PRIMA-1 cytotoxicity correlates with nucleolar localization and degradation of mutant p53 in breast cancer cells

PRIMA-1 has been identified as a compound that restores the transactivation function to mutant p53 and induces apoptosis in cells expressing mutant p53. Studies on subcellular distribution of the mutant p53 protein upon treatment with PRIMA-1^Met, a methylated form of PRIMA-1, have suggested that redistribution of mutant p53 to nucleoli may play a role in PRIMA-1 induced apoptosis. Here, we specifically investigated the influence of PRIMA-1 on cellular localization of mutated p53-R280K endogenously expressed in tumour cells. By using immunofluorescence staining, we found a strong nucleolar redistribution of mutant p53 following PRIMA-1 treatment. This subcellular localization was associated to p53 degradation via ubiquitylation. When cells were treated with adriamycin, neither nucleolar redistribution nor mutant p53 down modulation and degradation were observed. Interestingly, cells where p53-R280K was silenced were more sensitive to PRIMA-1 than the parental ones. These results indicate that in some cellular context, the cell sensitivity to PRIMA-1 could depend on the abolition of a gain-of-function activity of the mutated p53, through a protein degradation pathway specifically induced by this compound.     

A quantitative and statistically robust method for the determination of xylem conduit spatial distribution

? Premise of the study: Because of their limited length, xylem conduits need to connect to each other to maintain water transport from roots to leaves. Conduit spatial distribution in a cross section plays an important role in aiding this connectivity. While indices of conduit spatial distribution already exist, they are not well defined statistically. ? Methods: We used point pattern analysis to derive new spatial indices. One hundred and five cross-sectional images from different species were transformed into binary images. The resulting point patterns, based on the locations of the conduit centers-of-area, were analyzed to determine whether they departed from randomness. Conduit distribution was then modeled using a spatially explicit stochastic model. ? Key results: The presence of conduit randomness, uniformity, or aggregation depended on the spatial scale of the analysis. The large majority of the images showed patterns significantly different from randomness at least at one spatial scale. A strong phylogenetic signal was detected in the spatial variables. ? Conclusions: Conduit spatial arrangement has been largely conserved during evolution, especially at small spatial scales. Species in which conduits were aggregated in clusters had a lower conduit density compared to those with uniform distribution. Statistically sound spatial indices must be employed as an aid in the characterization of distributional patterns across species and in models of xylem water transport. Point pattern analysis is a very useful tool in identifying spatial patterns.     

Intrinsic choroidal neurons in the chicken eye: chemical coding and synaptic input

Intrinsic choroidal neurons (ICNs) exist in some primates and bird species. They may act on both vascular and non-vascular smooth muscle cells, potentially influencing choroidal blood flow. Here, we report on the chemical coding of ICNs and eye-related cranial ganglia in the chicken, an important model in myopia research, and further to determine synaptic input onto ICN. Chicken choroid, ciliary, superior cervical, pterygopalatine, and trigeminal ganglia were prepared for double or triple immunohistochemistry of calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), dopamine-β-hydroxylase, galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM), tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP), vesicular monoamine-transporter 2 (VMAT2), and α-smooth muscle actin. For documentation, light, fluorescence, and confocal laser scanning microscopy were used. Chicken ICNs express nNOS/VIP/GAL and do not express ChAT and SOM. ICNs are approached by TH/VMAT2-, CGRP-, and ChAT-positive nerve fibers. About 50% of the pterygopalatine ganglion neurons and about 9% of the superior cervical ganglion neurons share the same chemical code as ICN. SOM-positive neurons in the ciliary ganglion are GAL/NOS negative. CGRP-positive neurons in the trigeminal ganglion lack GAL/SOM. The neurochemical phenotype and synaptic input of ICNs in chicken resemble that of other bird and primate species. Because ICNs lack cholinergic markers, they cannot be readily incorporated into current concepts of the autonomic nervous system. The data obtained provide the basis for the interpretation of future functional experiments to clarify the role of these cells in achieving ocular homeostasis.     

Age related changes in T cell mediated immune response and effector memory to Respiratory Syncytial Virus (RSV) in healthy subjects

Respiratory syncytial virus (RSV) is the major pathogen causing respiratory disease in young infants and it is an important cause of serious illness in the elderly since the infection provides limited immune protection against reinfection. In order to explain this phenomenon, we investigated whether healthy adults of different age (20-40; 41-60 and > 60 years), have differences in central and effector memory, RSV-specific CD8+ T cell memory immune response and regulatory T cell expression status. In the peripheral blood of these donors, we were unable to detect any age related difference in term of central (CD45RA^-CCR7⁺) and effector (CD45RA^-CCR7^-) memory T cell frequency. On the contrary, we found a significant increase in immunosuppressive regulatory (CD4⁺25⁺FoxP3⁺) T cells (T_reg) in the elderly. An immunocytofluorimetric RSV pentamer analysis performed on these donors' peripheral blood mononuclear cells (PBMCs), in vitro sensitized against RSV antigen, revealed a marked decline in long-lasting RSV specific CD8+ memory T cell precursors expressing interleukin 7 receptor α (IL-7Rα), in the elderly. This effect was paralleled by a progressive switch from a Th1 (IFN-γ and TNF-α) to a Th2 (IL-10) functional phenotype. On the contrary, an increase in T_reg was observed with aging. The finding of T_reg over-expression status, a prominent Th2 response and an inefficient RSV-specific effector memory CD8+ T cell expansion in older donors could explain the poor protection against RSV reinfection and the increased risk to develop an RSV-related severe illness in this population. Our finding also lays the basis for new therapeutic perspectives that could limit or prevent severe RSV infection in elderly.     

