The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

Explicit Mentalizing Mechanisms and Their Adaptive Role in Memory Conformity

Memory conformity occurs when an individual endorses what other individuals remember about past events. Research on memory conformity is currently dominated by a ‘forensic’ perspective, which views the phenomenon as inherently undesirable. This is because conformity not only distorts the accuracy of an individual''s memory, but also produces false corroboration between individuals, effects that act to undermine criminal justice systems. There is growing awareness, however, that memory conformity may be interpreted more generally as an adaptive social behavior regulated by explicit mentalizing mechanisms. Here, we provide novel evidence in support of this emerging alternative theoretical perspective. We carried out a memory conformity experiment which revealed that explicit belief-simulation (i.e. using one''s own beliefs to model what other people believe) systematically biases conformity towards like-minded individuals, even when there is no objective evidence that they have a more accurate memory than dissimilar individuals. We suggest that this bias is functional, i.e. adaptive, to the extent that it fosters trust, and hence cooperation, between in-group versus out-group individuals. We conclude that memory conformity is, in more fundamental terms, a highly desirable product of explicit mentalizing mechanisms that promote adaptive forms of social learning and cooperation.     

Mathematical Modeling of Renal Tubular Glucose Absorption after Glucose Load

A partial differential Progressive Tubular Reabsorption (PTR) model, describing renal tubular glucose reabsorption and urinary glucose excretion following a glucose load perturbation, is proposed and fitted to experimental data from five subjects. For each subject the Glomerular Filtration Rate was estimated and both blood and urine glucose were sampled following an Intra-Venous glucose bolus. The PTR model was compared with a model representing the conventional Renal Threshold Hypothesis (RTH). A delay bladder compartment was introduced in both formulations. For the RTH model, the average threshold for glycosuria varied between 9.90±4.50 mmol/L and 10.63±3.64 mmol/L (mean ± Standard Deviation) under different hypotheses; the corresponding average maximal transport rates varied between 0.48±0.45 mmol/min (86.29±81.22 mg/min) and 0.50±0.42 mmol/min (90.62±76.15 mg/min). For the PTR Model, the average maximal transports rates varied between 0.61±0.52 mmol/min (109.57±93.77 mg/min) and 0.83±0.95 mmol/min (150.13±171.85 mg/min). The time spent by glucose inside the tubules before entering the bladder compartment varied between 1.66±0.73 min and 2.45±1.01 min.The PTR model proved much better than RTH at fitting observations, by correctly reproducing the delay of variations of glycosuria with respect to the driving glycemia, and by predicting non-zero urinary glucose elimination at low glycemias. This model is useful when studying both transients and steady-state glucose elimination as well as in assessing drug-related changes in renal glucose excretion.     

Essential Role of the EF-hand Domain in Targeting Sperm Phospholipase Cζ to Membrane Phosphatidylinositol 4,5-Bisphosphate (PIP2)

Sperm-specific phospholipase C-ζ (PLCζ) is widely considered to be the physiological stimulus that triggers intracellular Ca²⁺ oscillations and egg activation during mammalian fertilization. Although PLCζ is structurally similar to PLCδ1, it lacks a pleckstrin homology domain, and it remains unclear how PLCζ targets its phosphatidylinositol 4,5-bisphosphate (PIP₂) membrane substrate. Recently, the PLCδ1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLCδ1 are notably conserved in PLCζ. We investigated the potential role of these conserved cationic residues in PLCζ by generating a series of mutants that sequentially neutralized three positively charged residues (Lys-49, Lys-53, and Arg-57) within the mouse PLCζ EF-hand domain. Microinjection of the PLCζ EF-hand mutants into mouse eggs enabled their Ca²⁺ oscillation inducing activities to be compared with wild-type PLCζ. Furthermore, the mutant proteins were purified, and the in vitro PIP₂ hydrolysis and binding properties were monitored. Our analysis suggests that PLCζ binds significantly to PIP₂, but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the first EF-hand domain of PLCζ significantly alters in vivo Ca²⁺ oscillation inducing activity and in vitro interaction with PIP₂ without affecting its Ca²⁺ sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca²⁺ frequency and the binding ability of different PLCζ mutants to PIP₂. Moreover, a PLCζ mutant with mutations in the cationic residues within the first EF-hand domain and the XY linker region dramatically reduces the binding of PLCζ to PIP₂, leading to complete abolishment of its Ca²⁺ oscillation inducing activity.     

