The ontogeny of expression of murine metallothionein: comparison with the alpha-fetoprotein gene

The ontogeny of expression of mouse metallothionein was studied by RNA dot and Northern blot hybridization using a cloned cDNA probe. In some instances the synthesis of metallothionein was analyzed by cell-free translation of RNA as well as pulse-labeling of proteins in short-term organ cultures followed by polyacrylamide gel electrophoresis. Interesting parallels between metallothionein and alpha-fetoprotein gene expression during development were noted. Like alpha-fetoprotein mRNA ( Dziadek and Andrews, 1983), metallothionein mRNA was found to be abundant in developing liver as well as in visceral yolk sac endoderm. In addition, metallothionein mRNA was abundant in parietal yolk sac. During liver development metallothionein and alpha-fetoprotein mRNAs were abundant by Day 12 of gestation, increasing to maximal levels on Day 16 and decreasing during late fetal and neonatal life to basal levels in adult. Metallothionein mRNA increased in maternal liver and was also abundant in certain hepatomas. Synthesis of metallothionein and levels of metallothionein mRNA in visceral yolk sac increased from Day 9 of gestation to maximal levels on Days 11-12 and then decreased abruptly after Day 15. RNA from differentiated teratocarcinoma cells with primitive, parietal or visceral endoderm characteristics each contained high levels of metallothionein mRNA, whereas, levels of this mRNA varied widely among embryonal carcinoma stem cell lines. alpha-Fetoprotein mRNA was not detected in embryonal carcinoma cells but was expressed in visceral endoderm-like differentiated cells. These results indicate that parietal and visceral endoderm cells actively express the metallothionein gene and further suggest that expression may be initiated at the earlier stage of primitive endoderm.     

Control of the expression of interleukin-2 activity

Mitogen stimulation of lymphocytes activates phospholipase A₂, which in turn generates arachidonic acid by its action on phospholipids. Cyclooxygenases catalyze the conversion of arachidonic acid to prostaglandins and related cyclic compounds, whereas lipoxygenases direct the formation of straight-chain hydroxylated derivatives such as, for example, the leukotrienes. The studies in this report suggest a correlation between arachidonic acid metabolism and production of the lymphokine, interleukin-2 (IL-2). Inhibitors of phospholipase A₂ activation, mepacrine, tetracaine, glucocorticoids and estradiol, all inhibited the expression of IL-2 activity in concanavalin A-stimulated mouse spleen cells. Inhibition of cyclooxygenase and lipoxygenase activities also resulted in decreased IL-2 production. This was established by the use of the inhibitors 5,8,11,14-eicosatetraynoic acid (ETYA), indomethacin, and nordihydroguajaretic acid (NDGA). A more direct attempt at influencing the arachidonic acid metabolism by addition of the fatty acid to IL-2 production cultures demonstrated that arachidonic acid bound very tightly to IL-2. Extensive dialysis or partial purification of the lymphokine by reverse-phase high-performance liquid chromatography failed to remove the bound arachidonic acid. It was shown, however, that no covalent interactions were involved. In addition to an active arachidonic acid metabolism, continuous protein synthesis was required for expression of IL-2 activity. Thus incubation with puromycin inhibited IL-2 production.     

A simple method for the preparation of polyacrylamide gels containing thioglycoside ligands.

A simple method was developed for the preparation of acrylamide derivatives containing thioglycosides. The synthetic scheme involves the preparation of an O-acetylated 1-thiosugar via a pseudothiourea derivative, the controlled addition of the thiosugar to N,N′-bismethyleneacrylamide to form a monoaddition product in which one of the acrylamide groups remains unchanged, and finally de-O-acetylation. Similar reaction schemes using bisacrylamido derivatives of 1,2-diaminoethane and 1,6-diaminohexane lead to analogous compounds with longer aglycon chain lengths. The thioglycoside derivatives of acrylamide thus prepared could be copolymerized with acrylamide to form polymers or gels containing thioglycosides as ligands. These gels were successfully used as affinity materials for the purification of peanut lectin and in cell-adhesion studies.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Mitochondrial DNA-binding proteins that bind preferentially to supercoiled molecules containing the D-loop region of Xenopus laevis mtDNA

