Effect of transforming growth factor beta (TGF-β) on ATP citrate lyase in isolated hepatocytes

Summary Transforming growth factor beta (TGF-<img src="/content/h1314432126t4246/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">) activates ATP citrate lyase in freshly isolated rat liver hepatocytes in a time dependent manner. Maximal stimulation of the enzyme occurred with less than thirty minutes of incubation of the cells with TGF-<img src="/content/h1314432126t4246/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">. The half maximal effect on the enzyme determined in hepatocytes incubated with TGF-<img src="/content/h1314432126t4246/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"> for 10 min at 37°C was elicited by TGF-<img src="/content/h1314432126t4246/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"> concentrations in the 10<sup>–11</sup> – 10<sup>–12</sup> M range. The potential role of TGF-<img src="/content/h1314432126t4246/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"> stimulation of ATP citrate lyase activity in new membrane synthesis is discussed.     

Separation of chitosomes and secretory vesicles from the “slime” variant of Neurospora crassa

Cells from the <img src="/content/t18787603372n062/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">slime<img src="/content/t18787603372n062/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0"> variant of <i>Neurospora crassa</i> were broken in isotonic conditions by use of triethanolamine buffer plus EDTA. After removal of large membranous structures by low-speed centrifugation, chitosomes and secretory vesicles were separated by means of gel filtration, precipitation of membranous contaminants with Concanavalin A, and centrifugation in sucrose or glycerol gradients. Polypeptidic composition of fractions enriched in secretory vesicles or chitosomes was found to be distinct. By these criteria we concluded that chitosomes and secretory vesicles represent different populations of microvesicles. Both microvesicular populations appeared free of endoplasmic reticulum and vacuolar contaminants as demonstrated by determination of appropriate enzymatic markers.Abbreviations ER Endoplasmic reticulum - UDP-GlcNAc uridine-5<img src="/content/t18787603372n062/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-diphosphate N-acetyl glucosamine - GlcNAc N-acetyl glucosamine - SDS sodium dodecyl sulfate - PMSF phenyl methyl sulfonyl fluoride - EDTA ethylene diamino tetraacetic acid Investigador Nacional de Mexico. On leave from the Centro de Investigacion y Estudios Avanzados (IPN), and the Universidad de Guanajuato, Gto., Mexico     

The organization of repetitive sequences in the albumin and alpha-fetoprotein gene loci in the rat

Summary The distribution of middle repetitive sequences in the genic and extragenic regions of the rat albumin and <img src="/content/t7l55v7372129272/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-fetoprotein genes was analyzed. Their presence was determined by probing Southern blots of restriction fragments of albumin and <img src="/content/t7l55v7372129272/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-fetoprotein genomic subclones with <sup>32</sup>P-labeled total rat DNA. Repetitive sequences were detected in both genes. They were classified as weak, moderate and intense hybridizing elements according to the intensity of hybridization. Weak repetitive sequences were characterized as dG·dT repeats by using <sup>32</sup>P-labeled poly-(dG·dT)(dC·dA) oligomer probe. They occurred in 5<img src="/content/t7l55v7372129272/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> and 3<img src="/content/t7l55v7372129272/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> extragenic regions of the two genes and in introns 4 and 5 of the albumin gene. The moderate repetitive sequence present in intron 6 of the albumin gene was identified as the rat SINES element, 4D12. The intense repetitive sequence, localized in the 3<img src="/content/t7l55v7372129272/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> non-coding region of the albumin gene, corresponded to the terminal segment of a rat high repeat long interspersed DNA family, L1Rn. 4D12 and L1Rn sequences were also scattered throughout the <img src="/content/t7l55v7372129272/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-fetoprotein locus as moderate and intense repetitive elements, respectively, but their distribution was different from that of the albumin genomic region. These results indicate that repetitive sequences invaded the two loci in a non-conservative manner.     

