Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody

The principal iodinatable surface protein (P30) of our cloned RH strain of Toxoplasma gondii has an apparent molecular weight of 30,000, as measured by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Monoclonal antibody B specifically immunoprecipitated protein P30 from a detergent extract of surface radioiodinated T. gondii. Monoclonal antibody B in the presence of complement was also parasiticidal for T. gondii, and this parasiticidal effect could be blocked by protein P30. Monoclonal antibody B was purified from mouse ascitic fluid and linked to cyanogen bromide-activated Sepharose. The resulting immunoabsorbent was used to purify 1.7 mg of protein P30 from a large number of parasites. The efficiency of recovery of protein P30 was measured by assays of radioactivity and of parasiticidal blocking activity. Protein P30 represented 3 to 5% of the total protein. It is also present in a recently isolated strain of T. gondii. A convalescent human antitoxoplasma serum immunoprecipitated radiolabeled protein P30. Three convalescent antisera when quantitated by an ELISA test had a high anti-protein P30 titer. Charge shift electrophoresis showed that protein P30 has an extensive hydrophobic region and thus is probably an integral membrane protein. Electrophoresis under nonreducing conditions showed no evidence that protein P30 exists as a disulfide linked homo- or heterodimer, although it probably has intramolecular disulfide bonds.     

Anti-Oxidative Effects of Melatonin Receptor Agonist and Omega-3 Polyunsaturated Fatty Acids in Neuronal SH-SY5Y Cells: Deciphering Synergic Effects on Anti-Depressant Mechanisms

Omega-3 polyunsaturated fatty acids (n-3 or omega-3 PUFAs) and melatonin receptor agonist ramelteon (RMT) both display antidepressant effects, while their cellular effects on anti-oxidative and neuroprotective mechanisms might be different. In this study, we aimed to decipher the individual and synergistic actions of n-3 PUFAs and RMT, as compared with the conventional antidepressant fluoxetine (FLX), in a cellular model of oxidative stress, which might play an important role in the pathophysiology of depression and associated disorders. We investigated the rescue and prevention effects of FLX, RMT, and n-3 PUFAs, e.g., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), by using cell viability in SH-SY5Y cells under oxidative stress along with measurements of key cellular markers of oxidative stress, inflammatory, and neuroprotection. The results revealed that the RMT and EPA combination significantly increased the cell viability in a dose-dependent manner. RMT showed preventive effects, FLX and DHA possessed rescue effects, while EPA showed both rescue and preventive effects. We observed the dose-dependent activation and translocation of nuclear factor-κB to the nucleus augmented by the expressions of peroxisome proliferator activator receptor-gamma, tyrosine hydroxylase, c-Fos expression, and reactive oxygen species, implying that RMT and EPA combination reversed oxidative and neuroinflammatory pathophysiology and protected the neuronal cells from further damage. The results demonstrated that RMT and EPA synergistically provide effective neuroprotective, anti-oxidative/inflammatory effect against oxidative stress. Our study provides pre-clinical evidence to conduct future clinical trials of using n-3 PUFAs/RMT combination in depressive disorders.     

New Insight on Tuning Electrical Transport Properties via Chalcogen Doping in n‐type Mg3Sb2‐Based Thermoelectric Materials

n‐type Mg₃Sb_1.5Bi_0.5 has recently been discovered to be a promising thermoelectric material, yet the effective n‐type dopants are mainly limited to the chalcogens. This may be attributed to the limited chemical insight into the effects from different n‐type dopants. By comparing the effects of different chalcogen dopants Q (Q = S, Se, and Te) on thermoelectric properties, it is found that the chalcogen dopants Q become more efficient with decreasing electronegativity difference between Q and Mg, which is mainly due to the increasing carrier concentration and mobility. Using density functional theory calculations, it is shown that the improving carrier concentration originates from the increasing doping limit induced by the stabilizing extrinsic defect. Moreover, the increasing electron mobility with decreasing electronegativity difference between Q and Mg is attributed to the smaller effective mass resulting from the enhancing chemical bond covalency, which is supported by the decreasing theoretical density of states. According to the above trends, a simple guiding principle based on electronegativity is proposed to shed new light on n‐type doping in Zintl antimonides.     

Comparative studies on mammalian Muller (retinal glial) cells

Brain Cell Biology - Muller cells from 22 mammalian species were subjected to morphological and electrophysiological studies. In the ‘mid-periphery’ of retinae immunocytochemically...     

