Dynamics of chromosome compaction during mitosis

We have quantitatively studied the space-time dynamics of mitotic chromosome compaction in cultured amphibian cells. After collecting digital phase-contrast images we have done digital image analysis to study spatial correlations in density. We find a characteristic distance at which the strongest correlations occur, which provides a quantitative measure of the size of patches of dense chromatin during interphase and early prophase. Later in mitosis, this length corresponds to the thickness of prophase and metaphase chromosomes. We find that during interphase strong correlations exist at a few-micrometer length; during prophase this correlation length progressively drops as the chromosomes are compacted. Our data are explained by a model based on assembly of chromatin loops onto already fiberlike interphase chromosomes. To test this model we have microinjected cobalt hexamine trichloride into interphase nuclei and have observed the rapid condensation of the interphase chromatin into thick fibers with a spacing similar to the native-state interphase correlation length determined from our image analysis.     

Nonsteroidal compounds designed to mimic potent steroid sulfatase inhibitors

Chemical synthesis and enzyme inhibition results are reported for a series of nonsteroidal sulfatase inhibitors, 1-(p-sulfamoyloxyphenyl)-5-(p-t-butylbenzyl)-5-alkanols and the lower active phenolic analogues. These compounds conserve some structural elements from the previously reported potent steroidal inhibitor 3-O-sulfamate-17alpha-(p-t-butylbenzyl)-17beta-hydroxy-estra-1,3,5(10)-triene, while the C18-methyl group and the hydrocarbon backbone represented by the steroid rings B, C, and D were replaced with a free conformational chain. Using estrone sulfate (100 microM) as substrate and homogenate of transfected HEK-293 cells as source of steroid sulfatase activity, the IC(50) values of the best inhibitors, the undecanol derivatives, were 0.4+/-0.1 and >300 nM, respectively, in the sulfamate and phenolic series. Although these sulfamoylated nonsteroidal inhibitors appear a bit less active than their steroidal analogues, they are however more potent than known inhibitors estrone-3-O-sulfamate and p-(O-sulfamoyl)-N-tetradecanoyl tyramine. The optimal side-chain length for the inhibition of steroid sulfatase activity was found to be six carbons, which corresponds to the number of carbons that mimic the B, C and D steroid rings, between C6 and C17. Furthermore, compounds with only the t-butylbenzyl group or the alkyl chain of six carbons are less potent inhibitors compared to the one that include both of these hydrophobic substituents. Such results suggest that compound from this later category better mimic the steroidal inhibitor.     

Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem

The Arabidopsis mutant pho1 is deficient in the transfer of Pi from root epidermal and cortical cells to the xylem. The PHO1 gene was identified by a map-based cloning strategy. The N-terminal half of PHO1 is mainly hydrophilic, whereas the C-terminal half has six potential membrane-spanning domains. PHO1 shows no homology with any characterized solute transporter, including the family of H(+)-Pi cotransporters identified in plants and fungi. PHO1 shows highest homology with the Rcm1 mammalian receptor for xenotropic murine leukemia retroviruses and with the Saccharomyces cerevisiae Syg1 protein involved in the mating pheromone signal transduction pathway. PHO1 is expressed predominantly in the roots and is upregulated weakly under Pi stress. Studies with PHO1 promoter-beta-glucuronidase constructs reveal predominant expression of the PHO1 promoter in the stelar cells of the root and the lower part of the hypocotyl. There also is beta-glucuronidase staining of endodermal cells that are adjacent to the protoxylem vessels. The Arabidopsis genome contains 10 additional genes showing homology with PHO1. Thus, PHO1 defines a novel class of proteins involved in ion transport in plants.     

Merosin-integrin promotion of skeletal myofiber cell survival: Differentiation state-distinct involvement of p60Fyn tyrosine kinase and p38alpha stress-activated MAP kinase

Myofiber survival and suppression of anoikis depend in large part on the merosin (laminin-2/-4)-integrin alpha7beta1D cell adhesion system; however, the question remains as to the nature of the signaling molecules/pathways involved. In the present study, we investigated this question using the C2C12 cell model of myogenic differentiation and its merosin- and laminin-deficient derivatives. Herein, we report that: 1) of four members of the Src family of tyrosine kinases studied (p60Src, p53/56Lyn, p59Yes, or p60Fyn), the expression and activity of p60Fyn are found in myotubes exclusively; 2) a severe decrease of p60Fyn activity correlates with myotube apoptosis/anoikis induced by pharmocological compounds (herbimycin A or PP2) which inhibit tyrosine kinases of the Src family, by merosin deficiency and by beta1 integrin inhibition; 3) myoblast survival depends on Fak and the MEK/Erk pathway, in contrast to myotubes; 4) the PI3-K pathway is not involved in either myoblast or myotube survival; and 5) p38alpha SAPK stimulation and activity (but not that of p38beta) are required in the progression of myotube apoptosis/anoikis induced by p60Fyn inhibition, merosin deficiency or beta1 integrin-inhibition; however, p38 is not involved in myoblast apoptosis. Taken together, these results suggest that the promotion of myotube survival by the merosin-alpha7beta1D adhesion system involves p60Fyn, and that disruptions in this cell adhesion system induce myotube apoptosis/anoikis through a p38alpha SAPK-dependent pathway.     