Selective labeling of α-bungarotoxin with fluorescein isothiocyanate and its use for the study of toxin-acetylcholine receptor interactions

The main product of the reaction of fluorescein isothiocyanate (FITC) and bungarotoxin (Bgt) under near stoichiometric conditions is a monofluorescein derivative preferentially labeled at Lys 26, a highly conserved residue known to be involved in the binding (McDaniel, C. S., Manshouri, T., and Atassi, M. Z. (1987)<i>J. Prot. Chem.</i> <b>6</b>, 455–461; Garcia-Borron, J. C., Bieber, A. L., and Martinez-Carrion, M. (1987)<i>Biochemistry</i> <b>26</b>, 4295–4303) of postsynaptic neurotoxins specific for the nicotinic acetylcholine receptor (AcChR). The fluorescently labeled toxin retains a high affinity for the AcChR, and an unaltered specificity. Binding of FITC-Bgt to AcChR results in a significant decrease in the fluorescence intensity of the probe. This AcChR-mediated quenching of FITC-Bgt fluorescence allows for a continuous monitoring of the binding process. The quenching of free and bound FITC-Bgt by charged and neutral quenchers shows few fluorophore accessibility changes as induced by the toxin-bound state. The results are consistent with a model in which the positively charged concave surface of the toxin interacts with a negatively charged complementary surface in the receptor molecule.     

Core substructure in Mastigocladus laminosus phycobilisomes

A native high molecular complex (M<sub>r</sub> 850000) containing about 50% of the allphycocyanin of the phycobilisome but lacking allophycocyanin B was separated from isolated phycobilisomes by gel electrophoresis. It was designated AP<sub>CM</sub> since the large linker polypeptide L<sub>CM</sub> was exclusively localized in this complex. The complex exhibited a ?196°C fluorescence emission maximum at 673 nm (671 nm at 25°C). In addition, a core complex (designated AP<sub>C</sub>, M<sub>r</sub>≥1000000) consisting of both AP<sub>CM</sub> and AP 680 was isolated by combined gel filtration and linear gradient centrifugation. At 25°C this complex showed dual emission peaks at 670 and 680 nm demonstrating functional independence of the terminal emitters. A complex similar to AP<sub>CM</sub> can be isolated from phycobilisomes of <em>Anabaena variabilis</em>. This is evidence that AP<sub>CM</sub> is the constitutive center of the tricylindrical core of hemidiscoidal cyanobacterial phycobilisomes. Two models summarizing the structural and functional consequences of the results are presented in the discussion.     

The regions of alpha-neurotoxin binding on the extracellular part of the alpha-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the <img src="/content/k37851240158t29p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-subunit of human acetylcholine receptor were studied for their binding activity of<sup>125</sup>I-labeled <img src="/content/k37851240158t29p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide <img src="/content/k37851240158t29p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">122–138. In addition, low-binding activities were obtained with peptides <img src="/content/k37851240158t29p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">34–49 and <img src="/content/k37851240158t29p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">194–210. It is concluded that the region within residues <img src="/content/k37851240158t29p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

Expression sites and developmental regulation of genes encoding (1→3,1→4)-β-glucanases in germinated barley

