Measurement of cytosolic free magnesium ion concentration by 19F NMR

Fluorinated derivatives of the chelator o-aminophenol-N,N,O-triacetic acid (APTRA) have been developed, synthesized, and analyzed for use as 19F NMR indicators of free cytosolic magnesium concentration. Magnesium dissociation constants for the 4-fluoro, 5-fluoro, and 4-methyl-5-fluoro species were determined to be 3.1, 0.9, and 0.6 mM, respectively, on the basis of UV absorption measurements at 37 degrees C in 115 mM KCl and 20 mM NaCl, pH 7.1, buffered with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-tris-(hydroxymethyl)aminomethane. The corresponding pK values, which reflect protonation of the nitrogen atom, were determined by 19F NMR to be 4.15, 5.45, and 5.55, respectively, so that the chelators are insensitive to pH variations near the normal physiological range. The dissociation constants of these chelators for calcium ions are lower than those for magnesium but roughly 2-3 orders of magnitude above typical basal cytosolic free calcium levels, so that calcium ions will not interfere with the determinations of magnesium levels. 19F NMR studies carried out at 339.7 MHz indicate that magnesium ions are in slow exchange with the 5-fluoro and 4-methyl-5-fluoro APTRA derivatives and in fast exchange with the 4-fluoro APTRA derivative. In contrast, calcium ions were found to be in intermediate to fast exchange with all chelators. The apparent anomaly of higher thermodynamic stability of the APTRA complexes for calcium relative to magnesium but lower kinetic stability (higher k-1 values) for the calcium complexes reflects the very different association rates for the two ions. Thus, the magnesium association rates are 3 orders of magnitude slower than those for calcium ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

13C NMR of the bases of three DNA oligonucleotide duplexes: assignment methods and structural features

Natural abundance 13C NMR spectra of three DNA oligomers have been obtained. Most of the base resonances are well resolved from one another. A combination of two independent methods was used in making assignments: a one-dimensional spectral comparison method and a two-dimensional proton-detected 1H-13C correlated experiment for the protonated carbons. There are large shielding changes (between 1.62 and -1.40 ppm) upon thermal dissociation of the duplex. The shapes of the chemical shift vs temperature curves are largely independent of sequence. The base carbon resonance frequencies are sensitive to hydrogen bonding, base stacking, sugar conformation, and changes in the glycosyl torsion angle.     

A phenotypic and functional analysis of long-lived B and T lymphocytes

The lymphocyte composition of spleen, lymph nodes, bone marrow, and thymus of mice submitted to hydroxyurea treatments for four consecutive days was studied. The treatment selects for small lymphocyte populations that represent between 4 and 20% of control numbers in the various organs. Spleen and bone marrow contain the same B cell population with a low IgM, high IgD, low I-E phenotype, which respond to LPS at control clonal frequencies. The T cell compartment is equally depleted, and the lymphocytes remaining contain frequencies of clonable cells in response to mitogens and IL-2 that are comparable to those detected in normal spleen cells. Overall, the results suggest that only a minor fraction of all lymphocytes in a normal young adult mouse have life spans longer than 4 days.     

Identification of the hepatocyte Na+-dependent bile acid transport protein using monoclonal antibodies

Monoclonal antibodies have been utilized to characterize the hepatocyte Na+-dependent bile acid transport system. Sinusoidal plasma membrane proteins in the 49-54-kDa range, which are thought to be components of this transport system, based on photo-affinity labeling and reconstitution studies, have been partially purified by affinity chromatography and utilized as an immunogen for the production of a panel of monoclonal antibodies (mAb). One of these mAbs, 25A-3, recognized both a 49- and a 54-kDa protein as assessed by immunoprecipitation. In addition, it was shown to protect the bile acid transport system from inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a dose-dependent manner. DIDS covalently labeled membrane proteins of 49 and 54 kDa, and this process could be significantly inhibited when performed in the presence of mAb 25A-3. Furthermore, the DIDS-labeled membrane proteins were immunoprecipitated by 25A-3. These results establish that one of these membrane components is the bile acid carrier protein. Another mAb (25D-1) which immunoprecipitated only a 49-kDa protein was shown to block the protective effect of 25A-3 on DIDS inhibition of bile acid transport. In addition both antibodies effected each other's binding capacity to hepatocytes and reacted with the same 49-kDa protein as established by sequential immunoprecipitation. Binding studies indicated that there are approximately 3.3 X 10(6) 49-kDa transport molecules/hepatocyte. These results firmly establish that the 49-kDa protein is the Na+-dependent hepatocyte bile acid transporter.     