Proteomic analysis of a lymphoma-derived cell line (DG75) following treatment with a demethylating drug: modification of membrane-associated proteins

5'-azacytidine (AZC) is a potent DNA demethylating agent used clinically for treatment of patients with malignant hemopathies. We have previously shown that AZC induces a halt in cell growth and a decrease of cell activity, without affecting cell viability. We have also shown using proteomics, that 35 polypeptides were differentially expressed in a cytoplasmic fraction. The aim of this study was to provide a more complete picture of modifications in AZC-treated cells using cell membrane preparations. Therefore the protein pattern changes following AZC treatment of the cell line DG75 were studied on a detergent-solubilized fraction obtained from these membranes. Results showed that 49 proteins were differentially expressed in the membrane fraction. Seven polypeptides were down-regulated, while 42 were up-regulated. The identity of most of these differentially expressed proteins was determined by mass spectrometry (liquid chromatography-tandem mass spectrometry or matrix-assisted laser desorption/ionization-time of flight), and the identified proteins were grouped based on cellular function and participation in biochemical and signaling pathways.     

Apolipoprotein E and cholesterol metabolism in the pathogenesis and treatment of Alzheimer's disease

There is much evidence suggesting that there is a strong relationship between the deterioration of brain lipid homeostasis, vascular changes and the pathogenesis of Alzheimer's disease (AD). These associations include: (1). recognition that a key cholesterol transporter, apolipoprotein E type 4, acts a major genetic risk factor for both familial and sporadic AD; (2). epidemiological studies linking cardiovascular risk factors, such as hypertension and high plasma cholesterol, to dementia; (3). the discovery that small strokes can precipitate clinical dementia in cognitively normal elderly subjects; (4). the modulation of degradation of the amyloid precursor protein by cholesterol administration in cell culture and in animal models of beta-amyloid overproduction; and (5). the beneficial effect of cholesterol-lowering drugs, such as Probucol and statins, in combating common AD. The recent finding that there is a genetic association between the HMGR gene locus and sporadic AD further suggests that brain cholesterol metabolism is central to AD pathophysiology, and a potential therapeutic target for disease stabilization and primary disease prevention.     

3Beta-sulfamate derivatives of C19 and C21 steroids bearing a t-butylbenzyl or a benzyl group: synthesis and evaluation as non-estrogenic and non-androgenic steroid sulfatase inhibitors

A series of C19 and C21 steroids bearing one or two inhibiting groups (3beta-sulfamate and 17alpha- or 20(S)-t-butylbenzyl or benzyl) were synthesized and tested for inhibition of steroid sulfatase activity. When only a sulfamate group was added to dehydroepiandrosterone, androst-5-ene-3beta,17beta-diol, pregnenolone and 20-hydroxy-pregnenolone, no significant inhibition of steroid sulfatase occurred at concentrations of 0.3 and 3 microM. With only a t-butylbenzyl or a benzyl group, a stronger steroid sulfatase inhibition was obtained in the androst-5-ene than in the pregn-5-ene series. Comparative results from the screening tests and the IC50 values have shown that the effect of a sulfamate moiety as a second inhibiting group can be combined to the t-butylbenzyl or benzyl effect in the C19 and C21 steroid series. The 3beta-sulfamoyloxy-17alpha-t-butylbenzyl-5-androsten-17beta-ol (10) was thus found to be the most active compound with IC50 values of 46 +/- 8 and 14 +/- 1 nM, respectively for the transformations of E1S to E1 and DHEAS to DHEA. The IC50 values of compound 10 are similar to that of 17alpha-t-butylbenzyl-estradiol, which was previously reported by our group as a good steroid sulfatase reversible inhibitor, but remains higher than that of the potent inactivators estrone-3-O-sulfamate (EMATE) and 17alpha-t-butylbenzyl-EMATE. However, contrary to these two latter inhibitors, compound 10 did not induce any proliferative effect on estrogen-sensitive ZR-75-1 cells nor on androgen-sensitive Shionogi cells at concentrations tested, suggesting that this steroid sulfatase inhibitor is non estrogenic and non androgenic.     

