Adapting the γ-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects

The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.     

Structural Basis and Target-specific Modulation of ADP Sensing by the Synechococcus elongatus PII Signaling Protein

P_II signaling proteins comprise one of the most versatile signaling devices in nature and have a highly conserved structure. In cyanobacteria, PipX and N-acetyl-l-glutamate kinase are receptors of P_II signaling, and these interactions are modulated by ADP, ATP, and 2-oxoglutarate. These effector molecules bind interdependently to three anti-cooperative binding sites on the trimeric P_II protein and thereby affect its structure. Here we used the P_II protein from Synechococcus elongatus PCC 7942 to reveal the structural basis of anti-cooperative ADP binding. Furthermore, we clarified the mutual influence of P_II-receptor interaction and sensing of the ATP/ADP ratio. The crystal structures of two forms of trimeric P_II, one with one ADP bound and the other with all three ADP-binding sites occupied, revealed significant differences in the ADP binding mode: at one site (S1) ADP is tightly bound through side-chain and main-chain interactions, whereas at the other two sites (S2 and S3) the ADP molecules are only bound by main-chain interactions. In the presence of the P_II-receptor PipX, the affinity of ADP to the first binding site S1 strongly increases, whereas the affinity for ATP decreases due to PipX favoring the S1 conformation of P_II-ADP. In consequence, the P_II-PipX interaction is highly sensitive to subtle fluctuations in the ATP/ADP ratio. By contrast, the P_II-N-acetyl-l-glutamate kinase interaction, which is negatively affected by ADP, is insensitive to these fluctuations. Modulation of the metabolite-sensing properties of P_II by its receptors allows P_II to differentially perceive signals in a target-specific manner and to perform multitasking signal transduction.     

Diacylglycerols interact with the L2 lipidation site in TRPC3 to induce a sensitized channel state

Coordination of lipids within transient receptor potential canonical channels (TRPCs) is essential for their Ca²⁺ signaling function. Single particle cryo‐EM studies identified two lipid interaction sites, designated L1 and L2, which are proposed to accommodate diacylglycerols (DAGs). To explore the role of L1 and L2 in TRPC3 function, we combined structure‐guided mutagenesis and electrophysiological recording with molecular dynamics (MD) simulations. MD simulations indicate rapid DAG accumulation within both L1 and L2 upon its availability within the plasma membrane. Electrophysiological experiments using a photoswitchable DAG‐probe reveal potentiation of TRPC3 currents during repetitive activation by DAG. Importantly, initial DAG exposure generates a subsequently sensitized channel state that is associated with significantly faster activation kinetics. TRPC3 sensitization is specifically promoted by mutations within L2, with G652A exhibiting sensitization at very low levels of active DAG. We demonstrate the ability of TRPC3 to adopt a closed state conformation that features partial lipidation of L2 sites by DAG and enables fast activation of the channel by the phospholipase C‐DAG pathway.     

Cell cycles in human diploid and aneuploid strains

Summary Autoradiographic investigation of the cell cycle of 12 diploid and 12 abnormal human fibroblast strains was carried out. Two cell strains (trisomy 7 and monosomy 21) derived from spontaneous abortuses showed prolongation of the G₂ period accompanied by the shortening of the S period. Other cytogenetically abnormal embryonic strains (trisomy 9, 14 and triploidy) did not deviate from the diploid pattern. Three cell strains (LHC-1-70, LHC-6-70, LHC-411) derived from the patients with karyotypes 47,XXX, 47,XY,+18 and 46,XX,5p—respectivly had embryonic types of proliferation with a short G₂ period. In two other strains from the patients with Down's syndrom the G₂ period was prolonged. There was no statistically significant difference in the parameters of the cell cycle between the control and the strains derived from a patient with Klinefelter syndrom and from a male patient with karyotype 46,XX. The modificatory effect of the chromosomal abnormalities on the parameters of cell cycle is discussed.

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Zusammenfassung Der Zellcyclus von 12 normal diploiden und 12 abnormen menschlichen Fibroblasten-Zellinien wurde untersucht. Zwei Zellinien (Trisomie 7 und Monosomie 21) zeigten eine Verlängerung der G₂-Phase und eine Verkürzung der S-Phase. Zellinien (Trisomie 9, 14 und Triploidie) wichen nicht von der Norm ab. Drei Zellinien (47,XXX; 47,XY,+18; 46,XX,5p-) zeigten eine Verkürzung von G₂. G₂ war dagegen verlängert bei zwei Zellinien von Down-Syndrom. Keine Abweichung fand sich bei einem Klinefelter-Patienten und einem männlichen Probanden mit 46,XX.

