Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase

We have examined the effects of the "differentiating agent," sodium butyrate, on the induction of alkaline phosphatase in human colonic tumor cell line LS174T. Culture of these cells in the presence of 2 mM butyrate caused this activity to increase from less than 0.0001 unit/mg of protein to greater than 0.7 unit/mg of protein over an 8-day period. This induction proceeded in a nonlinear fashion with a lag time of 2-3 days occurring before enzymatic activity began to rise. These increases in activity were accompanied by elevations in the content of a placental-like isozyme of alkaline phosphatase as demonstrated by "Western" immunoblots. Dome formation, indicative of differentiation in cultured cells, also required 3 days treatment with butyrate before becoming evident. The rate of biosynthesis of the enzyme, examined using metabolic labeling with L-[35S]methionine and immunoprecipitation, was found to increase continuously between days 2 and 6 of butyrate treatment. "Northern" blot analysis indicated that treatment of these cells with butyrate caused greater than 20-fold induction of a 2700-base mRNA that hybridized to a cDNA probe for placental alkaline phosphatase. The mRNA for alkaline phosphatase produced by these cells upon butyrate treatment was approximately 300-400 bases smaller than the mRNA for alkaline phosphatase found in placenta. Human small intestine also contained two mRNAs that hybridized relatively weakly with the placental alkaline phosphatase probe. These results indicate that a placental alkaline phosphatase-like protein and mRNA are induced by butyrate in LS174T cells with a time course consistent with cellular differentiation preceding induction.     

Effect of mutation of cysteinyl residues in yeast Cu-metallothionein

Metallothioneins have been isolated from Saccharomyces cerevisiae CUP1 mutants generated by Wright et al. (Wright, C. F., Hamer, D. H., and McKenney, K. (1986) Nucleic Acids Res. 14, 8489-8499). In the mutant metallothioneins, pairs of cysteinyl residues have been converted to seryl residues. The mutant proteins differ only in the positions of the double substitutions; each mutant molecule contains 10 cysteinyl residues. Each mutant protein lacks the first 8 residues at the amino terminus from the decoded gene sequence of the CUP1 locus. Mutant molecules consist of 53 residues analogous to the wild-type metallothionein and are designated 9/11, 24/26, 36/38, and 49/50 (in reference to the sequence positions of the Cys----Ser conversions). The properties of the mutant metallothioneins are vastly different, and host cells harboring the different plasmid-encoded mutant molecules show marked differences in sensitivity to CuSO4. Growth inhibition was observed at CuSO4 concentrations up to mM in cells containing the 9/11, 24/26, and 36/38 molecules, but not for cells containing protein 49/50. A CuSO4 concentration of 5 mM was required to inhibit the growth of yeast containing either 49/50 or the wild-type metallothionein. In the purified proteins the copper binding stoichiometry of each molecule, except protein 24/26, was nearly 8 mol eq. Protein 24/26 bound 5.5 copper ions/molecule. The Cu(I) chelator bathocuproine disulfonate reacted with over 50% of the copper ions in proteins 9/11, 24/26, and 36/38, but less than 10% of the copper ions in proteins 49/50 and wild-type metallothionein were reactive. The thiolates in 9/11, 24/26, and 36/38 were also more reactive in a disulfide exchange reaction with dithiodipyridine compared with the sulfhydryls in 49/50 and the wild-type molecules. The four mutant copper proteins are luminescent and exhibit a similar quantum yield. The cluster structures contributing to the particular electronic transitions are markedly more sensitive to oxygen in proteins 9/11, 24/26, and 36/38 compared with 49/50 and the wild-type molecules. The air-sensitive proteins exhibit a tertiary fold not recognized by polyclonal antibodies directed to a conformational epitope on yeast Cu-metallothionein. Protein 49/50 cross-reacts with the antibody in a concentration-dependent fashion similar to the wild-type protein. Mutation of 2 cysteinyl residues in the carboxyl portion of metallothionein does not significantly alter properties of the molecule, whereas mutation of several cysteines in the amino-terminal portion of the molecule yields a different conformation.     

