation measured in the cytoplasm. This leads to the conclusion that diffusion in the cytomatrix is dominated not by steric effects but rather by binding of the diffusing species to elements of the cytomatrix. These diffusion rates were difficult to modulate; cytochalasin, colchicine (except at 5 degrees C), and taxol treatments had little effect. The diffusion rates were not dependent on cellular energy metabolism. However, hypotonic treatment increased the D for bovine serum albumin by nearly 2-fold, whereas hypertonic treatment halved D. Withdrawing the free water from the cell by using 44% polyethylene glycol treatment stopped the translational mobility of the test molecules. A survey of the recent literature is presented, which shows that major differences in the cytomatrix of different cell types exist with respect to the translational diffusion of injected probes. Finally, the spectrum of cytoplasmic translational mobilities ranging from small molecules to organelles is discussed.
Calcium dependency of antigen-specific (T3-Ti) and alternative (T11) pathways of human T-cell activation.
Authors: Weiss M J , Daley J F , Hodgdon J C , Reinherz E L
Published: May 2006

Human T lymphocytes are activated by two lineage-specific surface components: the antigen/major histocompatibility complex receptor (T3-Ti) and the unrelated T11 molecule. Interaction of either of these with their respective ligands leads to T-cell proliferation via an interleukin 2(IL-2) dependent autocrine mechanism. To begin to characterize the molecular details of the activation process, the role of Ca2+ was examined using human T-cell clones and monoclonal antibodies directed against their surface components. Here, we show that within minutes of triggering either the T3-Ti or T11 molecule, there is a large increase in intracellular Ca2+ concentration, as measured by quin-2 fluorescence. This is essential for induction of T-cell proliferation in inducer, suppressor, and cytotoxic clones and therefore presumably is required at an early step in the autocrine growth pathway. Thus, chelating exogenous Ca2+ with EGTA specifically inhibits proliferation triggered by anti-T3-Ti or anti-T11 monoclonal antibodies, but it does not affect triggering by exogenous IL-2. In addition, the Ca2+ ionophore A23187 can, by itself, initiate clonal proliferation.
Evolutionary conservation of key structures and binding functions of neural cell adhesion molecules.
Authors: Hoffman S , Chuong C M , Edelman G M
Published: May 2006

The neural cell adhesion molecule N-CAM is a sialic acid-rich, cell surface glycoprotein that mediates cell adhesion by a homophilic mechanism. Its binding function has been implicated in both morphogenesis and histogenesis; during development it changes in amount at the cell surface and perinatally it undergoes a decrease in sialic acid content (embryonic--adult conversion) with an increase in binding efficacy. In the present study, salient aspects of the structure and the mutual binding specificities of N-CAMs from a variety of vertebrate species were examined to determine whether (N-CAM)-mediated adhesion mechanisms have been conserved during evolution. N-CAM immunoreactivity was detected in a series of polypeptides of characteristic molecular weight extracted from brain tissues of all vertebrate species tested, including mammals, birds, reptiles, amphibia, and bony and cartilaginous fish. Adhesion mediated by N-CAM occurred across species lines as indicated by the co-aggregation of chicken and mouse neural cells. By using a quantitative membrane vesicle aggregation assay, the efficacy of cross-species brain membrane vesicle adhesion in various pairings (chicken-mouse, chicken-frog, mouse-frog) was found to be similar to the efficacy of intra-species adhesion. Effective cross-species aggregation of brain membrane vesicles also occurred in embryonic-embryonic, adult-adult, and embryonic-adult pairings. In a control experiment, embryonic chicken liver membrane vesicles (which do not contain N-CAM) did not co-aggregate with embryonic chicken brain membrane vesicles. Cross-species co-aggregation could be inhibited by Fab' fragments of antibodies of N-CAM and was most effectively inhibited in the presence of mixtures made from the Fab' fragments of specific antibodies prepared against the N-CAMs from each of the animal species constituting a co-aggregating pair. These results suggest that, in accord with the proposed role of N-CAM as a regulator of morphogenesis, both the specificity of the binding region of the molecule and its basic chemical structure have been highly conserved during evolution.
Chromosomal mapping of a middle-repetitive DNA sequence in a cluster of five species of Hawaiian Drosophila.
Authors: Hunt J A , Bishop J G , Carson H L
Published: May 2006

