Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells

Neuritic alterations are a major feature of many neurodegenerative disorders. Methylation of protein phosphatase 2A (PP2A) catalytic C subunit by the leucine carboxyl methyltransferase (LCMT1), and demethylation by the protein phosphatase methylesterase 1, is a critical PP2A regulatory mechanism. It modulates the formation of PP2A holoenzymes containing the Bα subunit, which dephosphorylate key neuronal cytoskeletal proteins, including tau. Significantly, we have reported that LCMT1, methylated C and Bα expression levels are down-regulated in Alzheimer disease-affected brain regions. In this study, we show that enhanced expression of LCMT1 in cultured N2a neuroblastoma cells, which increases endogenous methylated C and Bα levels, induces changes in F-actin organization. It promotes serum-independent neuritogenesis and development of extended tau-positive processes upon N2a cell differentiation. These stimulatory effects can be abrogated by LCMT1 knockdown and S-adenosylhomocysteine, an inhibitor of methylation reactions. Expression of protein phosphatase methylesterase 1 and the methylation-site L309Δ C subunit mutant, which decrease intracellular methylated C and Bα levels, block N2a cell differentiation and LCMT1-mediated neurite formation. Lastly, inducible and non-inducible knockdown of Bα in N2a cells inhibit process outgrowth. Altogether, our results establish a novel mechanistic link between PP2A methylation and development of neurite-like processes.     

Effects on the conformation of FVIIa by sTF and Ca binding: Studies of fluorescence resonance energy transfer and quenching

The apparent length of FVIIa in solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein (Fl-FPR) and a rhodamine derivative (TMR), were covalently attached to FVIIa. The binding site of Fl-FPR was in the protease domain whereas TMR was positioned in the Gla domain, thus allowing a length measure over virtually the whole extension of the protein. From the FRET measurements, the distances between the two probes were determined to be 61.4 for free FVIIa and 65.5 Å for FVIIa bound to soluble tissue factor (sTF). These seemingly short distances, compared to those anticipated based on the complex crystal structure, require that the probes stretch towards each other. Thus, the apparent distance from the FRET analysis was shown to increase with 4 Å upon formation of a complex with sTF in solution. However, considering how protein dynamics, based on recent molecular dynamics simulations of FVIIa and sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044–1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF.It is known from amidolytic activity measurements that Ca²⁺ binding leads to activation of FVIIa, but we have for the first time directly demonstrated conformational changes in the environment of the active site upon Ca²⁺ binding. Interestingly, this Ca²⁺-induced conformational change can be noted even in the presence of an inhibitor. Forming a complex with sTF further stabilized this conformational change, leading to a more inaccessible active-site located probe.     

M-Track: detecting short-lived protein-protein interactions in vivo

We developed a protein-proximity assay in yeast based on fusing a histone lysine methyltransferase onto a bait and its substrate onto a prey. Upon binding, the prey is stably methylated and detected by methylation-specific antibodies. We applied this approach to detect varying interaction affinities among proteins in a mitogen-activated protein kinase pathway and to detect short-lived interactions between protein phosphatase 2A and its substrates that have so far escaped direct detection.     

Éjaculation provoquée par le vibromassage ou l’électrostimulation endorectale chez le blessé médullaire: injection intracytoplasmique de spermatozoïdes obtenus par stimulation endorectale chez neuf couples

800 to 1,000 cases of traumatic spinal cord injury (S.C.I.) are observed in France each year. At present time, there are more than 35,000 survivors of S.C.I. in France. Studies show that 80% of injuries occur in men and 82% occur in individuals between the ages of 16 and 45 years. It is well known that more than 80% of men with S.C.I. suffer from ejaculation dysfunction. Since most of these couples are young, many desire a family and seek help to remedy their infertility. Two methods are mainly used to retrieve sperm from S.C.I. men: penile vibratory stimulation and rectal electrostimulation. Penile vibratory stimulation to induce ejaculation is recommended as first-line treatment, and S.C.I. men should only be referred for electroejaculation after failure of this technique. More than 70% of patients respond to penile vibratory stimulation with anterograde ejaculation. Patients who fail to ejaculate by penile vibratory stimulation are treated by electroejaculation. Their ejaculates often have normal sperm counts, but with a higher proportion of immotile sperm than in men without S.C.I. The authors report the pregnancy outcome of a series of 9 couples undergoing combined electroejaculation and in vitro fertilization with intracytoplasmic sperm injection: 25% pregnancies per cycle and 33% pregnancies per couple.     