Acrosomal status evaluation in human ejaculated sperm with monoclonal antibodies

An important question in mammalian gamete physiology concerns how capacitation and the occurrence of acrosome reactions in motile sperm relate to fertility. Evaluation of these relationships has been restricted by practical limitations because rapid, quantitative assays are unavailable. We have developed a rapid, reproducible assay for the evaluation of acrosomal status utilizing monoclonal antibodies specific to antigens localized in the acrosomal cap region of the sperm head. Mice were immunized with human ejaculated sperm preparations and the resultant hybridomas producing antisperm antibody were selected by solid-phase radioimmunoassay and indirect immunofluorescence (IIF). Two monoclonal antibodies (HS-19, HS-21) recognized target antigens restricted to the acrosomal cap by IIF, and 87 +/- 8.5% of the sperm in fresh ejaculates from 10 different sperm donors showed positive cap fluorescence with these reagents. Loss of HS-21 binding as measured by IIF was correlated with disappearance of the acrosomal cap as observed directly by transmission electron microscopy. Acrosomal disappearance, artificially induced in vitro using the calcium ionophore A23187, also resulted in a loss of HS-21 binding. The induction of acrosomal loss by ionophore was dependent upon extracellular calcium. The data presented suggest that specific monoclonal antibodies can be used for the rapid evaluation of acrosomal status in mammalian sperm.     

Alkyl phosphotriester modified oligodeoxyribonucleotides. VI. NMR and UV spectroscopic studies of ethyl phosphotriester (Et) modified Rp-Rp and Sp-Sp duplexes, (d[GGAA(Et)TTCC])2.

1H NMR chemical shift assignments for the title compounds were made for all but a few H5' and H5" signals using two-dimensional nuclear Overhauser effect (2D-NOE) data, which was also used for the first time to assign absolute configuration at phosphorus. The chemical shifts were, in general, similar to those reported [Broido, M.S., et al. (1985) Eur. J. Biochem. 150, 117-128] for the B-like conformation of the unmodified, parent duplex, [d(GGAATTCC)]2. Differences in chemical shifts for corresponding protons were mostly localized to the AA(Et)TT region, and showed some stereochemical dependence. Unambiguous assignment of the phosphotriester 31P signals was achieved in a novel way using selective insensitive nucleus enhancement by polarization transfer (selective INEPT) NMR. The Rp-Rp duplex melted ca. 11 degrees C lower than either the Sp-Sp or parent duplexes, as evidenced by Tm and variable temperature 1H/31P NMR measurements. The 2D-NOE data for the Rp-Rp duplex suggested possible steric interactions between the ethyl group and the H3' of the flanking A residue. At low ionic strength, the Sp-Sp and parent duplexes had similar stability but at high ionic strength the Sp-Sp duplex was less stable.     

Molecular cloning of rat intestinal mucin. Lack of conservation between mammalian species

We have prepared antisera to deglycosylated rat intestinal mucin and used it to obtain immunoreactive clones from a rat jejunum cDNA library. Four of these clones were sequenced, and all were found to be partial cDNAs that contained 18-base pair tandem repeats characteristic of a mucin. These cDNAs encoded a repetitive peptide with a consensus sequence of TTTPDV. Thus, they bear little resemblance to either of the two human intestinal mucin cDNAs isolated previously (Gum, J. R., Byrd, J. C., Hicks, J. W., Toribara, N. W., Lamport, D. T. A., and Kim, Y. S. (1989) J. Biol. Chem. 264, 6480-6487 and Gum, J. R., Hicks, J. W., Swallow, D. M., Lagace, R. E., Byrd, J. C., Lamport, D. T. A., Siddiki, B., and Kim, Y. S. (1990) Biochem. Biophys. Res. Commun. 171, 407-415). One of these rat mucin clones, designated RMUC 176, was chosen for further analysis. This clone recognized a band of approximately 9 kilobases when used to probe RNA blots. A strong hybridization band was present using rat small intestine and colon RNA but was not detectable when RNA isolated from heart, liver, or kidney was tested. The RMUC 176 clone and the two previously isolated human intestinal mucin cDNA clones were used to probe blots prepared from BamHI-digested DNA of various species. Here, the human probes detected fragments present only in human and chimpanzee DNA, whereas the RMUC 176 clone recognized fragments only in rat and mouse DNA. Thus, the repetitive portions of intestinal mucin genes are apparently not well conserved between phylogenetically distant species.     

Organization and nucleotide sequence of the rat T cell receptor beta-chain complex.

