Decreased expression of J11d antigen during monocytic differentiation of M1 myeloid leukemic cells.

Four myeloid cell lines (M1, WEHI-3B D+, FDC-P1, and 32D) were screened for the presence of J11d antigen. One of these cell lines, the myeloid leukemia M1, was found to express a high level of J11d antigen on the cell surface. Recombinant mouse leukemic inhibitory factor (rm-LIF), recombinant human LIF (rh-LIF), and steroids (hydrocortisone and dexamethasone) could induce M1 cells to undergo monocytic differentiation. The level of J11d antigen was greatly reduced after treatment of the cells with LIF or steroids. Western blotting revealed that the apparent molecular weight of the J11d antigen on M1 cells was 45-48 kDa. Furthermore, the level of J11d mRNA was also reduced during LIF-induced differentiation of M1 cells.     

Assay of 1L-myo-inositol-1-phosphatase using a fluorometric method.

1L-myo-Inositol-1-phosphatase, an enzyme purified from brain tissues, catalyzes the dephosphorylation of 1L-myo-inositol 1-phosphate. This enzyme has become the subject of intense research interest, since myo-inositol is needed for the resynthesis of phosphatidylinositol. We have developed a sensitive fluorometric assay for detecting the activity of 1L-myo-inositol-1-phosphatase. The assay is based on o-aminobenzoyl beta-glycerophosphate fluorescence, according to the following principles: (I) The fluorescence yield of o-aminobenzoyl beta-glycerophosphate is increased by 2.75-fold in the presence of saturating concentrations of bovine serum albumin. (II) o-Aminobenzoyl beta-glycerophosphate has the same fluorescence yield as o-aminobenzoyl glycerol, but the latter does not bind to bovine serum albumin. (III) Dephosphorylation of the substrate, catalyzed by the monophosphatase, makes less o-aminobenzoyl beta-glycerophosphate available for binding to bovine serum albumin, thereby producing a decrease in the fluorescence intensity.     

Alterations in high-affinity binding characteristics and levels of opioids in invertebrate ganglia during aging: Evidence for an opioid compensatory mechanism

In Mytilus and Leucophaea the high-affinity binding site density is significantly lower in old animals than in young animals, whereas the low-affinity site density remains unchanged. In Mytilus the estimated met-enkephalin and met-enkephalin-Arg6-Phe7 levels are significantly higher in old than in young animals. In Leucophaea only the met-enkephalin level can be determined, and it is also higher in old animals. The decrease in the high-affinity binding site density and the corresponding increase in endogenous enkephalin levels suggest the existence of an opioid compensatory mechanism associated with the aging process. In Mytilus there is a demonstrated decrease with age in intraganglionic dopamine levels in response to applied opiates. In addition, the inhibition of dopamine-stimulated adenylate cyclase activity by opiates also decreases in older animals. In Leucophaea the sex difference in opioid binding densities diminishes with age.     

Turnover of hepatic phosphofructokinase in normal and diabetic rats. Role of insulin and peptide stabilizing factor.

Earlier work demonstrated that the activity of liver phosphofructokinase (PFK-L2) and immunoreactive PFK-L2 were decreased in diabetic rats and increased to normal or super-normal amounts following insulin treatment (Dunaway, G.A., and Weber, G., (1974) Arch. Biochem. Biophys. 162, 629-637). This report indicates that the decrease in levels of PFK-L2 in diabetic rats is a result of an accelerated degradation rate while the synthetic rate remains nearly normal. Following insulin treatment, the rate of PFK-L2 synthesis is enhanced 2-fold, whereas the rate of degradation appears to be greatly diminished. An inverse relationship is shown to exist between the PFK-L2 levels and the rates of PFK-L2 degradation, suggesting that the levels of PFK-L2 are primarily regulated by degradation rate. In addition, the levels of the PFK-L2 peptide stabilizing factor are inversely proportional to rates of PFK-L2 degradation. These results indicate that insulin mediates the rate of degradation of PFK-L2 by controlling the level of the peptide stabilizing factor.     

Identity between the novel integrin beta 7 subunit and an antigen found highly expressed on intraepithelial lymphocytes in the small intestine.

A cDNA clone encoding the N-terminal sequence of the murine integrin beta 7 subunit, a novel member of the leukocyte cell adhesion molecule subset (Leu-CAM), has been isolated. An N-terminal region of 13 contiguous amino acids deduced from the cDNA shows complete identity with the N-terminus of the 120 kDa subunit of the M290 antigen, a surface molecule found highly expressed on mouse intestinal intraepithelial lymphocytes (IEL). This unexpected result focuses two previously unconnected areas of research and suggests that integrins may have a special role to play in the defence of the gut mucosa.     