Alteration of poly(ADP-ribose) glycohydrolase nucleocytoplasmic shuttling characteristics upon cleavage by apoptotic proteases

Poly(ADP-ribosyl)ation is an important post-translational modification which mostly affects nuclear proteins. The major roles of poly(ADP-ribose) synthesis are assigned to DNA damage signalling during base excision repair, apoptosis and excitotoxicity. The transient nature and modulation of poly(ADP-ribose) levels depend mainly on the activity of poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG), the key catabolic enzyme of poly(ADP-ribose). Given the fact that PARG substrate, poly(ADP-ribose), is found almost exclusively in the nucleus and that PARG is mainly localized in the cytoplasm, we wanted to have a closer look at PARG subcellular localization in order to better understand the mechanism by which PARG regulates intracellular poly(ADP-ribose) levels. We examined the subcellular distribution of PARG and of its two enzymatically active C-terminal apoptotic fragments both biochemically and by fluorescence microscopy. Green fluorescent protein (GFP) fusion proteins were constructed for PARG (GFP-PARG), its 74 kDa (GFP-74) and 85 kDa (GFP-85) apoptotic fragments and transiently expressed in COS-7 cells. Localization experiments reveal that all three fusion proteins localize predominantly to the cytoplasm and that a fraction also co-localizes with the Golgi marker FTCD. Moreover, leptomycin B, a drug that specifically inhibits nuclear export signal (NES)-dependent nuclear export, induces a redistribution of GFP-PARG from the cytoplasm to the nucleus and this nuclear accumulation is even more pronounced for the GFP-74 and GFP-85 apoptotic fragments. This observation confirms our hypothesis for the presence of important regions in the PARG sequence that would allow the protein to engage in CRM1-dependent nuclear export. Moreover, the altered nuclear import kinetics found for the apoptotic fragments highlights the importance of PARG N-terminal sequence in modulating PARG nucleocytoplasmic trafficking properties.     

Analysis of ADP-ribose polymer sizes in intact cells

Poly(ADP-ribose) is a polymer (pADPr) that is synthesized by poly (ADP-ribose) polymerases in response to DNA damaging agents. For instance, chemical alkylating agents such as MNNG [1] or physical stimulation of cells by -rays [2] are well known to induce pADPr synthesis. PARPs are members of a growing family of enzymes which includes PARP-1, PARP-2, S-PARP-1, tankyrase and V-PARP [3]. The association of PARP-1 and PARP-2 in DNA damage signaling pathways has been characterized, but tankyrase and V-PARP seem to be independent of DNA repair mechanisms.Poly(ADP-ribosyl)ation leads to heterogenous chain lengths of up to 200 units (mers) in vitro [3]. While most of these will be covalently bound to proteins, they may be released under alkaline conditions for analysis. Previous immunological methods such as immunoblots [4] showed that about 60–70% of the 6–8 mers pADPr were lost during fixation and that the very short pADPr (2–5 mers) were very weakly bound to the membrane [5]. Furthermore, detection of cellular pADPr using enzyme-linked immunosorbent assay (ELISA) revealed that some molecules of pADPr are also lost during fixation and washings. This phenomenon leads to underestimation of the short pADPr population in cells. Thus, evaluating which pADPr sizes are present in cells and tissues becomes critical.We report here the development of a new highly sensitive immunological method to detect synthesized pADPr sizes distribution in intact cells.     

Regulation of CD45-induced signaling by galectin-1 in Burkitt lymphoma B cells

It has been well established that Galectin-1 (GAL1), a beta-galactoside-binding protein, regulates the viability of lymphoid cells. However, the signaling pathway governed by the binding of GAL1 to the cell membrane is not understood. As a first step towards the elucidation of GAL1-initiated signaling events leading to a reduced viability of Burkitt lymphoma B cells, we tried to characterize the initial events induced by the binding of GAL1 to its receptor. This characterization was performed in BL36 cells, a Burkitt lymphoma cell line sensitive to GAL1. The results were as follows: (1) when solubilized cell membrane lysates were affinity bound to immobilized GAL1 and eluted by competition, the tyrosine phosphatase glyco-protein CD45 was found in the eluate, highlighting the role of CD45 as a receptor of GAL1; (2) the phosphatase activity of cell membranes diminished after incubation with GAL1; (3) immunoprecipitation experiments demonstrated that the phosphotyrosine kinase Lyn was dysregulated in cells that have been cultured in medium containing 700 nM GAL1, and (4) that the ratio between two isoforms of Lyn was modified during the treatment with GAL1. The regulation of Lyn therefore seems to be a key event in the action of GAL1.