Chromosome aberration and environmental physical activity: Down syndrome and solar and cosmic ray activity, Israel, 1990–2000

The possibility that environmental effects are associated with chromosome aberrations and various congenital pathologies has been discussed previously. Recent advances in the collection and computerization of data make studying these potential associations more feasible. The aim of this study was to investigate a possible link between the number of Down syndrome (DS) cases detected prenatally or at birth yearly in Israel over a 10-year period compared with the levels of solar and cosmic ray activity 1 year before the detection or birth of each affected child. Information about 1,108,449 births was collected for the years 1990–2000, excluding 1991, when data were unavailable. A total of 1,310 cases of DS were detected prenatally or at birth—138 in the non-Jewish community and 1,172 in the Jewish population. Solar activity indices—sunspot number and solar radio flux 2,800 MHz at 10.7 cm wavelength for 1989–1999—were compared with the number of DS cases detected. Pearson correlation coefficients (r) and their probabilities (P) were established for the percentage of DS cases in the whole population. There was a significant inverse correlation between the indices of solar activity and the number of cases of DS detected—r=–0.78, P=0.008 for sunspot number and r=–0.76, P=0.01 for solar flux. The possibility that cosmophysical factors inversely related to solar activity play a role in the pathogenesis of chromosome aberrations should be considered. We have confirmed a strong trend towards an association between the cosmic ray activity level and the incidence of DS.     

Cleavage site selection within a folded substrate by the ATP-dependent lon protease

Mechanistic studies of ATP-dependent proteolysis demonstrate that substrate unfolding is a prerequisite for processive peptide bond hydrolysis. We show that mitochondrial Lon also degrades folded proteins and initiates substrate cleavage non-processively. Two mitochondrial substrates with known or homology-derived three-dimensional structures were used: the mitochondrial processing peptidase alpha-subunit (MPPalpha) and the steroidogenic acute regulatory protein (StAR). Peptides generated during a time course of Lon-mediated proteolysis were identified and mapped within the primary, secondary, and tertiary structure of the substrate. Initiating cleavages occurred preferentially between hydrophobic amino acids located within highly charged environments at the surface of the folded protein. Subsequent cleavages proceeded sequentially along the primary polypeptide sequence. We propose that Lon recognizes specific surface determinants or folds, initiates proteolysis at solvent-accessible sites, and generates unfolded polypeptides that are then processively degraded.     

Caspase-dependent and -independent activation of acid sphingomyelinase signaling

Recent evidence suggests clustering of plasma membrane rafts into ceramide-enriched platforms serves as a transmembrane signaling mechanism for a subset of cell surface receptors and environmental stresses (Grassme, H., Jekle, A., Riehle, A., Schwarz, H., Berger, J., Sandhoff, K., Kolesnick, R., and Gulbins, E. (2001) J. Biol. Chem. 276, 20589-20596; Cremesti, A., Paris, F., Grassme, H., Holler, N., Tschopp, J., Fuks, Z., Gulbins, E., and Kolesnick, R. (2001) J. Biol. Chem. 276, 23954-23961). Translocation of the secretory form of acid sphingomyelinase (ASMase) into microscopic rafts generates therein the ceramide that drives raft coalescence. This process serves to feed forward Fas activation, with approximately 2% of full caspase 8 activation sufficient for maximal ASMase translocation, leading to death-inducing signaling complex formation within ceramide-rich platforms, and apoptosis. Here we report that treatment of Jurkat T cells with UV-C also induces ASMase translocation into rafts within 1 min, catalyzing sphingomyelin hydrolysis to ceramide and raft clustering. In contrast to Fas, UV-induced ASMase translocation and activation were caspase-independent. Nonetheless, ceramide-rich platforms promoted UV-C-induced death signaling, because ASMase inhibition or raft disruption inhibited apoptosis, improving clonogenic cell survival. These studies thus define two distinct mechanisms for biologically relevant ASMase activation within rafts; a Fas-mediated mechanism dependent upon caspase 8 and FADD, and a UV-induced mechanism independent of caspase activation. Consistent with this notion, genetic depletion or pharmacologic inhibition of caspase 8 or FADD, which render Jurkat cells incapable of sphingolipid signaling and apoptosis upon Fas ligation, did not impair these events upon UV-C stimulation.     