Keeping homologous recombination in check

Pathway choice is a critical event in the repair of DNA double-strand breaks. In a recent paper published in Nature, Orthwein et al. define a mechanism by which homologous recombination is controlled in G1 cells to favor non-homologous end joining.Homologous recombination (HR) is an essential process that produces genetic variation during meiosis and protects the genome during mitotic cell division¹. Inherited mutations in various HR factors, including the BRCA1, BRCA2 and PALB2 tumor suppressors, predispose to the development of cancer. Although HR is generally beneficial for maintaining genome integrity, HR events between homologous chromosomes can also be deleterious and lead to loss of genetic information. HR is therefore suppressed during G1 phase and in non-dividing cells, yet, the exact mechanism behind this phenomenon has remained elusive. New work from the laboratory of Daniel Durocher describes a mechanism that is both necessary and sufficient for the suppression of HR in G1 cells².DNA double-strand breaks (DSBs) are one of the most dangerous types of DNA lesion and need to be eliminated to prevent the accumulation of mutations. DSB repair is carried out by two main pathways, HR and non-homologous end joining (NHEJ)¹. Whereas NHEJ is an error-prone process that simply fuses the two broken ends together, HR is essentially error-free as it uses the genetically identical sister chromatid as a template for repair. Due to the cell cycle-dependent availability of sister chromatids, HR is restricted to the S and G2 phases of the cell cycle.In the HR repair pathway, the DSB ends are first resected to produce extended single-stranded DNA (ssDNA) tails by the coordinated actions of a series of helicase and nuclease activities (e.g., MRN, CtIP and EXO1)¹. CtIP plays a particularly important role in regulating resection, which is mediated through its interaction with BRCA1³. In the following cascade of events, BRCA1 interacts directly with the BRCA2-PALB2 complex, which in turn is recruited to the ssDNA where it acts as a chaperone that stimulates the formation of RAD51 nucleoprotein filaments that drive homology-directed HR repair to restore the integrity of the DNA^4,5.Whereas most HR events take place between the newly replicated sister chromatids, recombination between homologous chromosomes can result in loss of heterozygosity, a potentially mutagenic event that can lead to the inactivation of tumor suppressors or activation of oncogenes. HR must therefore be tightly regulated and effectively suppressed in G1 phase, at the time when only homologous chromosomes are available for repair. At such times, NHEJ is the favored mechanism for DSB repair.A number of mechanisms regulate HR to a specific phase of the cell cycle. For example, CtIP is activated for interaction with BRCA1 by CDK-dependent phosphorylation, which occurs in the S and G2 phases of the cell cycle. Conversely, HR is suppressed in G1 phase by the pro-NHEJ factors 53BP1⁶, RIF1⁷ and REV7⁸, which impair the recruitment of BRCA1 and thereby inhibit DNA end resection. Consequently, disruption of 53BP1 leads to the recruitment of BRCA1 to DSBs in G1 phase. In the recent Nature paper from Durocher''s laboratory, Orthwein et al.² discovered that although BRCA1 is localized to DSBs during G1 phase in 53BP1-deficient cells, it fails to recruit the BRCA2-PALB2 complex, which is consistent with the lack of HR activity in these cells.Through immunoprecipitation experiments Orthwein et al. showed that while BRCA2 and PALB2 interact throughout the cell cycle, BRCA1 and PALB2 only interact efficiently in S phase, suggesting that there might be a mechanism that restricts their interaction to S and G2 phases, while also blocking it in G1 phase. The region of PALB2 that is responsible for its cell cycle-regulated interaction with BRCA1 was localized to its N-terminal domain, which corresponds to a known interaction site for KEAP1, a substrate adaptor for the CUL3-RING (CRL3) ubiquitin ligase. Remarkably, they found that deletion of the KEAP1 gene using CRISPR-Cas9 technology restored the BRCA1-PALB2 interaction in G1 cells, and led to the recruitment of BRCA2-PALB2 to sites of DNA damage in 53BP1-deficient G1 cells.Since KEAP1 is involved in protein ubiquitylation, Orthwein et al. hypothesized that ubiquitylation of PALB2 in the BRCA1-interacting region might block their interaction. Indeed, mutation of lysines in the interacting region of PALB2 restored its interaction with BRCA1 in G1 cells. Furthermore, pull-down experiments showed that ubiquitylation of PALB2 on Lysine-20 by KEAP1-CRL3 prevented its interaction with BRCA1. However, as neither the activity of the KEAP1-CRL3 ubiquitin ligase nor its interaction with BRCA1 is cell cycle regulated, Orthwein et al. reasoned that a deubiquitylation step could be the rate-limiting regulator of the BRCA1-PALB2 interaction. They highlighted the deubiquitylating enzyme USP11 as a potential candidate for this activity due to its interaction with BRCA1, BRCA2 and PALB2, and indeed found that USP11 disruption impaired the interaction between BRCA1 and PALB2. Moreover, they found that USP11 was unstable and interacted poorly with PALB2 in G1 cells, and that USP11 was rapidly lost by proteasomal degradation in G1 phase after DNA damage. By contrast, expression of USP11 in S-phase was high and insensitive to DNA damage. Taken together, these data led the authors to propose that the opposing activities of USP11 and KEAP1-CRL3 regulate cell cycle-dependent interactions between BRCA1 and PALB2 (Figure 1).Open in a separate windowFigure 1Schematic representation indicating how the opposing activities of USP11 and KEAP1-CRL3 regulate cell cycle-dependent interactions between BRCA1 and PALB2, and thereby mediate pathway choice in DSB repair.To extend these remarkable observations, Orthwein et al. disrupted this regulatory network to allow HR in G1 cells. They expected that depletion of KEAP1 in 53BP1-deficient cells might be sufficient for RAD51 foci formation following ionizing radiation (IR), but this was not the case because end resection remained a limiting factor. To counteract this, the authors expressed a constitutively active form of CtIP (T847E)⁹, which augmented resection and led to the efficient formation of IR-induced RAD51 foci in 53BP1- and KEAP1-deficient G1 cells. To address whether these RAD51 foci in G1 cells corresponded to productive HR events, they used a fluorescent-based gene-targeting assay. Whereas CtIP (T847E)expressed in 53BP1-deficient cells alone was insufficient to induce productive HR, depletion of KEAP1 or expression of a non-ubiquitylable version of PALB2 led to a robust increase in gene-targeting events. Collectively, this study therefore demonstrates that activation of DNA end resection, combined with the recruitment of BRCA2 to DSBs, are both necessary and sufficient to produce HR in G1 cells.Gene targeting has great potential for therapeutic purposes, but the fact that most cells in the body are non-dividing has so far limited its use¹⁰. We suspect that the new knowledge highlighted in this work will further improve gene-targeting therapies to help fight human diseases.