We describe the properties of a repetitive transposable element isolated from a chromosomal site close to the Adh (alcohol dehydrogenase gene) region of the Hawaiian Drosophila species, D. heteroneura. The cloned element is less than 2 kilobases in length. Although its polytene chromosome sites are constant in an individual, it shows a pattern of in situ hybridization that varies both within and between five species of the D. planitibia subgroup. These species are closely related, having diverged at various times from 0.5 to 5 million years ago. The distribution of the element appears to reflect the evolutionary relationships of the species except that the differences between D. planitibia and D. differens are ambiguous. Evidence of ragged excision of the element is found in one species.
Internal duplication in human alpha 1 and beta 1 interferons.
Authors: Erickson B W , May L T , Sehgal P B
Published: May 2006

Metric analysis of the nucleotide sequence of the intron-free human interferon beta 1 (IFN-beta 1) gene by using the Sellers TT algorithm revealed that this gene contains two major repeated segments, which span the entire coding region. These repeats are each approximately 300 nucleotides in length and have 45% identical aligned nucleotides (common bases). When these metrically aligned DNA repeats were translated into amino acids, 9 (19%) of the 47 in-phase amino acid residues were identical (common acids). This internal duplication was also apparent on visual inspection of the amino acid sequence of IFN-beta 1. In addition, metric analysis of the nucleotide sequence of the intron-free IFN-alpha 1 gene showed that this gene also contains two repeats, each approximately 300 nucleotides long, having 47% common bases and 19% common acids. Since the IFN-alpha 1 and -beta 1 genes are known to be related (by the present metric analysis they contain 53% common bases and 45% common acids), a consensus DNA sequence was derived from all four of these repeats. Manual alignment of the separate metric alignments corresponding to the two halves of the IFN-alpha 1 and -beta 1 genes provided a composite alignment with 58% of the alignment positions having the same nucleotide in at least three of the four repeats. When this composite nucleotide alignment was translated to define a composite alignment of the four protein segments, 10 (31%) of the 32 in-phase amino acid residues contained the same amino acid in at least three of the four segments. These sequences relationships provide insight into the origin of the IFN-alpha 1 and -beta 1 genes and furnish an additional basis for comparing them with other related genes.
A "convertible pore" model of neural membrane conductance.
Authors: Wooldridge D E
Published: May 2006

Instead of the single-channel pore proposed earlier [Wooldridge, D. E. (1984) Proc. Natl. Acad. Sci. USA 81, 5609-5612] for the transit of conductance ions through the neural membrane, a pore with a second channel for "influence ions" of calcium or magnesium is considered in this paper. By entering trapping centers at the closed inner ends of the new channels, the influence ions are postulated to alter the rates of chemical reactions that change the configurational state of the gates guarding the inner ends of the nearby conductance channels. This makes the permeability of the conductance channels strongly dependent on voltage. Using a four-state reaction scheme for both the sodium and potassium pore systems, a computer model of the membrane conductance is constructed. When suitable values are assigned to its parameters, the model closely reproduces the results of the Hodgkin-Huxley voltage-clamp and action potential experiments.
Primary structure of the L subunit of the reaction center from Rhodopseudomonas sphaeroides.
Authors: Williams J C , Steiner L A , Feher G , Simon M I
Published: May 2006

The reaction center is an integral membrane protein that, together with several cofactors, mediates the primary photochemical events in bacterial photosynthesis. The amino-terminal sequences of the three subunits, L, M, and H, of the reaction center protein and the sequence of the structural gene encoding the M subunit have been reported previously. In the present study, we found that the 3' end of the structural gene encoding the L subunit overlaps by eight bases the 5' end of the gene encoding the M subunit. The primary structure of the L subunit has been determined from the nucleotide sequence of the gene and from analyses of the amino and carboxyl termini of the protein. The sequences of a number of tryptic and chymotryptic peptides were used to corroborate the nucleotide sequence. The L subunit was found to be composed of 281 amino acids (Mr 31,319) and to contain five hydrophobic segments. It is homologous to the M subunit and to a plant thylakoid protein referred to as the QB or Mr 32,000 protein.
Rat liver asialoglycoprotein receptor lacks a cleavable NH2-terminal signal sequence.
Authors: Holland E C , Leung J O , Drickamer K
Published: May 2006