Purification and reconstitution of the bile acid transport system from hepatocyte sinusoidal plasma membranes

The taurocholic acid transport system from hepatocyte sinusoidal plasma membranes has been studied using proteoliposome reconstitution procedures. Membrane proteins were initially solubilized in Triton X-100. Following detergent removal, the resultant proteins were incorporated into lipid vesicles prepared from soybean phospholipids (asolectin) using sonication and freeze-thaw procedures. The resultant proteoliposomes demonstrated Na+-dependent transport of taurocholic acid which could be inhibited by bile acids. Greatly reduced amounts of taurocholic acid were associated with the phospholipid or membrane proteins alone prior to proteoliposome formation. Membrane proteins were fractionated on an anionic glycocholate-Sepharose 4B affinity column which was prepared by coupling (3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholan-24-oyl)-N alpha-lysine to activated CH-Sepharose 4B via the epsilon-amino group of lysine resulting in the retention of a free carboxyl group. The adsorbed proteins enriched in components in the 54 kDa zone, which were originally identified by photoaffinity labeling to be components of the bile acid transport system, were also incorporated into liposomes. This vesicle system showed almost a 4-fold increase in Na+-dependent taurocholic acid uptake when compared to proteoliposomes formed from total membrane protein, as well as sensitivity to inhibition by bile acids. These results demonstrate that the bile acid carrier system can be reconstituted in proteoliposomes and that utilizing proteins in the 54 kDa zone leads to a significant enhancement in the transport capacity of the reconstituted system, consistent with the role of 54 kDa protein(s) as component(s) of the bile acid carrier system.     

Synergistic antitumor effect of interferon and anti-idiotype monoclonal antibody in murine lymphoma

Both IFN-alpha and anti-idiotype monoclonal antibody therapy have significant antitumor activity in vivo in a murine B cell lymphoma model. Combination therapy with syngeneic anti-idiotype antibody of the IgG2a or IgG2b isotype (a single i.p. injection of 100 micrograms) and recombinant human hybrid interferon-alpha A/D (10(4) to 10(6) U three times weekly for 3 wk) synergistically increased median survival time in mice challenged with a lethal dose of tumor cells compared with the sum of the median survival times of the two individual treatments. IFN-alpha has direct antiproliferative activity against 38C13 in vitro and enhances in vitro macrophage anti-idiotype antibody-specific cytolysis for IgG2a, IgG2b, and IgG1 isotypes.     

Subcellular Compartmentation of Opioid Receptors: Modulation by Enkephalin and Alkaloids

Abstract: A subclone of NG108–15 neuroblastoma-glioma hybrid cells was used to study the intracellular distribution of opioid receptors. Subcellular organelles were separated on self-generating Percoll-sucrose gradients and the enzymes β-glucuronidase, galactosyltransferase, 5′-nucleotidase, and glucose-6-phosphatase were used as markers to localize the various structures. Analysis of the receptor distribution from untreated cells shows that the plasma membranes contained the highest receptor density, but a significant portion of the opioid binding sites was unevenly distributed between the lysosomes, microsomes, and Golgi elements. The enzyme markers indicated that appearance of opioid receptors in these intracellular structures does not result merely from contamination with plasma membranes. About 11% of the receptors appeared in a fraction lighter than plasma membranes. The antilysosomal agent chloroquine altered the intracellular compartmentation of the receptors, possibly by blocking their translocation in the cells. Leu-enkephalin induced time-dependent loss of receptors from all four intracellular compartments examined, but a kinetic analysis showed that the rate of receptor loss in these fractions was not identical. Thus, the percent of receptors appearing in the lysosomal fraction that could still bind [³H]-D-Ala²D-Leu⁵-enkephalin in vitro was increased on treatment with Leu-enkephalin. As an additional approach to follow the intracellular fate of the receptors, cells were labeled with [³H]diprenorphine, chased with various unlabeled opiates, and the distribution of ³H-ligand-receptors in the cells was monitored. Leu-enkephalin and etorphine altered the distribution of receptor-bound [³H]diprenorphine between the plasma membranes, lysosomes, and Golgi elements, whereas morphine had no such effect. The study sheds light on the role of intracellular structures in the metabolism of opioid receptors in untreated and opioid-treated cells.     

Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

The immune response to mouse hepatitis virus: expression of monocyte procoagulant activity and plasminogen activator during infection in vivo

After infection with 10(3) plaque-forming units of mouse hepatitis virus strain 3 (MHV-3) in vivo, peripheral blood mononuclear cells and splenic cells expressed procoagulant activity (PCA) in a pattern directly correlating with susceptibility to disease. Mononuclear cells from BALB/cJ mice, a strain which is fully susceptible to MHV-3, expressed a greater than 500-fold increase in PCA. PCA was first detected within 12 hr of infection; prior to histologic evidence of disease and viral replication, it reached maximal levels 48 hr post-infection (p.i.) and persisted until the death of the animals 5 to 7 days p.i. Mononuclear cells from C3HeB/FeJ mice expressed a significant but lesser titer of PCA, with elevated PCA persisting throughout the chronically infected state until death of the animals 4 to 6 mo p.i. Basal levels of PCA were detected in mononuclear cells from fully resistant A/J mice despite the presence of large amounts of virus in livers, spleens, and sera from these animals. When mononuclear cells expressing high PCA were subfractionated, monocytes were found to be the cellular source of greater than 96% of the PCA activity. Increased plasminogen activator activity was found in monocytes from resistant A/J mice at the time when PCA was markedly elevated in BALB/cJ and C3HeB/FeJ mice. This activity persisted for 5 to 7 days p.i., but was undetectable 10 days p.i. at a time when the mice had cleared the virus from their blood streams. These observations suggest that monocyte PCA may be important in the pathogenesis of MHV-3 disease, whereas the production of monocyte plasminogen activators may contribute to resistance of A/J mice to MHV-3-induced liver disease.     

Properties and compartmentalization of the testicular receptor for 1,25-dihydroxyvitamin D3

Adult rat testis contains a specific, high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) with properties similar to 1,25-(OH)2D3 receptors in other tissues. The receptor sediments at 3.5 +/- 0.2 S20,w in high-salt sucrose density gradients, but aggregates in low-salt gradients. Binding of 1,25-(OH)2D3 was abolished by trypsin, but not by DNase or RNase. Binding was also heavily reduced by the sulfhydryl alkylating agent, N-ethylmaleimide, and by the mercurial reagent, mersalyl, showing that free, reduced SH-groups are necessary for hormone-binding activity. The receptor shows high affinity for 1,25-(OH)2D3 (Kd = 3 X 10(-11) M), but low capacity (Nmax = 8 fmol/mg protein) and is specific for 1,25-(OH)2D3 (Affinity: 1,25-(OH)2D3 greater than 1,24(R),25-(OH)3D3 greater than 25-OH-D3 greater than 1 alpha-OH-D3 greater than 24(R),25-(OH)2D3 much greater than 17 beta-estradiol, testosterone, dexamethasone, R5020, progesterone). With 0.6 nM [3H]1,25-(OH)2D3 and at 0 degrees C, maximum specific binding was achieved after 4 h, and the occupied receptors were stable for more than 24 h. The dissociation of hormone-receptor complexes was temperature-dependent and very slow at low temperature (t1/2 (0 degrees C) much greater than 48 h). At 0 degrees C, the second order association rate constant and the pseudo-first order dissociation rate constant were 2.7 X 10(7) M-1 min-1 and 2 X 10(-5) min-1, respectively. Receptors for 1,25-(OH)2D3 are present in similar amounts in isolated seminiferous tubules and interstitial tissue of adult rats. No specific binding of [3H]1,25-(OH)2D3 could be detected in cultured immature Sertoli cells, cultured immature peritubular (myoid) cells or crude germ cells.