From the bulk of the Xenopus laevis mitochondrial proteins insoluble in 1% Triton X-100 + 1M NaCl, we have isolated, by DNA-cellulose chromatography, a protein fraction enriched in DNA-binding proteins. This fraction contains proteins showing a specific affinity for supercoiled DNA molecules containing the mitochondrial DNA displacement-loop region, as measured by filter binding and competition assays.     

Characterization of canine T-lymphocyte subpopulations: the detection of T mu and T gamma lymphocytes with homologous and heterologous immunoglobulins and the requirements for Fc receptor expression

Erythrocyte antibody (EA) rosette techniques employing sheep red blood cells sensitized with canine (homologous) and rabbit (heterologous) IgM and IgG antibodies were used to determine the number of cells with Fc receptors for IgM (Tμ) and IgG (Tγ) among T lymphocytes isolated from peripheral blood and lymph nodes of dogs. The percentages of Tμ and Tγ lymphocytes detected were found to be independent of the species origin of sensitizing antibody. Among peripheral blood T lymphocytes there were 53.0 ± 2.7% Tμ cells and 18.4 ± 3.6% Tγ cells. T lymphocytes obtained from lymph nodes were 62.1 ± 5.4% Tγ and 15.7 ± 2.6% Tγ. The number of Tμ cells detected increased from 20.0% when freshly isolated to 49.1 ± 4.1% after in vitro culture for 2–16 hr. The expression of the Fc-μ receptor in culture was inhibited by cycloheximide, demonstrating a requirement for active protein synthesis. In contrast, the number of Tγ lymphocytes detected did not vary between freshly isolated cells and those which had been cultured for 16 hr. Expression of the Fc-γ receptor during this time period was not inhibited by cycloheximide.     

Sustained oscillations in a reconstituted enzyme system containing phosphofructokinase and fructose 1,6-bisphosphatase

In a reconstituted open and homogeneous enzyme system containing phosphofructokinase, fructose 1,6-bisphosphatase, pyruvate kinase, adenylate kinase, and glucose-6-phosphate isomerase sustained oscillations could experimentally be generated. The approach is based on a stirred flow-through reaction chamber. The periodic motions of the reactants are mainly caused by the antagonistic allosteric effects of the adenine nucleotides on the activities of the phosphofructokinase and fructose 1,6-bisphosphatase.     

Multiple dioxygenation by lipoxygenase of lipids containing all-cis-1, 4, 7-octatriene moieties.

Soybean lipoxygenase-1 acting upon polyunsaturated fatty acids containing a suitably positioned all-cis-1,4,7-octatriene moiety generates bishydroperoxy derivatives while consuming approximately 2 mol of O₂ per mole of substrate. The reaction has been separated into two steps: The first is marked by the rapid monohydroperoxidation of the starting material and the second is marked by the much slower hydroperoxidation of the first product. All of the compounds which are successfully converted to bishydroperoxy derivatives contain an ω6,9,12 grouping of cis double bonds. α-Linolenic acid (ω3,6,9) cannot serve as a substrate because its preferred site of oxygenation is in the center of the octatriene moiety. The K_m for arachidonic acid in the reaction leading to the singly hydroperoxidized monohydroperoxide is 8.6× 10^?5, m, which is approximately 200 times smaller than the K_m for the monohydroperoxide in the second step leading to the bishydroperoxide. All of the substrates which undergo double dioxygenation form conjugated trienes.     