     

Kinetics of protein degradation in diploid and trisomic human fibroblasts

The degradation rate of long-lived and short-lived proteins was determined in diploid fibroblasts and fibroblasts with trisomy 7 derived from human embryos. Two fractions of proteins were detected in the exponentially growing diploid fibroblasts with half-lives (T 1/2) 37 and 19 hours. The rate of protein degradation increases in diploid fibroblasts as they approach confluence and protein fractions with T 1/2 30, 18 and 12 hours appear. The rate of protein degradation in trisomic fibroblasts does not change for the long-lived and short-lived proteins and is the same in both exponential (T 1/2 31 and 14 hours) and stationary phase (T 1/2 33 and 17 hours). The relative amount of the short-lived proteins in trisomic fibroblasts in the stationary phase decreased as compared with the one in diploid fibroblasts. It is apparent that a mechanism of regulation of protein catabolism in trisomic fibroblasts is impaired.     

Optopharmacological control of TRPC channels by coumarin-caged lipids is associated with a phototoxic membrane effect

Photouncaging of second messengers has been successfully employed to gain mechanistic insight of cellular signaling pathways. One of the most enigmatic processes of ion channel regulation is lipid recognition and lipid-gating of TRPC channels, which represents pivotal mechanisms of cellular Ca~(2+) homeostasis. Recently, optopharmacological tools including caged lipid mediators became available, enabling an unprecedented level of temporal and spatial control of the activating lipid species within a cellular environment. Here we tested a commonly used caged ligand approach for suitability to investigate TRPC signaling at the level of membrane conductance and cellular Ca~(2+) handling. We report a specific photouncaging artifact that is triggered by the cage structure coumarin at UV illumination. Electrophysiological characterization identified a light-dependent membrane effect of coumarin. UV light(340 nm) as used for photouncaging, initiated a membrane conductance specifically in the presence of coumarin as low as 30 μmol L~(-1) concentrations. This conductance masked the TRPC3 conductance evoked by photouncaging, while TRPC-mediated cellular Ca~(2+) responses were largely preserved. The observed light-induced membrane effects of the released caging moiety may well interfere with certain cellular functions, and prompt caution in using coumarin-caged second messengers in cellular studies.     

Complex study of human cell strains with karyotype anomalies. II. Mitotic cycle parameters]

The object of this investigation were the parameters of the mitotic cycle in 14 fibroblasts-like cell strains with chromosome aberrations obtained from skin biopsies of patients and from spontaneous human abortuses. In two strains of embryonal origin (trisomic for chromosome and monosomic for chromosome 21) increased duration of stage G2 of the cell cycle accompanied by a shorter period of DNA synthesis was observed. In the other 5 strains of embryonal origin (two strains trisomic for chromosome 7, strains trisomic for chromosome 9, trisomic for chromosome 14 and triploid strains) no deviations from the normal duration of the stages of the cell cycle were observed. Two types of changes of the mitotic cycle parameters were observed in the cell strains obtained from patients with chromosome aberrations. A considerably prolonged G2 stage was observed in two strains obtained from patients affected by Down's syndrome. Three strains with the karyotypes 47, XXX, 47 XY+18 and 46, XX, 5p-were characterized by a complex of features typical of the strains of embryonal origin. A considerable decrease of the stage G2 duration was observed in these strains. In the strains obtained from a proband with Kleinfelter's syndrome and from a patient with the karyotype 46XX no deviations in the parameters of the cell cycle were observed.     

Impact of low‐intensity pulsed ultrasound on the growth of Schizochytrium sp. for omega‐3 production

Schizochytrium sp. is a microalga that is known for its high content of oils or lipids. It has a high percentage of polyunsaturated fatty acids in the accumulated oil, especially docosahexaenoic acid (DHA). DHA is an important additive for the human diet. Large‐scale production of Schizochytrium sp. can serve as an alternative source of DHA for humans as well as for fish feed, decreasing the burden on aqua systems. Therefore, research on improving the productivity of Schizochytrium attracts a lot of attention. We studied the potential of using low‐intensity pulsed ultrasound (LIPUS) in the growth cycle of Schizochytrium sp. in shake flasks. Different intensities and treatment durations were tested. A positive effect of LIPUS on biomass accumulation was observed in the Schizochytrium sp. culture. Specifically, LIPUS stimulation at the ultrasound intensity of 400 mW/cm² with 20 min per treatment 10 times a day with equal intervals of 2.4 h between the treatments was found to enhance the growth of Schizochytrium biomass most effectively (by up to 20%). Due to the nature of cell division in Schizochytrium sp. which occurs via zoospore formation, LIPUS stimulation was inefficient if applied continuously during all 5 days of the growth cycle. Using microscopy, we studied the interval between zoospore formation in the culture and selected the optimal LIPUS application days (Days 0–1 and Days 4–5 of the 5‐day growth cycle). Microscopic images have also shown that LIPUS stimulation enhances zoospore formation in Schizochytrium sp., leading to more active cell division in the culture. This study shows that LIPUS can serve as an additional tool for cost‐efficiency improvement in the large‐scale production of Schizochytrium as a sustainable and environmentally friendly source of omega‐3 (DHA).