Two cDNA clones encoding the predominant form of the asialoglycoprotein receptor from rat liver (the major rat hepatic lectin; RHL-1) were identified by screening a rat liver cDNA library with a mixed oligonucleotide probe 35 nucleotides long. One clone was a nearly full-length copy of the mRNA for RHL-1, while the other was shortened at both ends. The sequences of these clones demonstrate that this transmembrane receptor is not synthesized with an NH2-terminal signal sequence. The only proteolytic processing occurring in the biosynthesis of RHL-1 is the removal of the NH2-terminal initiator methionine residue. Insertion of RHL-1 into the membrane is postulated to occur by the recognition of the internal transmembrane region as a signal sequence.
Serological cross-reactivity between envelope gene products of type I and type II human T-cell leukemia virus.
Authors: Lee T H , Coligan J E , McLane M F , Sodroski J G , Popovic M , Wong-Staal F , Gallo R C , Haseltine W , Essex M
Published: May 2006

People exposed to type I human T-cell leukemia virus (HTLV-I) develop antibodies to an antigen at the surface of virus-infected cells, designated human T-cell leukemia virus membrane antigen (HTLV-MA). In an earlier study, we demonstrated that the major component of HTLV-MA is gp61, a glycoprotein encoded by the HTLV env gene. In the current study, we found that human antibodies that react with HTLV-MA on cells infected with HTLV-I react equally well with HTLV-MA on C3-44/MO, a target cell infected with type II HTLV. A glycoprotein with an approximate size of 67 kDa, gp67, was identified in C3-44/MO using immunoprecipitation and NaDodSO4/PAGE analysis. The positions of serine and cysteine residues were determined in the amino terminus of gp67 by radiolabel sequencing analysis. Comparison with the amino acid sequence deduced from the primary nucleotide sequence of HTLV-IIMO virus reveals that gp67 is also encoded, at least in part, by the env gene. The gp67 of HTLV-IIMO, like the env gene product of HTLV-ICR, gp61, is recognized both by antibodies from a HTLV-IIMO-infected patient with a variant form of hairy cell leukemia, and by antibodies from patients with HTLV-I-associated adult T-cell leukemia/lymphoma. These results indicate that, despite the divergence between HTLV-I and HTLV-II, the major env gene products of the two types of HTLV are conserved to the degree that they are serologically cross-reactive.
Expression of differentiation and murine leukemia virus antigens on cells of primary tumors and cell lines derived from chemically induced lymphomas of RF/J mice.
Authors: Goodenow M M , Lilly F
Published: May 2006

3-Methylcholanthrene (MCA) skin painting rapidly induced a 100% incidence of thymic lymphomas in RF/J mice. Tumors developed exclusively in cells of the T-lymphocyte lineage and displayed the surface phenotype: Thy-1+, Lyt-1+, Lyt-2+, Qa-1+, H-2K+, H-2D+, TL-, Ig-, Ia-. This phenotype resembled that of cortisone-resistant, medullary thymocytes, whereas the phenotype of spontaneous AKR thymomas resembles that of cortisone-sensitive, cortical thymocytes. The phenotype differed very little from one tumor to another and was maintained during syngeneic passage in vivo and adaptation to growth in culture. Infectious ecotropic murine leukemia virus (MuLV) was produced in a minority of primary tumors and cell lines, but no xenotropic or mink cell focus-inducing virus (MCF) MuLV production was reliably detected. MuLV-related antigen expression on normal thymocytes increased in an age-dependent manner, and levels on thymoma cells were similar to those on thymocytes from age-matched normal controls. MuLV nonproducer tumor cells expressed few or no epitopes of AKR ecotropic gp70, but they were positive when tested with an antiserum prepared against MCFenv glycoprotein.