Sequence and expression of the discoidin I gene family in Dictyostelium discoideum

We have isolated recombinant phage and plasmids containing the four developmentally regulated discoidin I genes of Dictyostelium discoideum. Two of the genes are linked within 0.5 × 10³ bases with the same polarity. S¹ nuclease mapping shows that at least three members of the gene family are expressed and that the 5′ ends of the mRNAs start at equivalent sites. The genes have homologous 5′ untranslated regions and extremely divergent 3′ untranslated regions. In addition, some of the genes are flanked by homologous repeat sequences. The genes encode three different isoelectric forms of the protein. Examination of nucleotide sequences in the protein coding region shows that most nucleotide changes in the 5′ half of the gene result in amino acid substitutions while most base substitutions in the 3′ half are neutral.     

Regional differences in the expression of myosin light chains and tropomyosin subunits during development of chicken breast muscle

Types of myosin light chains and tropomyosins present in various regions and at different developmental stages of embryonic and posthatched chicken breast muscle (pectoralis major) have been characterized by two-dimensional gel electrophoresis. In the embryonic muscle all areas appear to accumulate both slow and fast forms of mysoin light chains in addition to α and β forms of tropomyosin. During development regional differences in myosin and tropomyosin expression become apparent. Slow myosin subunits become gradually restricted to areas of the anterior region of the muscle and finally become localized to a small red strip found on its anterior deep surface. This red region is characterized by the presence of slow and fast myosin light chains, α-fast, α-slow, and β-tropomyosin. In all other areas of the muscle examined only fast myosin light chains, β-tropomyosin and the α-fast form of tropomyosin, are found. In addition, β-tropomyosin also gradually becomes lost in the posterior regions of the developing breast muscle. In the adult, the red strip area represents less than 1% of the total pectoralis major mass and of the myosin extracted from this area approximately 15% was present as an isozyme that comigrated on nondenaturing gels with myosin from a slow muscle (anterior latissimus dorsi). The red region accumulates therefore fast as well as slow muscle myosin. Thus while the adult chicken pectoralis major is over 99% fast white muscle, the embryonic muscle displays a significant and changing capacity to accumulate both fast and slow muscle peptides.     

Incorporation of a fatty acid containing a photosensitive group into lipids of cultured cardiac cells from chick embryo

Radioactive 12-(4-azido-2-nitrophenoxy)-stearic acid (NAP-stearate) was synthetized; it behaves as a competitive inhibitor of long-chain fatty acids for the entry into cultured cardiac cells from chick embryo. After uptake, [3H] NAP-stearate was incorporated by an energy-dependent process into neutral and polar lipids. Photoactivation as a function of time leads to a covalent labelling of the cells: up to 31 per cent of the radioactivity was recovered in the 105 000 g cell pellet, mainly in proteins. These experiments show that fatty acids containing photosensitive groups would potentially allow to localize the proteins involved in the binding and/or in the transport of fatty acids.     

Anti-mutagenic chalcones: antagonizing the mutagenicity of benzo(a)pyrene on Salmonella typhimurium

Several chalcone derivatives; e.g. Ro 09-0204, Ro 09-0323 and Ro 09-0501 were found to reduce markedly the revertant increase of Salmonella typhimurium TA100 by benzo(a)pyrene during the incubation with S-9 Mix. The antimutagenic activity was 100 - 700 times stronger than that of L-ascorbic acid. Effect on other mutagens, the structure activity relationship and the possible mechanism of action are briefly discussed.     

Studies on the reduction of cytochrome c by thiols : A preliminary report

The reduction of cytochrome c by reduced glutathione is catalyzed by an impurity or impurities present in oxidized glutathione and cystine. In contrast to the conclusions arrived at by V. Massey, C. H. Williams, and G. Palmer [Biochem. Biophys. Res. Commun. 42, 730 (1971)], we conclude that this catalysis is not primarily caused by trisulfides. Heating of cystine or GSSG solutions results in a marked increase in the catalytic properties of the solutions. The formation of the catalytic compounds by heating is favored at alkaline pH, with an apparent pK value of about 9.0. The catalytic compounds appear to possess absorption spectra with maxima at 285 nm for GSSG solutions and 300 nm for cystine solutions. The compounds appear to be stable at pH values between 1.3 and 11. The identity of these compounds is presently being investigated.