Expression sites of genes encoding (1<img src="/content/t7845513235851r1/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">3,1<img src="/content/t7845513235851r1/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">4)-<i><img src="/content/t7845513235851r1/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"></i>-glucan 4-glucanohydrolase (EC 3.2.1.73) have been mapped in germinated barley grains (<i>Hordeum vulgare</i> L.) by hybridization histochemistry. A<sup>32</sup>P-labelled cDNA (copy DNA) probe was hybridized to cryosections of intact barley grains to localize complementary mRNAs. No mRNA encoding (1<img src="/content/t7845513235851r1/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">3,1<img src="/content/t7845513235851r1/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">4)-<i><img src="/content/t7845513235851r1/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"></i>-glucanase is detected in ungerminated grain. Expression of (1<img src="/content/t7845513235851r1/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">3,1<img src="/content/t7845513235851r1/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">4)-<i><img src="/content/t7845513235851r1/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"></i>-glucanase genes is first detected in the scutellum after 1 d and is confined to the epithelial layer. At this stage, no expression is apparent in the aleurone. After 2 d, levels of (1<img src="/content/t7845513235851r1/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">3,1<img src="/content/t7845513235851r1/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">4)-<i><img src="/content/t7845513235851r1/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"></i>-glucanase mRNA decrease in the scutellar epithelium but increase in the aleurone. In the aleurone layer, induction of (1<img src="/content/t7845513235851r1/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">3,1<img src="/content/t7845513235851r1/xxlarge8594.gif" alt="rarr" align="BASELINE" BORDER="0">4)-<i><img src="/content/t7845513235851r1/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"></i>-glucanase gene expression, as measured by mRNA accumulation, progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum.Abbreviations cDNA copy DNA - RNase ribonuclease     

Regulation of polyphosphate metabolism in Acinetobacter strain 210A grown in carbon- and phosphate-limited continuous cultures

The response of <i>Acinetobacter</i> strain 210A to low phosphate concentrations was investigated in P- or C-limited chemostat cultures. The organism accumulated poly-<img src="/content/x572u11560t8068n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-hydroxybutyric acid under P-deprivation, at phosphate concentrations ranging from 0.1 to 0.7 mM. The amount of biomass was proportional to the phosphate concentration in the medium and no polyphosphate was formed. When shifting a culture from P- to C-limitation phosphate was accumulated as polyphosphate. No poly-<img src="/content/x572u11560t8068n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-hydroxybutyrate could be detected in these cells. The amount of polyphosphate in the cell showed a hysteresis. When cultures were shifted from low to high phosphate concentrations, polyphosphate reached a maximum of about 60 mg P per gram of dry weight at about 3 times excess phosphate (ca. 2.5 mM P<sub>i</sub>). It decreased to 45 mg P per gram dry weight at approximately 5 times the phosphate needed for growth (ca. 3.5 mM P<sub>i</sub>). In the reverse case (high to low) polyphosphate did never exceed 45 mg P per gram dry weight. The specific activities of alkaline phosphatase and the phosphate uptake system were induced at residual P<sub>i</sub> concentrations below the detection limit (<10 <img src="/content/x572u11560t8068n/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M). The specific uptake rate followed also a hysteresis. The specific activities of polyphosphatase and polyphosphate: AMP phosphotransferase increased when polyphosphate formation was possible.Abbreviations HPP High polymeric polyphosphates - PHB Poly-<img src="/content/x572u11560t8068n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-hydroxybutyric acid - PP<sub>n</sub> Polyphosphate - PQQ Pyrrolo-quinoline quinone - U 1 <img src="/content/x572u11560t8068n/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">mol product formed · min<sup>-1</sup>     

Anaerobic degradation of trans-cinnamate and ω-phenylalkane carboxylic acids by the photosynthetic bacterium Rhodopseudomonas palustris: evidence for a β-oxidation mechanism