Mycovirus cryphonectria hypovirus 1 elements cofractionate with trans-Golgi network membranes of the fungal host Cryphonectria parasitica

The mycovirus cryphonectria hypovirus 1 (CHV1) causes proliferation of vesicles in its host, Cryphonectria parasitica, the causal agent of chestnut blight. These vesicles have previously been shown to contain both CHV1 genomic double-stranded RNA (dsRNA) and RNA polymerase activity. To determine the cellular origins of these virus-induced membrane structures, we compared the fractionation of several cellular and viral markers. Results showed that viral dsRNA, helicase, polymerase, and protease p29 copurify with C. parasitica trans-Golgi network (TGN) markers, suggesting that the virus utilizes the fungal TGN for replication. We also show that the CHV1 protease p29 associates with vesicle membranes and is resistant to treatments that would release peripheral membrane proteins. Thus, p29 behaves as an integral membrane protein of the vesicular fraction derived from the fungal TGN. Protease p29 was also found to be fully susceptible to proteolytic digestion in the absence of detergent and, thus, is wholly or predominantly on the cytoplasmic face of the vesicles. Fractionation analysis of p29 deletion variants showed that sequences in the C terminal of p29 mediate membrane association. In particular, the C-terminal portion of the protein (Met-135-Gly-248) is sufficient for membrane association and is enough to direct p29 to the TGN vesicles in the absence of other viral elements.     

Correlation between induction of DNA fragmentation in lung cells from rats and humans and carcinogenic activity

Six chemicals, known to induce lung tumors in rats, were examined for their ability to induce DNA fragmentation in primary cultures of rat and human lung cells, and in the lung of intact rats. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the single-cell gel electrophoresis (Comet) assay, were obtained in primary lung cells from male rats with the following, minimally toxic, concentrations of the six test compounds: N-nitrosodimethylamine (NDMA; 2.5-10mM), hydrazine (HZ; 0.5-4mM), cadmium sulfate (CD; 31.2 and 62.5muM), 4,4'-methylene bis (2-chloroaniline) (MOCA; 31.2-125muM), isobutyl nitrite (IBN; 7.8-31.2muM) and tetranitromethane (TNM; 1.9-15.6muM). Similar degrees of DNA fragmentation were obtained in primary human lung cells; however, due to inter-donor differences, the minimum effective concentrations were in some donors lower and in others higher than in rats, and IBN induced DNA damage only in one of three donors. The DNA-damaging potency of HZ was higher in rats than in humans, and the opposite was true for MOCA. In agreement with these findings, statistically significant increases in the average frequency of DNA breaks were obtained in the lung of rats given a single oral dose (1/2 LD50) of the six test compounds. These findings give evidence that genotoxic lung carcinogens may be identified by use of the DNA fragmentation/Comet assay on rat lung cells as targets cells, and show that the six compounds tested produce in primary cultures of lung cells from human donors DNA-damaging effects substantially similar to those observed in rats.     

Partial purification and characterization of sperm receptor hydrolase,a cortical granule proteoesterase,from eggs of the sea urchin Strongylocentrotus purpuratus

Sperm receptor hydrolase, one of two classes of cortical granule proteoesterases (E.C.3.4.4.4) was purified approximately 30-fold with 80% yield from Strongylocentrotus purpuratus cortical granule exudate. Sperm receptor hydrolase preparations were free of vitelline delaminase activity (the other class of cortical granule proteoesterase) and had less than 1% of the starting levels of cortical granule peroxidase and β-1,3-glucanohydro-lase activities. Native polyacrylamide gel electrophoresis coupled with a protease activity stain showed that three proteases were present in the most highly purified preparations of sperm receptor hydrolase. Each of the three proteases has the same molecular weight of 60,000, but different isoelectric points of 2.4, 3.8, and 5.5. The K_m value of the mixture of proteases for α-N-benzoyl-L-arginine ethyl ester as substrate was 263 μM at pH 8.4 and 30°C; the pH dependence of V_m showed a single prototrophic group with a pK of 6.7 and an enthalpy of ionization of 8.6 kcal-mol^?1. The values of these kinetic parameters are consistent with an enzyme-active site containing histidine. Phenylmethyl sulfonyl fluoride, tosyl lysine chloromethyl ketone, several proteinaceous trypsin inhibitors, and p-aminobenzamidine inhibited the esterase activity of the proteases. These data suggest that sperm receptor hydrolases are serine proteases.     

Calmodulin Kinase II in Pure Cultured Astrocytes

Calcium- and calmodulin-dependent protein kinase activity was studied in pure neuronal and glial cultures. The addition of calcium and calmodulin stimulated 32P incorporation into several neuronal proteins including two in the 50- and 60-kilodalton (kD) region which comigrated with purified forebrain calmodulin kinase II subunits (CaM kinase II). In mature astrocytes, CaM kinase activity was also present, and was inhibited by trifluoroperazine and diazepam. Again in homogenates of these cells, two phosphoproteins of apparent molecular masses of 50 and 60 kD comigrated with purified CaM kinase. CaM kinase activity was absent in immature mixed glia and oligodendrocytes. The presence of CaM kinase in neurons and mature astrocytes was confirmed using monoclonal antibodies specific for the 50-kD subunit of the enzyme. No immunoreactivity was observed in oligodendrocytes. The presence of CaM kinase in astrocytes suggests a more ubiquitous role of this enzyme in regulating cellular processes than was previously recognized.