8-Azaguanine versus 6-thioguanine: influence on frequency and expression time of induced HGPRT- mutations in Chinese hamster V79 cells

Chinese hamster V79 cells were mutagenized with ethyl methanesulfonate at various concentrations. Clones resistant to 8-azaguanine (20 and 80 micrograms/ml) or 6-thioguanine (4 micrograms/ml) were selected at different times after the treatments. The total yield of induced mutations was only slightly affected by the kind and concentration of purine analog used in the selection. However, full phenotypic expression of the mutants selected with 8-azaguanine was achieved earlier than that of mutants resistant to 6-thioguanine. This result seems to be best explained by the reported lower affinity of 8-azaguanine for the wild-type HGPRT enzyme, thus providing evidence that, in this gene-mutation assay, the phenotypic expression time has a physiological component.     

The radiosensitivity of spermatogonial stem cells in C3H/101 F1 hybrid mice

The radiosensitivity of spermatogonial stem cells of C3H/HeH × 101/H F₁ hybrid mice was determined by counting undifferentiated spermatogonia at 10 days after X-irradiation. During the spermatogenic cycle, differences in radiosensitivity were found, which were correlated with the proliferative activity of the spermatogonial stem cells. In stage VIII_irr, during quiescence, the spermatogonial stem cells were most radiosensitive with a D₀ of 1.4 Gy. In stages XI_irr−V_irr, when the cells were proliferatively active, the D₀ was about 2.6 Gy. Based on the D₀ values for sensitive and resistant spermatogonia and on the D₀ for the total population, a ratio of 45:55% of sensitive to resistant spermatogonial stem cells was estimated for cell killing.

When the present data were compared with data on translocation induction obtained in mice of the same genotype, a close fit was obtained when the translocation yield (Y; in % abnormal cells) after a radiation dose D was described by Y = e^τD, with τ = 1 for the sensitive and τ = 0.1 for the resistant spermatogonial stem cells, with a maximal e^τD of 100.     

Optimization of in vitro protein synthesis by isolated mouse adrenal mitochondria

The requirements for in vitro mitochondrial protein synthesis have been studied using isolated mitochondria from cultured adrenal Y-1 tumor cells from mice. By reducing the reaction volume to 50 microliter we were able to assay in replicate the requirements for various reaction components using trichloroacetic acid (TCA)-precipitable counts for a quantitative evaluation with time of incubation. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography was also used for a qualitative and quantitative evaluation of the translation products. With the optimized system, 1 to 3% of added [35S]methionine was incorporated. The products of mitochondrial protein synthesis range from 70,000 to 5000 molecular weight. Major autoradiographic bands were observed at 38,000, 31,000, 23,000, 20,000, and 5600 molecular weight as separated on 10 to 20% gradient SDS-polyacrylamide gels; however, 20 to 30 protein products of various molecular weights were discernible. Mitochondrial concentrations of 0.8 to 1.4 mg/ml of incubation gave the better incorporation of [35S]methionine per milligram of protein. Total [35S]methionine incorporated into mitochondrial protein was greatest at 25 degrees C after 90 min. Chloramphenicol at 10 micrograms/ml inhibited mitochondrial protein synthesis by more than 50% and at 100 micrograms/ml inhibited incorporation by more than 95%. Cycloheximide had no effect on incorporation at less than 1.0 mg/ml. Magnesium and ATP in a molar ratio of one to one at 5 mM gave optimal incorporation. Other energy generating systems using oxidative phosphorylation to supply ATP for protein synthesis were not as effective as ATP and 5 mM phosphoenol pyruvate, 20 micrograms/ml pyruvate kinase and 5 mM a-ketoglutarate. In contrast to in vitro yeast mitochondrial protein synthesis, no enhancement of in vitro adrenal cell mitochondrial protein synthesis was found with GTP or its analogs. The buffers N,N-bis(2-hydroxyethyl)glycine, N-(tris(hydroxymethyl)methyl)glycine, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were superior to Tris-HCl for mitochondrial protein synthesis. Optimal pH for [35S]methionine incorporation into mitochondrial proteins was pH 7.0 to 7.6. Potassium at 50 to 90 mM gave the best incorporation of [35S]methionine, and the higher molecular weight products of translation were enhanced at these concentrations. Sodium at 10 to 40 mM had no effect; however, 100 mM sodium inhibited label incorporation by 30%. Calcium at 100 microM inhibited mitochondrial protein synthesis by approximately 50%, and at 1.0 mM little if any incorporation occurred.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enucleation eliminates a differentiation-specific surface marker from normal and leukemic murine erythroid cells

Mammalian erythropoiesis includes a step in which the nucleus is extruded through the cell membrane. We have investigated the relationship between concanavalinA (conA) plasma membrane receptors, which are known to leave the incipient reticulocyte during enucleation, and regions of the plasma membrane which bind merocyanine 540, a differentiation-specific marker of hematopoietic cells. The distribution of these two fluorescent probes was examined on living cells from the spleens of neonatal mice and on erythroleukemia cells induced to enucleate in culture. In both cases, the region of the membrane extruded with the nucleus preferentially binds conA and merocyanine 540, whereas the plasma membrane which is left behind retains the capacity to bind another lectin, wheat germ agglutinin (WGA). The implications of these findings are discussed with respect to the mechanism by which markers are eliminated from the erythrocyte cell surface.     

