SKOV-3 and Me45 cell response to cisplatin-based chemotherapy: an in vitro study.

We studied malignant melanoma cell line Me45 and human ovarian carcinoma cell line SKOV-3 (resistant to cisplatin, adriamycin and diphtheria toxin), assessing their expression level of p53, HSP70 and glutathione S-transferase GST-π before and after chemotherapy with cisplatin. These proteins may be responsible for the occurrence of chemoresistance in cancer patients. To assess protein expression we used the immunocytochemical Avidin-Biotin-peroxidase Complex (ABC) method. Before application of chemotherapy, proteins p53, HSP70 and GST-π were present in 100 % of the examined melanoma cells. After the treatment, the intensity of the immunocytochemical reaction for p53 increased, whereas the intensity of immunocytochemical staining for HSP70 and GST-π decreased. In SKOV-3 cells, p53 and HSP70 were present in 100 % of the examined cells both prior to chemotherapy and after it. However, the intensity of the immunocytochemical reaction for p53 decreased, while that of HSP70 increased. As regards GST-π, only 5 % of all examined SKOV-3 cells revealed its expression before chemotherapy. Incubation with cisplatin caused an elevation in the number of ovarian cancer cells expressing GST-π up to 50 %. Moreover, the intensity of the immunocytochemical reaction for GST-π significantly increased.

Trim17-mediated ubiquitination and degradation of Mcl-1 initiate apoptosis in neurons.

Short-term proteasome inhibition has been shown to prevent neuronal apoptosis. However, the key pro-survival proteins that must be degraded for triggering neuronal death are mostly unknown. Here, we show that Mcl-1, an anti-apoptotic Bcl-2 family member, is degraded by the proteasome during neuronal apoptosis. Using primary cultures of cerebellar granule neurons deprived of serum and KCl, we found that ubiquitination and proteasomal degradation of Mcl-1 depended on its prior phosphorylation by GSK3, providing the first insight into post-translational regulation of Mcl-1 in neurons. In a previous study, we have reported that the E3 ubiquitin-ligase Trim17 is both necessary and sufficient for neuronal apoptosis. Here, we identified Trim17 as a novel E3 ubiquitin-ligase for Mcl-1. Indeed, Trim17 co-immunoprecipitated with Mcl-1. Trim17 ubiquitinated Mcl-1 in vitro. Overexpression of Trim17 decreased the protein level of Mcl-1 in a phosphorylation- and proteasome-dependent manner. Finally, knock down of Trim17 expression reduced both ubiquitination and degradation of Mcl-1 in neurons. Moreover, impairment of Mcl-1 phosphorylation, by kinase inhibition or point mutations, not only decreased ubiquitination and degradation of Mcl-1, but also blocked the physical interaction between Trim17 and Mcl-1. As this stabilization of Mcl-1 increased its neuroprotective effect, our data strongly suggest that Trim17-mediated ubiquitination and degradation of Mcl-1 is necessary for initiating neuronal death.

Trim17, a novel E3 ubiquitin-ligase, initiates neuronal apoptosis.

Accumulating data indicate that the ubiquitin-proteasome system controls apoptosis by regulating the level and the function of key regulatory proteins. In this study, we identified Trim17, a member of the TRIM/RBCC protein family, as one of the critical E3 ubiquitin ligases involved in the control of neuronal apoptosis upstream of mitochondria. We show that expression of Trim17 is increased both at the mRNA and protein level in several in vitro models of transcription-dependent neuronal apoptosis. Expression of Trim17 is controlled by the PI3K/Akt/GSK3 pathway in cerebellar granule neurons (CGN). Moreover, the Trim17 protein is expressed in vivo, in apoptotic neurons that naturally die during post-natal cerebellar development. Overexpression of active Trim17 in primary CGN was sufficient to induce the intrinsic pathway of apoptosis in survival conditions. This pro-apoptotic effect was abolished in Bax(-/-) neurons and depended on the E3 activity of Trim17 conferred by its RING domain. Furthermore, knock-down of endogenous Trim17 and overexpression of dominant-negative mutants of Trim17 blocked trophic factor withdrawal-induced apoptosis both in CGN and in sympathetic neurons. Collectively, our data are the first to assign a cellular function to Trim17 by showing that its E3 activity is both necessary and sufficient for the initiation of neuronal apoptosis.

Efficient parallel tempering for first-order phase transitions.

We present a Monte Carlo algorithm that facilitates efficient parallel tempering simulations of the density of states g(E) . We show that the algorithm eliminates the supercritical slowing down in the case of the Q=20 and Q=256 Potts models in two dimensions, typical examples for systems with extreme first-order phase transitions. As recently predicted, and shown here, the microcanonical heat capacity along the calorimetric curve has negative values for finite systems.

The budding yeast proteins Spc24p and Spc25p interact with Ndc80p and Nuf2p at the kinetochore and are important for kinetochore clustering and checkpoint control.

Here, we show that the budding yeast proteins Ndc80p, Nuf2p, Spc24p and Spc25p interact at the kinetochore. Consistently, Ndc80p, Nuf2p, Spc24p and Spc25p associate with centromere DNA in chromatin immunoprecipitation experiments, and SPC24 interacts genetically with MCM21 encoding a kinetochore component. Moreover, although conditional lethal spc24-2 and spc25-7 cells form a mitotic spindle, the kinetochores remain in the mother cell body and fail to segregate the chromosomes. Despite this defect in chromosome segregation, spc24-2 and spc25-7 cells do not arrest in metaphase in response to checkpoint control. Furthermore, spc24-2 cells showed a mitotic checkpoint defect when microtubules were depolymerized with nocodazole, indicating that Spc24p has a function in checkpoint control. Since Ndc80p, Nuf2p and Spc24p are conserved proteins, it is likely that similar complexes are part of the kinetochore in other organisms.

Serum level of soluble intercellular adhesion molecule-1 in patients with acute viral hepatitis A or acute viral hepatitis B: lack of correlation with severity of the disease.

Serum soluble ICAM-1 (sICAM-1) was found to be increased in patients with chronic hepatitis. The aim of the study was the evaluation of sICAM-1 dynamics in patients with acute hepatitis A or hepatitis B. Serum level of sICAM-1 was determined in 13 patients with acute hepatic A, 19 patients with acute hepatitis B, and 20 healthy controls. Measurements were done four times, at the beginning of jaundice, in the 14th day of jaundice, at release from the hospital, and when biochemical indices of the liver function became normal. An increase in sICAM-1 was shown in the patients. There was no difference between patients with hepatitis A and those with hepatitis B. The sICAM-1 decreased in the course of recovery but the values in the last measurements were still higher than in the controls. There was no correlation of sICAM-1 and duration of hospitalization. The obtained results do not suggest sICAM-1 as diagnostic or prognostic marker in patients with acute hepatitis A or B.

A new gonadotropin treatment for idiopathic moderate oligoasthenospermia.

Twenty-three idiopathic oligoasthenospermic patients were subdivided into three classes according to degree of sperm concentration and typical sperm motility percentage. These patients were treated with 75 I.U. of Urofollitropin every other day for three months. The patients with moderate oligoasthenospermia responded more favorably than patients with severe or more severe oligoasthenospermia.