Acid-mediated mutagenicity of tobacco snuff: its possible mechanism

Polar solvent extracts of tobacco snuff under acidic conditions were mutagenic in Salmonella typhimurium. Using the Griess reagent test, nitrite ranging from approximately 1.8 to 5.4 mg/g of snuff was found in the polar fraction of extracts. After acid treatment, nitroso compounds in the amount corresponding to the nitrite concentration were detected. The mutagenic potency of the acid-treated extracts was consistent with the content of nitroso compounds generated. Formation of nitroso compounds and the mutagenic activity under acidic conditions was inhibited by ascorbic acid. The results indicate that a nitrosation process was involved in snuff extracts during acid treatment. Studies related to the source of nitrite in tobacco snuff demonstrated that snuff contained bacteria which were able to reduce nitrate to nitrite and that the amount of nitrite in snuff extracts could be further increased by incubation of the extracts with the bacteria. Since snuff contains a considerable amount of nitrate, it seems that reduction of nitrate in snuff to nitrite by bacteria, and nitrosation of certain constituents in snuff by nitrite under acidic conditions to form mutagenic nitroso compounds are possible mechanisms responsible for the acid-mediated mutagenicity of snuff extracts.     

Binding specificities of cellular retinol-binding protein and cellular retinol-binding protein, type II

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein, type ii (CRBP(II] are cytoplasmic proteins that bind trans-retinol as an endogenous ligand. These proteins are structurally similar having greater than 50% sequence homology. Employing fluorescence, absorbance, and competition studies, the ability of pure preparations of CRBP(II) and CRBP to bind various members of the vitamin A family has been examined. In addition to trans-retinol, CRBP(II) was able to form high affinity complexes (K'd less than 5 X 10(-8) M) with 13-cis-retinol, 3-dehydroretinol, and all-trans-retinaldehyde. CRBP bound those retinol isomers with similar affinities, but did not bind trans-retinaldehyde. Neither protein bound retinoic acid nor 9-cis- and 11-cis-retinol. The spectra of 13-cis-retinol and 3-dehydroretinol, when bound, were shifted and displayed fine structure compared to their spectra in organic solution. However, the lambda max and fluorescent yield of a particular ligand were different when bound to CRBP(II) versus CRBP. It appears that CRBP(II) and CRBP bind trans-retinol, 13-cis-retinol, and 3-dehydroretinol in a planar configuration. However, the binding sites of CRBP(II) and CRBP are clearly distinct based on the observed spectral differences of the bound ligands and the observations that only CRBP(II) could bind trans-retinaldehyde. The ability of CRBP(II) to bind trans-retinaldehyde suggests a physiological role for the protein in accepting retinaldehyde generated from the cleavage of beta-carotene in the absorptive cell.     

Trifluoperazine Activates and Releases Latent ATP-Generating Enzymes Associated with the Synaptic Plasma Membrane

Neurone-specific enolase (NSE) and the brain form of creatine phosphokinase (CPK-BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two-dimensional gel (2-D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be "cryptic" unless the SPM were treated with Triton X-100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for "unmasking" the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK-BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2-D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70-kilodalton heat-shock cognate protein. Tubulin, synapsin I, and a 35-kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase on the basis of 2-D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.     

Effect of the uvs-2 allele of Neurospora crassa on the mutagenic potency of two N-hydroxylaminopurines and 2-aminopurine in the ad-3 forward-mutation test

The mutagenic potencies of 3 purine analogs were determined in the ad-3 forward-mutation test in growing cultures of heterokaryon 59 (H-59), a nucleotide excision repair-deficient (uvs-2/uvs-2) 2-component heterokaryon of Neurospora crassa. Two N-hydroxylaminopurines, 2-amino-6-N-hydroxylaminopurine (AHA) and 6-N-hydroxylaminopurine (HAP), were potent and strong mutagens, respectively, whereas 2-aminopurine (AP) was a moderate mutagen. Dose-response curves showed that AHA and HAP were about equally mutagenic at low doses but that AHA was more mutagenic than HAP at high doses. Comparison of these results in H-59 with our earlier results in heterokaryon 12 (H-12) of N. crassa, which is identical to H-59 except for being DNA-repair-proficient (uvs-2+/uvs-2+), shows that the defect in nucleotide excision repair due to uvs-2 has little or no effect on the mutagenic potencies of these 3 purine analogs. Therefore, the nucleotide excision-repair pathway in N. crassa that is deficient in H-59 does not appear to have a major role in the repair of pre-mutational lesions induced by these 3 purine analogs. On the other hand, based on the controls of these experiments, the frequency of spontaneous ad-3 mutants was 4 greater in H-59 than in H-12. This result suggests that the nucleotide excision-repair pathway in N. crassa that is inactivated by the uvs-2 mutation has a major role in the repair of lesions that would lead to spontaneous mutation at the ad-3+ region if they were not repaired.