Lesion bypass by human DNA polymerase mu reveals a template-dependent, sequence-independent nucleotidyl transferase activity

DNA polymerase mu (pol mu), which is related to terminal deoxynucleotidyl transferase and DNA polymerase beta, is thought to be involved in non-homologous end joining and V(D)J recombination. Pol mu is induced by ionizing radiation and exhibits low fidelity. Analysis of translesion replication by purified human pol mu revealed that it bypasses a synthetic abasic site with high efficiency, using primarily a misalignment mechanism. It can also replicate across two tandem abasic sites, using the same mechanism. Pol mu extends primers whose 3'-terminal nucleotides are located opposite the abasic site. Most remarkably, this extension occurs via a mode of nucleotidyl transferase activity, which does not depend on the sequence of the template. This is not due to simple terminal nucleotidyl transferase activity, because pol mu is unable to add dNTPs to an oligo(dT)29 primer or to a blunt end duplex oligonucleotide under standard conditions. Thus, pol mu is a dual mode DNA-synthesizing enzyme, which can act as either a classical DNA polymerase or as a non-canonical, template-dependent, but sequence-independent nucleotidyl transferase. To our knowledge, this is the first report on a DNA-synthesizing enzyme with such properties. These activities may be required for its function in non-homologous end joining in the processing of DNA ends prior to ligation.     

Biochemical characterization of the Methanothermobacter thermautotrophicus minichromosome maintenance (MCM) helicase N-terminal domains

Minichromosome maintenance helicases are ring-shaped complexes that play an essential role in archaeal and eukaryal DNA replication by separating the two strands of chromosomal DNA to provide the single-stranded substrate for the replicative polymerases. For the archaeal protein it was shown that the N-terminal portion of the protein, which is composed of domains A, B, and C, is involved in multimer formation and single-stranded DNA binding and may also play a role in regulating the helicase activity. Here, a detailed biochemical characterization of the N-terminal region of the Methanothermobacter thermautotrophicus minichromosome maintenance helicase is described. Using biochemical and biophysical analyses it is shown that domain C of the N-terminal portion, located adjacent to the helicase catalytic domains, is required for protein multimerization and that domain B is the main contact region with single-stranded DNA. It is also shown that although oligomerization is not essential for single-stranded DNA binding and ATPase activity, the presence of domain C is essential for helicase activity.     

Quantitative analysis of translesion DNA synthesis across a benzo[a]pyrene-guanine adduct in mammalian cells: the role of DNA polymerase kappa

Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized, low fidelity DNA polymerases. We studied translesion DNA synthesis (TLS) across a benzo[a]pyrene-guanine (BP-G) adduct, a major mutagenic DNA lesion generated by tobacco smoke. This was done using a quantitative assay that measures TLS indirectly, by measuring the recovery of gapped plasmids transfected into cultured mammalian cells. Analysis of PolK(+/+) mouse embryo fibroblasts (MEFs) showed that TLS across the BP-G adduct occurred with an efficiency of 48 +/- 4%, which is an order of magnitude higher than in Escherichia coli. In PolK(-/-) MEFs, bypass was 16 +/- 1%, suggesting that at least two-thirds of the BP-G adducts in MEFs were bypassed exclusively by polymerase kappa (polkappa). In contrast, poleta was not required for bypass across BP-G in a human XP-V cell line. Analysis of misinsertion specificity across BP-G revealed that bypass was more error-prone in MEFs lacking polkappa. Expression of polkappa from a plasmid introduced into PolK(-/-) MEFs restored both the extent and fidelity of bypass across BP-G. Polkappa was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both polkappa and poleta, suggesting that the biological role of polkappa in TLS across BP-G is due to regulation of TLS and not due to an exclusive ability to bypass this lesion. These results indicate that BP-G is bypassed in mammalian cells with relatively high efficiency and that polkappa bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.     