The de-ubiquitylating enzymes USP26 and USP37 regulate homologous recombination by counteracting RAP80

The faithful repair of DNA double-strand breaks (DSBs) is essential to safeguard genome stability. DSBs elicit a signaling cascade involving the E3 ubiquitin ligases RNF8/RNF168 and the ubiquitin-dependent assembly of the BRCA1-Abraxas-RAP80-MERIT40 complex. The association of BRCA1 with ubiquitin conjugates through RAP80 is known to be inhibitory to DSB repair by homologous recombination (HR). However, the precise regulation of this mechanism remains poorly understood. Through genetic screens we identified USP26 and USP37 as key de-ubiquitylating enzymes (DUBs) that limit the repressive impact of RNF8/RNF168 on HR. Both DUBs are recruited to DSBs where they actively remove RNF168-induced ubiquitin conjugates. Depletion of USP26 or USP37 disrupts the execution of HR and this effect is alleviated by the simultaneous depletion of RAP80. We demonstrate that USP26 and USP37 prevent excessive spreading of RAP80-BRCA1 from DSBs. On the other hand, we also found that USP26 and USP37 promote the efficient association of BRCA1 with PALB2. This suggests that these DUBs limit the ubiquitin-dependent sequestration of BRCA1 via the BRCA1-Abraxas-RAP80-MERIT40 complex, while promoting complex formation and cooperation of BRCA1 with PALB2-BRCA2-RAD51 during HR. These findings reveal a novel ubiquitin-dependent mechanism that regulates distinct BRCA1-containing complexes for efficient repair of DSBs by HR.     

Application of a bioenergetics growth model for European anchovy (<Emphasis Type="Italic">Engraulis encrasicolus</Emphasis>) linked with a lower trophic level ecosystem model

A bioenergetics model is implemented for European anchovy (Engraulis encrasicolus) and applied to the north-eastern Aegean Sea (eastern Mediterranean Sea). The model reproduces the growth of anchovy in a one-way linked configuration with a lower trophic level (LTL) ecosystem model. The LTL model provides densities for three zooplankton functional groups (heterotrophic flagellates, microzooplankton and mesozooplankton) which serve as available energy via consumption for the anchovy model. Our model follows the basic structure of NEMURO.FISH type models (North Pacific Ecosystem Model for Understanding Regional Oceanography for Including Saury and Herring). Several model parameters were specific for the Mediterranean or the Black Sea anchovy and some others were adopted from related species and NEMURO.FISH due to lack of biological information on E. encrasicolus. Simulation results showed that the fastest growth rate occurs during spring and the slowest growth rate from August to December. Zooplankton abundance during autumn was low implying that decreased prey density lead to a reduction in anchovy weight, especially for the age-3 class. Feeding parameters were adjusted to adequately fit the model growth estimates to available weight-at-age data. A detailed sensitivity analyses is conducted to evaluate the importance of the biological processes (consumption, respiration, egestion, specific dynamic action, excretion and egg production) and their parameters to fish growth. The most sensitive parameters were the intercept and exponent slope of the weight-dependent consumption and respiration process equations. Fish weight was fairly sensitive to temperature-dependent parameters.     

Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract

Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.     

Embryoid bodies from mouse stem cells express oxytocin receptor, Oct-4 and DAZL

Oxytocin is a nine amino acid peptide involved in a wide spectrum of physiological functions; predominantly those concerning reproduction and differentiation are of interest. Oxytocin receptors are expressed at early developmental stages of mammals, suggesting that oxytocin might be involved in the determination of the germ stem cell line, at the very early stages of mammalian development. In this respect, the proximate aim of the present study was to confirm and further analyze the existence of oxytocin receptors at a very early level of cell commitment, that is, the determination of germ cells derived from embryoid bodies. To achieve our purpose we have cultured mouse embryonic stem cells under conditions inducing formation of embryoid bodies. In this work, ES cells were allowed to aggregate in a novel medium, “Stefanidis medium” from day 0 to day 14 until formed EBs. RNA was isolated from EBs and using RT-PCR we showed that EBs expressed Oct-4, OTR, OT, and DAZL. To demonstrate simultaneous expression immunocytochemistry was preformed, in which EBs showed strong immunoreactivity for both, OTR and DAZL molecular markers. We found that 35% of the cells displayed OTR, using flow cytometry. In addition, this novel medium showed to increase OTR mRNA. We propose, that at least in murine induced embryoid bodies there is simultaneous expression of oxytocin receptors and germ cell markers (DAZL) in many cells (expressing Oct-4). We thus conclude that, the oxytocin might indeed be a molecule playing a leading role in germ cell determination.     

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Monoclonal anti‐HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L ‐Phe/β‐Ala bi‐substituted 1,3,5‐triazine (Trz) scaffold (β‐Ala‐Trz‐L ‐Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10‐binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K_D) of 0.41 ± 0.05 µM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60–80%). Analysis of the antibody preparation by SDS‐PAGE, enzyme‐linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids. Copyright © 2009 John Wiley & Sons, Ltd.