Id genes and proteins as promising targets in cancer therapy

Since the identification of Id proteins more than a decade ago, much work has demonstrated their regulatory roles in development, cell fate and lineage determination, proliferation, differentiation, angiogenesis, invasion and migration. Recent studies reveal not only that Id protein expression is significantly correlated both with cancer progression and with overall prognosis, but also that it can be exploited as a therapeutic target. This review will focus on the recent advances in our understanding of the relationships between Id expression and cancer, as well as providing a rationale for developing therapeutic strategies using Ids as targets to treat metastatic cancers.     

The cellular protein level of parkin is regulated by its ubiquitin-like domain

Parkin is a ubiquitin-protein isopeptide ligase (E3) involved in ubiquitin/proteasome-mediated protein degradation. Mutations in the parkin gene cause a loss-of-function and/or alter protein levels of parkin. As a result, the toxic build-up of parkin substrates is thought to lead to autosomal recessive juvenile Parkinsonism. To identify a role for the ubiquitin-like domain (ULD) of parkin, we created a number of hemagglutinin (HA)-tagged parkin constructs using mutational and structural information. Western blotting and immunocytochemistry showed a much stronger expression level for HA-parkin residues 77-465 (without ULD) than HA-parkin full-length (with ULD). The deletion of ULD in Drosophila parkin also caused a sharp increase in expression of the truncated form, suggesting that the function of the ULD of parkin is conserved across species. By progressive deletion analysis of parkin ULD, we found that residues 1-6 of human parkin play a crucial role in controlling the expression levels of this gene. HA-parkin residues 77-465 showed ubiquitination in vivo, demonstrating that the ULD is not critical for parkin auto-ubiquitination; ubiquitination seemed to cluster on the central domain of parkin (residues 77-313). These effects were specific for the ULD of parkin and not transfection-, toxic-, epitope tag-, and/or vector-dependent. Taken together, these data suggest that the 76 most NH(2)-terminal residues (ULD) dramatically regulate the protein levels of parkin.     

Orexin/hypocretin neurons: chemical phenotype and possible interactions with melanin-concentrating hormone neurons

We showed earlier that a specific neuron population of the rat lateral hypothalamus, differing from the codistributed melanin-concentrating hormone (MCH) neurons, express both dynorphin (DYN) and secretogranin II (SgII) genes. We demonstrated later that this population corresponds in fact to the newly identified orexin/hypocretin (OX/Hcrt) neurons. In the present study, by revisiting the chemical phenotype of these neurons, we confirm that all of them contain DYN B- and SgII-immunoreactive materials. The roles played by these peptide/protein in OX/Hcrt neurons are still unclear.Double immunocytochemical stainings highlight putative somasomatic, axosomatic and axodendritic contacts between OX/Hcrt and MCH neurons. Adding OX/Hcrt to the culture medium of hypothalamic slices from 8-day-old rats results either in a significant increase of MCH mRNA after 24 h survival or a strong fall after 10 days culture. These results taken together suggest that OX/Hcrt can directly and/or indirectly affect MCH expression, and that both OX/Hcrt and MCH neuron populations interact to respond in a coordinated manner to central and peripheral signals.     

Non-invasive detection of respiratory muscles activity during assisted ventilation

The instantaneous pressure applied by the respiratory muscles [Pmus(t)] of a patient under ventilatory support may be continuously assessed with the help of a model of the passive respiratory system updated cycle by cycle. Inspiratory activity (IA) is considered present when Pmus goes below a given threshold. In six patients, we compared IA with (i) inspiratory activity (IAref) obtained from esophageal pressure and diaphragmatic EMG and (ii) that (IAvent) detected by the ventilator. In any case, a ventilator support onset coincides with an IA onset but the opposite is not true. IA onset is always later than IAref beginning ((0.21 +/- 0.10 s) and IA end always precedes IAref end (0.46 +/- 0.16 s). These results clearly deteriorate when the model is not updated.     

Unique ligand-protein interactions in a new truncated hemoglobin from Mycobacterium tuberculosis

A new truncated hemoglobin (HbO) from Mycobacterium tuberculosis has been expressed and purified. Sequence alignment of HbO with other hemoglobins suggests that the proximal F8 residue is histidine and the distal E7 and the B10 positions are occupied by alanine and tyrosine, respectively. The highly conserved residue at the CD1 position, surprisingly, is tyrosine, making HbO the first exception in the hemoglobin family that does not contain phenylalanine at this position. Resonance Raman data suggest that a strong hydrogen bonding network, involving the B10 Tyr and the CD1 Tyr, stabilizes the heme-bound O2 and CO as evidenced by the relatively low frequency of the Fe-O2 stretching mode (559 cm(-1)) and the high frequency of the Fe-CO stretching mode (527 cm(-1)). The presence of this hydrogen bonding network is supported by mutagenesis studies with the B10 tyrosine or the CD1 tyrosine mutated to phenylalanine. Taken together, these data demonstrate a rigid and polar distal pocket in HbO, which is significantly different from that of HbN, the other hemoglobin from M. tuberculosis. The distinct features in the heme active site structures and the temporal expression patterns of HbO and HbN suggest that these two hemoglobins may have very different physiological functions.     

Spectral decomposition of intracellular complex fluorescence using multiple-wavelength phase modulation lifetime determination: technical approach and preliminary applications

Lifetime-based spectral decomposition using a frequency-domain phase/modulation technique is developed on a microspectrofluorimeter prototype. In a fluorescent mixture with strongly overlapping components, such measurements enable us to not only obtain excited state lifetimes of each fluorescent component but also determine the specific spectral contribution of each species without the use of any model spectra. Examples of such applications are first given for complex mixtures of highly overlapping fluorescent components in solution. Preliminary results concerning cellular applications are also reported. This allows us to follow the cellular uptake and intracellular stability of fluorescent labeled modified oligonucleotides in the context of antisense strategy studies. Indeed, the intracellular signal from the fluorescent label bound to oligonucleotides can be distinguished from those of the free label by its specific excited state lifetime.     

Dual influence of aldosterone on AQP2 expression in cultured renal collecting duct principal cells

In the renal collecting duct (CD) the major physiological role of aldosterone is to promote Na+ reabsorption. In addition, aldosterone may also influence CD water permeability elicited by vasopressin (AVP). We have previously shown that endogenous expression of the aquaporin-2 (AQP2) water channel in immortalized mouse cortical CD principal cells (mpkCCDC14) grown on filters is dramatically increased by administration of physiological concentrations of AVP. In the present study, we investigated the influence of aldosterone on AQP2 expression in mpkCCDC14 cells by RNase protection assay and Western blot analysis. Aldosterone reduced AQP2 mRNA and protein expression when administered together with AVP for short periods of time (< or =24 h). For longer periods of time, however, aldosterone increased AQP2 protein expression despite sustained low expression levels of AQP2 mRNA. Both events were dependent on mineralocorticoid receptor occupancy because they were both induced by a low concentration of aldosterone (10-9 m) and were abolished by the mineralocorticoid receptor antagonist canrenoate. Inhibition of lysosomal AQP2 protein degradation increased AQP2 protein expression in AVP-treated cells, an effect that was potentiated by aldosterone. Finally, both aldosterone and actinomycin D delayed AQP2 protein decay following AVP washout, but in a non-cumulative manner. Taken together, our data suggest that aldosterone tightly modulates AQP2 protein expression in cultured mpkCCDC14 cells by increasing AQP2 protein turnover while maintaining low levels of AQP2 mRNA expression.