Adenylyl cyclase type-VIII activity is regulated by G(betagamma) subunits

The Ca2+-activated adenylyl cyclase type VIII (AC-VIII) has been implicated in several forms of neural plasticity, including drug addiction and learning and memory. It has not been clear whether Gi/o proteins and G-protein coupled receptors regulate the activity of AC-VIII. Here we show in intact mammalian cell system that AC-VIII is inhibited by mu-opioid receptor activation and that this inhibition is pertussis toxin sensitive. Moreover, we show that G(betagamma) subunits inhibit AC-VIII activity, while constitutively active alphai/o subunits do not. Different Gbeta isoforms varied in their efficacies, with Gbeta1gamma2 or Gbeta2gamma2 being more efficient than Gbeta3gamma2 and Gbeta4gamma2, while Gbeta5 (transfected with gamma2) had no effect. As for the Ggamma subunits, Gbeta1 inhibited AC-VIII activity in the presence of all gamma subunits tested except for gamma5 that had only a marginal activity. Moreover, cotransfection with proteins known to serve as scavengers of Gbetagamma dimers, or to reduce Gbetagamma plasma membrane anchorage, markedly attenuated the mu-opioid receptor-induced inhibition of AC-VIII. These results demonstrate that Gbetagamma (originating from agonist activation of these receptors) and probably not Galphai/o subunits are involved in the agonist inhibition of AC-VIII.     

p53 and p21 regulate error-prone DNA repair to yield a lower mutation load

Regulation of mutation rates is critical for maintaining genome stability and controlling cancer risk. A special challenge to this regulation is the presence of multiple mutagenic DNA polymerases in mammals. These polymerases function in translesion DNA synthesis (TLS), an error-prone DNA repair process that involves DNA synthesis across DNA lesions. We found that in mammalian cells TLS is controlled by the tumor suppressor p53, and by the cell cycle inhibitor p21 via its PCNA-interacting domain, to maintain a low mutagenic load at the price of reduced repair efficiency. This regulation may be mediated by binding of p21 to PCNA and via DNA damage-induced ubiquitination of PCNA, which is stimulated by p53 and p21. Loss of this regulation by inactivation of p53 or p21 causes an out of control lesion-bypass activity, which increases the mutational load and might therefore play a role in pathogenic processes caused by genetic instability.     

PSMA PET/CT to evaluate response to SBRT for prostate cancer bone metastases

BackgroundIn the current study we evaluated ⁶⁸Ga PSMA PET/ CT to measure local control of bone metastasis in oligometastatic prostate cancer patients treated with SBRT.Materials and methodsAfter the institutional review board approval, a retrospective review of medical records of consecutive prostate cancer patients treated between 2014 and 2018 was conducted. Only medical records of patients that were treated with SBRT for bone metastasis and had pre-and post-SBRT ⁶⁸Ga PSMA PET/CT scans were included in our study. Data extracted from the medical files included patient-related (age), disease-related (Gleason score, site of metastasis), and treatment-related factors and outcomes.ResultsDuring the study period, a total of 12 patients (15 lesions) were included, with a median age of 73 years. The median follow-up was 26.5 months (range 13–45 months). Median time of ⁶⁸Ga PSMA PET/ CT follow up was 17.0 months (range 3–39 months). The median pre-treatment PSA was 2 ng/mL (range 0.56–44 ng/mL) vs. post treatment PSA nadir of 0.01 ng/mL (0.01–4.32) with a median time to nadir of 7 months (range, 2–12). Local control was 93% during the follow up period and there was correlation with PS MA avidity on PE T. None patients developed recurrences in the treated bone. None of the patients had grade 3 or more toxicities during follow-up.ConclusionsSBRT is a highly effective and safe method for treatment of prostate cancer bone metastases. More studies are required to determine if SBRT provides greater clinical benefit than standard fractionation for oligometastatic prostate cancer patients. ⁶⁸Ga PSMA PET/CT should be further investigated for delineation and follow-up.