In vivo evidence for translesion synthesis by the replicative DNA polymerase δ

The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass.     

A defective conditioned behavior and a defective adenylate cyclase in theDrosophila mutantrutabaga

Summary TheDrosophila X-linked mutantrutabaga (Duerr and Quinn 1982) fails to perform normally in olfactory conditioning paradigms, in spite of being able to sense odorants and shock (Figs. 1–3).rut is capable of forming an association between shock and odorant, but memory decays rapidly; the memory of the mutant following intensive training resembles that of normal flies following very brief training (Fig. 4).rut flies also display in vitro a defective adenylate cyclase activity (Fig. 6). The enzyme in the mutant is responsive to stimulation by a putative neurotransmitter and by a guanyl nucleotide (Fig. 8) but the activity is lower than normal even in the presence of forskolin (Fig. 8) and MnATP (Fig. 9), suggesting that the lesion is closely associated with the function of the catalytic subunit.rut/ + heterozygotes are semi-recessive with regard to both the behavioral defect and the biochemical defect (Figs. 5, 7). The behavioral and the biochemical lesions detected inrut flies are discussed in light of current molecular models of learning.     

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.     

Lipophylic compounds from Euphorbia peplis L.--a halophytic plant from the Bulgarian Black Sea coast

The chemical composition of the lipophylic fraction from the halophytic plant Euphorbia peplis L. was investigated. Compared to other terrestrial higher plants an increase of triacylglycerols and especially of glycolipids was observed. The main phospholipid was phosphatidyl choline, followed by almost equal concentrations of phosphatidyl ethanolamine and phosphatidyl glycerol. A relatively high concentration of phosphatidic acids (6.5% of the total phospholipids) was found. The main sterol appeared to be sitosterol and significant amounts of tetracyclic triterpene alcohols were found. The composition of the volatile compounds is relatively simple and only one chlorinated compound, identified as 2,2-diethoxy-1-chloroethane, was found. There was a strong toxicity of the total lipophylic extract towards Artemia salina.     

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.     

Voltage-induced inhibition of antigen-antibody binding at conducting optical waveguides

Optical waveguides coated with electrically conducting indium-tin oxide (ITO) are demonstrated here as a new class of substrate for fluorescent immunosensors. These waveguides combine electrochemical control with evanescent excitation and image-based detection. Presented here are preliminary results utilizing these waveguides that demonstrate influence of waveguide voltage on antigen binding. Specifically, waveguide surfaces were bisected into electrically addressable halves, anti-ovalbumin immobilized in patterns on their surfaces, and a 1.3 V bias applied between waveguide halves in the presence of Cy5-labeled ovalbumin in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl and 0.05% Tween-20. Fluorescence imaging indicated that binding of the antigen to positively biased waveguide halves was inhibited nearly 10-fold compared with negatively biased waveguide halves and unbiased controls. Furthermore, it is shown that ovalbumin binding to positively biased waveguide regions is regenerated after removal of applied voltage. These results suggest that electrochemical control of immunosensor substrates can be used as a possible strategy toward minimizing cross-reactive binding and/or nonspecific adsorption, immunosensor regeneration, and controlled binding.     

Backbone Cyclization of the C-terminal Part of Substance P. Part 1: The Important Role of the Sulphur in Position 11

Novel backbone-to-side chain and backbone-to-backbone cyclic analogues of substance P (SP) were prepared by solid-phase synthesis and screened for biological activity. An analogue containing a thioether- lactam ring between positions 9 and 11 showed an EC₅₀ value of 20nM toward the neurokinin 1 (NK-1) and was inactive toward the NK-2 and NK-3 receptors. On the other hand, in a multiple backbone cyclic peptide library of similar analogues, in which the sulphur was excluded from the ring, very low activity was detected. The activity was re-evaluated and was found to be even lower (EC₅₀=0.11 mM ) than the previously published data. These results indicate that the thioether moiety has a crucial role in receptor activation. The results also show tolerance of the NK-1 receptor, but not NK-2 or NK-3, to cyclization of the C-terminal portion of the SP_6–11 hexapeptide.     

CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-¹⁴C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.