Simple emission-reducing measures in an open biological waste treatment plant

In the course of composting biological waste, concentrations of various thermophilic and thermotolerant microorganisms increase. Moving piles of compost results in increased emissions of Actinomycetes and fungi. The present investigation deals with the reduction of airborne microorganism emission and immission in large-scale composting plants with open piles. Simple measures were introduced in order to reduce the release of bioaerosols when turning piles and the release of dust and bioaerols at large. These measures included the sealing of turning machinery with rubber mats, the wetting of piles before and after turning and regular cleaning and wetting driveways during the dry season.Concentrations of airborne microorganisms during the summer season were determined on 5 days before and after the introduction of emission-reducing measures using the six-stage Andersen cascade sampler. The investigation showed that following the introduction of emission-reducing measures there was, at all locations, a highly significant reduction not only of all culturable indicator organisms (thermophilic actinomycetes and Aspergillus fumigatus) but also of total microorganism concentrations (p < 0,001). The introduction of the simple emission-reducing measures mentioned above, however, reduced the immission in the vicinity of the plant to such a degree that the natural background levels were reached at a distance of 150 m.     

Functional status of hip joint after surgical and conservative treatment of acetabular fracture

Both conservative and surgical treatment of acetabular fractures alter biomechanical conditions in the hip joint resulting in various degenerative changes in the acetabulum and the femur head. These changes may progress to secondary coxarthrosis, causing dysfunction of the hip joint. The aim of this paper is to compare the outcomes of both conservative and surgical treatments in different types of acetabular fractures, so that clear indications for either conservative or surgical treatment could be determined. This paper is based on retrospective study of 103 patients with acetabular fracture (21 surgically treated and 82 conservatively treated). In this study the incidence of particular types of acetabulum fractures, the treatment period, the incidence of complications and the functional status of the hip after the treatment were analyzed. In patients with anterior fracture surgical treatment lasted three times less than conservative treatment and resulted in far better functional status of the hip joint compared to conservatively treated patients. However, in patients with transverse fractures the functional status was better and the treatment period shorter after the conservative treatment. We believe that the present treatment indications should be corrected so that in anterior column fracture the surgical method should be preferred, whereas the transverse fracture should be treated conservatively. In other types of acetabular fracture, with the radiographic roof arc angle of 45 degrees or less, the surgical method should be preferred to conservative method.     

Monitoring Cell Death in Regorafenib-Treated Experimental Colon Carcinomas Using Annexin-Based Optical Fluorescence Imaging Validated by Perfusion MRI

Objective

To investigate annexin-based optical fluorescence imaging (OI) for monitoring regorafenib-induced early cell death in experimental colon carcinomas in rats, validated by perfusion MRI and multiparametric immunohistochemistry.

Materials and Methods

Subcutaneous human colon carcinomas (HT-29) in athymic rats (n = 16) were imaged before and after a one-week therapy with regorafenib (n = 8) or placebo (n = 8) using annexin-based OI and perfusion MRI at 3 Tesla. Optical signal-to-noise ratio (SNR) and MRI tumor perfusion parameters (plasma flow PF, mL/100mL/min; plasma volume PV, %) were assessed. On day 7, tumors underwent immunohistochemical analysis for tumor cell apoptosis (TUNEL), proliferation (Ki-67), and microvascular density (CD31).

Results

Apoptosis-targeted OI demonstrated a tumor-specific probe accumulation with a significant increase of tumor SNR under therapy (mean Δ +7.78±2.95, control: -0.80±2.48, p = 0.021). MRI detected a significant reduction of tumor perfusion in the therapy group (mean ΔPF -8.17±2.32 mL/100 mL/min, control -0.11±3.36 mL/100 mL/min, p = 0.036). Immunohistochemistry showed significantly more apoptosis (TUNEL; 11392±1486 vs. 2921±334, p = 0.001), significantly less proliferation (Ki-67; 1754±184 vs. 2883±323, p = 0.012), and significantly lower microvascular density (CD31; 107±10 vs. 182±22, p = 0.006) in the therapy group.

Conclusions

Annexin-based OI allowed for the non-invasive monitoring of regorafenib-induced early cell death in experimental colon carcinomas, validated by perfusion MRI and multiparametric immunohistochemistry.     

Bioclipse: an open source workbench for chemo- and bioinformatics

Background  

There is a need for software applications that provide users with a complete and extensible toolkit for chemo- and bioinformatics accessible from a single workbench. Commercial packages are expensive and closed source, hence they do not allow end users to modify algorithms and add custom functionality. Existing open source projects are more focused on providing a framework for integrating existing, separately installed bioinformatics packages, rather than providing user-friendly interfaces. No open source chemoinformatics workbench has previously been published, and no sucessful attempts have been made to integrate chemo- and bioinformatics into a single framework.