Stimulation of δ-Opioid Receptors Reduces the In Vivo Binding of the Cholecystokinin (CCK)-B-Selective Agonist [3H]pBC 264: Evidence for a Physiological Regulation of CCKergic Systems by Endogenous Enkephalins

Cholecystokinin (CCK) and enkephalins appear to be colocalized in several brain structures, and a physiological interaction between these peptides has been suggested by a large number of pharmacological studies. In this work we have shown, by in vivo binding experiments, that the endogenous enkephalins, protected from degrading enzymes by mixed inhibitors such as kelatorphan and N-[(R,S)-2-benzyl-3-[(S)-2-amino-4-methylthiobutyldithio]-1-oxo pro pyl]- L-phenylalanine benzyl ester (RB 101), a systemically active prodrug, modulate CCK release in mouse brain, leading to an overall increase in the extracellular levels of CCK. This was quantified by measuring the effects of both inhibitors on the in vivo binding of [3H]propionyl-Tyr(SO3H)-gNle-mGly-Trp-(N-Me)Nle-Asp-Phe-NH2 ([3H]pBC 264), a selective and highly potent CCK-B agonist. Thus, intracerebroventricular injection of kelatorphan produced a dose-dependent inhibition of the in vivo binding of [3H]pBC 264 with a maximal effect (40%) at 50 nmol. A similar response was observed after intravenous injection of RB 101 (40 mg/kg). The specific binding of [3H]pBC 264 was also inhibited (25%) by intravenous injection of the selective delta-opioid agonist H-Tyr-D-Cys(StBu)-Gly-Phe-Leu-Thr(OtBu)-OH (BUBUC; 2 mg/kg) but not by the mu-agonist H-Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol (5 mg/kg), suggesting a preferential involvement of delta-opioid receptors in the modulation of CCK release. This was confirmed by using the selective delta-opioid antagonist naltrindole, which prevented the inhibitory effects of BUBUC and of enkephalin-degrading enzyme inhibitors on [3H]pBC 264 binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Small-molecule active pharmaceutical ingredients of approved cancer therapeutics inhibit human aspartate/asparagine-β-hydroxylase

Human aspartate/asparagine-β-hydroxylase (AspH) is a 2-oxoglutarate (2OG) dependent oxygenase that catalyses the hydroxylation of Asp/Asn-residues of epidermal growth factor-like domains (EGFDs). AspH is reported to be upregulated on the cell surface of invasive cancer cells in a manner distinguishing healthy from cancer cells. We report studies on the effect of small-molecule active pharmaceutical ingredients (APIs) of human cancer therapeutics on the catalytic activity of AspH using a high-throughput mass spectrometry (MS)-based inhibition assay. Human B-cell lymphoma-2 (Bcl-2)-protein inhibitors, including the (R)-enantiomer of the natural product gossypol, were observed to efficiently inhibit AspH, as does the antitumor antibiotic bleomycin A₂. The results may help in the design of AspH inhibitors with the potential of increased selectivity compared to the previously identified Fe(II)-chelating or 2OG-competitive inhibitors. With regard to the clinical use of bleomycin A₂ and of the Bcl-2 inhibitor venetoclax, the results suggest that possible side-effects mediated through the inhibition of AspH and other 2OG oxygenases should be considered.     

Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoglobin

Functional and structural properties of protoglobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the globin fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0 Å resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misfolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

ATG13

During the past 20 years, autophagy signaling has entered the main stage of the cell biological theater. Autophagy represents an intracellular degradation process that is involved in both the bulk recycling of cytoplasmic components and the selective removal of organelles, protein aggregates, or intracellular pathogens. The understanding of autophagy has been greatly facilitated by the characterization of the molecular machinery governing this process. In yeast, initiation of autophagy is controlled by the Atg1 kinase complex, which is composed of the Ser/Thr kinase Atg1, the adaptor protein Atg13, and the ternary complex of Atg17-Atg31-Atg29. In vertebrates, the orthologous ULK1 kinase complex contains the Ser/Thr kinase ULK1 and the accessory proteins ATG13, RB1CC1, and ATG101. Among these components, Atg1/ULK1 have gained major attention in the past, i.e., for the identification of upstream regulatory kinases, the characterization of downstream substrates controlling the autophagic flux, or as a druggable target for the modulation of autophagy. However, accumulating data indicate that the function of Atg13/ATG13 has been likely underestimated so far. In addition to ensuring proper Atg1/ULK1 recruitment and activity, this adaptor molecule has been implicated in ULK1-independent autophagy processes. Furthermore, recent data have identified additional binding partners of Atg13/ATG13 besides the components of the Atg1/ULK1 complex, e.g., Atg8 family proteins or acidic phospholipids. Therefore, in this review we will center the spotlight on Atg13/ATG13 and summarize the role that Atg13/ATG13 assumes in the autophagy stage play.