We have characterized four overlapping genomic clones containing the DA rat TCR C beta complex, which span a total of 23 kb and bear two closely related complexes of gene segments. The D beta 1-J beta 1-C beta 1 and the D beta 2-J beta 2-C beta 2 complexes each contain a single diversity segment, six joining segments and four exons that encode the C region. All gene segments appear to be functional except J beta 2.5, which has a 5-bp frame-shifting deletion. This organizational pattern is identical to that of the mouse, and the homologous rat and mouse coding regions share about 92% nucleotide sequence identity. Our sequence comparisons indicate that a localized gene correction event has homogenized the sequences of the first exons of C beta 1 and C beta 2 in the evolutionary time since rats and mice became separate species. We have identified three repetitive elements, each flanked by short direct repeats, present in the region "brain-specific" identifier (ID) sequences, another is a truncated member of the LINE I class of repetitive elements, and the third is a member of the Alu type 2 family. The insertion of at least two, and probably all, of these elements has occurred since the time of rat/mouse divergence. We have identified a substantial number of "cryptic" rearrangement signals (heptamer/nonamer) in the C beta locus, which match the consensus sequence as well or better than authentic signals, and may represent sites of nonfunctional rearrangements.     

Effect of Abx, Bx and Pbx Mutations on Expression of Homeotic Genes in Drosophila Larvae

We have examined the patterns of expression of the homeotic gene Ubx in imaginal discs of Drosophila larvae carrying mutations in the abx, bx and pbx regulatory domains. In haltere discs, all five bx insertion mutations examined led to a general reduction in Ubx expression in the anterior compartment; for a given allele, the strength of the adult cuticle phenotype correlated with the degree of Ubx reduction. Deletions mapping near or overlapping the sites of bx insertions, including three abx alleles and the bx34e-prv(bx-prv) allele, showed greatly reduced Ubx expression in parts of the anterior compartment of the haltere disc; however, anterior patches of strong Ubx expression often remained, in highly variable patterns. As expected, the pbx1 mutation led to reduced Ubx expression in the posterior compartment of the haltere disc; surprisingly, pbx1 also led to altered expression of the en protein near the compartment border in the central region of the disc. In the metathoracic leg, all the bx alleles caused extreme reduction in Ubx expression in the anterior regions, with no allele-specific differences. In contrast, abx and bx-prv alleles resulted in patchy anterior reductions in third leg discs. In the larval central nervous system, abx but not bx alleles affected Ubx expression; the bx-prv deletion gave a wild-type phenotype, but it could not fully complement abx mutations. In the posterior wing disc, the bx-prv allele, and to a much lesser extent the bx34e chromosome from which it arose, led to ectopic expression of Ubx. Unlike other grain-of-function mutations in the BX-C, this phenotype appeared to be partially recessive to wild type. Finally, we asked whether the ppx transformation, which results from early lack of Ubx+ function in the mesothorax and is seen in abx animals, is due to ectopic Scr expression. Some mesothoracic leg and wing discs from abx2 larvae displayed ectopic expression of Scr, which was variable in extent but always confined to the posterior compartment.     

Molecular cloning of cDNAs derived from a novel human intestinal mucin gene

A human small intestinal lambda gt11 cDNA library was screened with antibodies to deglycosylated small intestinal mucin. Four partial cDNA clones were isolated that define a novel human mucin gene. These include two partial cDNA clones, SIB 124 and SIB 139, that contain 51 nucleotide tandem repeats which encode a seventeen amino acid repetitive peptide with a consensus sequence of HSTPSFTSSITTTETTS. SIB 139 hybridized to messages produced by small intestine, colon, colonic tumors and also by high mucin variant LS174T colon cancer cells. The gene from which cDNAs SIB 124 and SIB 139 are derived (proposed name MUC 3) maps to chromosome 7, distinct from other known human mucin genes.     

The relationship between plasma free fatty acids and liver mitochondrial function in vivo

P/O ratio, state 3 and 4 respiration rates, and acceptor control index (ACI) were assessed in rat liver mitochondria following an overnight fast and single bout of treadmill exercise of 30-180 min. P/O was unaffected by fasting and 30 min of exercise; however, ACI was reduced because of an increase in state 4 respiration. Fasting, followed by running for 1 h or more decreased P/O approx. 40% and ACI by 50%, an effect that could be attributed to a reduction in state 3 respiration. The decrease in P/O was reversed 15 min after the cessation of exercise, whereas ACI remained depressed. All these functional alterations were mimicked by incubation of isolated mitochondria with palmitate and reversed by washing them with albumin. No direct correlation between plasma free fatty acids and the alterations in mitochondrial respiration was apparent. These data demonstrate that the decrease in the normal coupling of oxidation and phosphorylation in liver mitochondria produced by fasting/exercise is reversed rapidly in vivo. Furthermore, it is apparent that, if fatty acids act as a regulatory agent under these conditions, they do not do so solely on the basis of their plasma concentration.