The mechanism responsible for the initial steps in the anaerobic degradation of <i>trans</i>-cinnamate and <img src="/content/t846t1801r316232/xxlarge969.gif" alt="ohgr" align="BASELINE" BORDER="0">-phenylalkane carboxylates by the purple non-sulphur photosynthetic bacterium <i>Rhodopseudomonas palustris</i> was investigated. Phenylacetate did not support growth and there was a marked CO<sub>2</sub> dependence for growth on acids with greater side-chain lengths. Here, CO<sub>2</sub> was presumably acting as a redox sink for the disposal of excess reducing equivalents. Growth on benzoate did not require the addition of exogenous CO<sub>2</sub>. Aromatic acids with an odd number of side-chain carbon atoms (3-phenylpropionate, 5-phenylvalerate, 7-phenylheptanoate) gave greater apparent molar growth yields than those with an even number of side-chain carbon atoms (4-phenylbutyrate, 6-phenylhexanoate, 8-phenyloctanoate). HPLC analysis revealed that phenylacetate accumulated and persisted in the culture medium during growth on these latter compounds. Cinnamate and benzoate transiently accumulated in the culture medium during growth on 3-phenylpropionate, and benzoate alone accumulated transiently during the course of <i>trans</i>-cinnamate degradation. The transient accumulation of 4-phenyl-2-butenoic acid occurred during growth on 4-phenylbutyrate, and phenylacetate accumulated to a 1:1 molar stoichiometry with the initial 4-phenylbutyrate concentration. It is proposed that the initial steps in the anaerobic degradation of <i>trans</i>-cinnamate and the group of acids from 3-phenylpropionate to 8-phenyloctanoate involves <img src="/content/t846t1801r316232/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-oxidation of the side-chain.Abbreviation 3-PP 3-phenylpropionic acid - 4-PB 4-phenylbutyric acid - 5-PV 5-phenylvaleric acid - 6-PH 6-phenylhexanoic acid - 7-PH 7-phenylheptanoic acid - 8-PO 8-phenyloctanoic acid - 4-P2B 4-phenyl-2-butenoic acid - GC/MS Gas chromatography/Mass spectrometry - HPLC High-pressure liquid chromatography     

Comparative efficiencies of bispecific F(ab′γ)2 and chimeric mouse/human IgG antibodies in recruiting cellular effectors for cytotoxicity via Fcγ receptors

Summary The three forms of Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0"> receptor carried by monocytes (Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">RI, II) and natural killer (NK) cells (Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">RIII) are all capable of mediating cell lysis. Here we compare the use of F(ab<img src="/content/j8718xt0312t7050/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"><img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">)<sub>2</sub> bispecific antibodies, specifically targetting individual Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">R, and chimeric IgG mouse/human antibodies which are capable of targetting all Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">R, for their ability to mediate target cell destruction. The derivatives are prepared by linking hinge sulphydryl residues via tandem thioether bonds, using a bismaleimide crosslinker: Fab<img src="/content/j8718xt0312t7050/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> from an anti-Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">R mAb linked to Fab<img src="/content/j8718xt0312t7050/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> from a common anti-target mAb (BsAb), or Fab<img src="/content/j8718xt0312t7050/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> from the common anti-target mouse antibody linked to human Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0"> (FabFc or bisFabFc). All the derivatives targetting chick red blood cells gave efficient lysis, although different effector cell donors yielded differences in both the lytic levels achieved and the comparative efficiencies of derivatives. In contrast, significant lysis of the guinea pig lymphoblastic leukaemia, L<sub>2</sub>C, regularly resulted only via the anti-Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">RIII BsAb and the chimeric derivatives. These results suggest that the chimeric, Fc-containing derivatives mediate tumour cell lysis principally through Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">RIII on NK cells. This is in contrast to the situation with the chick red blood cells where the chimeric derivatives appear capable of lysing erythrocytes by utilizing either monocytes or NK cells, because significant (<img src="/content/j8718xt0312t7050/xxlarge8776.gif" alt="ap" align="MIDDLE" BORDER="0">50%) lysis occurred with effector cell populations magnetically depleted through either Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">RII or Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">RIII. A major difference between these two types of antibody derivative was their ability to function in the presence of high concentrations of normal human Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">. The lysis mediated by BsAb reactive with Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">RI or II was unaffected by the presence of human Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0"> at 2.5 mg/ml (a concentration comparable with that yielded by IgG in plasma) whereas the BsAb recognizing Fc<img src="/content/j8718xt0312t7050/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">RIII and all the Fc-containing derivatives were completely inhibited.This work has been supported by Tenovus, the Cancer Research Campaign, the Leukaemia Research Fund, Italfarmaco, Milano, Italy and the Imperial Cancer Research Fund