Serum withdrawal induces a redistribution of intracellular gelsolin towards F-actin in NIH 3T3 fibroblasts preceding apoptotic cell death

The intracellular distribution of gelsolin in NIH 3T3 cells was examined by immunostaining using affinity-purified polyclonal gelsolin antibodies before and after induction of apoptosis by serum withdrawal. Serum deprivation induced detachment of an increasing number of NIH 3T3 cells, but also apoptosis in attached cells as verified morphologically by chromatin condensation, nuclear fragmentation and labelling of their periphery by FITC-annexin V. Ongoing apoptosis was also demonstrated by activation of caspase-3 activity and chromatin cleavage into high-molecular-mass fragments, although no internucleosomal chromatin degradation (DNA-ladder formation) was detected. When cells were maintained in the presence of 10% foetal calf serum, gelsolin immunoreactivity was evenly distributed in the cytoplasm. No obvious co-localisation of gelsolin and the actin-containing stress fibres was detected under these conditions. At day one after serum withdrawal, a redistribution of gelsolin to actin filaments was detected within a few attached cells by double fluorescence staining. The number of cells exhibiting this redistribution increased at days two to four. In addition, the stress fibres increased in thickness and their length was continuously reduced. At day four, many cells contained shortened stress fibres, which had lost their longitudinal orientation. Additionally, the cytoplasm of a number of attached cells was highly condensed around their nuclei and a homogenous distribution of both gelsolin and actin was detected in the remaining cytoplasmic rim. Up to day two, these effects were reversible after re-addition of serum to attached cells. A similar redistribution of gelsolin immunore-activity was observed after induction of apoptosis by cycloheximide, but not after initiation of necrosis by hydrogen peroxide. In NIH 3T3 cells no alteration in the expression of gelsolin at the level of protein (Western blot) or specific mRNA (Northern blot) was observed after serum withdrawal. Using Western blotting, no proteolysis of gelsolin was detected up to day 4, although caspase-3 activity was found to have increased fivefold after serum withdrawal. These results suggested that in these cells F-actin severing might occur in the absence or advance of gelsolin cleavage by caspases. Intact gelsolin on its own may be sufficient for the dissolution of the microfilaments, since micro-injection of gelsolin into primary bovine lens cells led to a transient disappearance of the stress fibres and to a reduction of their attachment area to the substratum. In NIH 3T3 cells similar effects of micro-injected gelsolin were only observed at day one after serum withdrawal.     

Micellar lipid composition profoundly affects LXR-dependent cholesterol transport across CaCo2 cells

Intraluminal phospholipids affect micellar solubilization and absorption of cholesterol. We here study cholesterol transport from taurocholate-phospholipid-cholesterol micelles to CaCo2 cells, and associated effects on ABC-A1 mediated cholesterol efflux. Micellar incorporation of egg-yolk-phosphatidylcholine markedly increased apical retention of the sterol with decreased expression of ABC-A1, an effect that is prevented by synthetic liver X receptor (LXR) or retinoid X receptor (RXR) agonists. On the other hand, incorporation of lyso-phosphatidylcholine (LysoPC) increased ABC-A1-HDL-dependent basolateral cholesterol efflux, an effect that is abated when LXR is silenced. Thus, the modulation of cholesterol metabolism via intraluminal phospholipids is related to the activity of the oxysterol nuclear receptor LXR.     

Gene expression induced by interleukin-17 in fibroblast-like synoviocytes of patients with rheumatoid arthritis: upregulation of hyaluronan-binding protein TSG-6

Interleukin-17 (IL-17) has been characterized as a proinflammatory cytokine produced by CD4+ CD45RO+ memory T cells. Overproduction of IL-17 was detected in the synovium of patients with rheumatoid arthritis (RA) compared with patients with osteoarthritis. This study examines differentially expressed genes after the stimulation of fibroblast-like synoviocytes of RA patients by IL-17. Among these genes we identified the following: tumor necrosis factor-stimulated gene-6 (TSG-6), IL-6, IL-8, GRO-beta, and bone morphogenetic protein-6 with an expression 3.6-10.6-fold that in the unstimulated control. IL-17 augmented the expression of TSG-6, a hyaluronan-binding protein, in a time- and dose-dependent manner. IL-17 showed additive effects with IL-1beta and tumour necrosis factor-alpha on the expression of TSG-6, IL-6 and IL-8. The mitogen-activated protein kinase p38 seems to be necessary for the regulation of TSG-6 expression by IL-17, as shown by inhibition with SB203580. Our results support the hypothesis that IL-17 is important in the pathogenesis of RA, contributing to an unbalanced production of cytokines as well as participating in connective tissue remodeling.     

Rapid assembly of diverse and potent allosteric Akt inhibitors

This paper describes the rapid assembly of four different classes of potent Akt inhibitors from a common intermediate. Among them, a pyridopyrimidine series displayed the best intrinsic and cell potency against Akt1 and Akt2. This series also showed a promising pharmacokinetic profile and excellent selectivity over other closely related kinases.     

Neural networks: models and neurons show hybrid vigor in real time

Hybrid networks in which living neurons interact with digital or analog model neurons are providing insights into the role of neural and synaptic properties in shaping neural network activity.     

Acyclic N-(azacycloalkyl)bisindolylmaleimides: isozyme selective inhibitors of PKCbeta

The synthesis and structure-activity relationship (SAR) trends of a new class of N-(azacycloalkyl)bisindolylmaleimides 1, acyclic derivatives of staurosporine, is described. The representative compound for this series (1e) exhibits an IC(50) of 40-50 nM against the human PKCbeta(1) and PKCbeta(2) isozymes and selectively inhibits the PKCbeta isozymes in comparison to other PKC isozymes (alpha, gamma, delta, epsilon, lambda, and eta). The series is also kinase selective for PKC in comparison to other ATP-dependent kinases. A comparison of the PKC isozyme and kinase activity of the series is made to the kinase inhibitor staurosporine.     

Uncoupling Kapbeta2 substrate dissociation and ran binding

Karyopherinbeta2 (Kapbeta2) imports a variety of mRNA binding proteins into the nucleus. Release of import substrates in the nucleus involves formation of a high-affinity Kapbeta2-RanGTP complex and concomitant dissociation of import substrates. The crystal structure of the Kapbeta2-RanGppNHp complex shows that Ran binds in the Kapbeta2 N-terminal arch and substrate most likely binds its C-terminal arch. The structure suggested a mechanism for Ran-mediated substrate dissociation where a long internal acidic loop in Kapbeta2 transmits structural information between the GTPase and substrate sites, leading to displacement of substrate by the loop when Ran is bound. To study the molecular mechanism of substrate dissociation, we have cleaved the acidic loop of Kapbeta2 proteolytically (cl-Kapbeta2) and also constructed a mutant of Kapbeta2 with a truncated loop (TL-Kapbeta2). Both modified Kapbeta2s are unable to undergo Ran-mediated substrate dissociation. We have also mapped the boundaries of the Kapbeta2 binding site of substrate mRNA binding protein A1 using a widely applicable method employing NMR spectroscopy. This has allowed design of reagents to quantitate the affinities of the Kapbeta2 proteins for Ran and substrate. cl-Kapbeta2, TL-Kapbeta2, and native Kapbeta2 have comparable affinities for both RanGppNHp and import substrates, indicating that perturbation of the loop has not altered the strength of binary Kapbeta2-Ran or Kapbeta2-substrate interactions. The TL-Kapbeta2 mutant also binds RanGppNHp and substrate simultaneously to form a ternary complex, indicating that in addition to the loss of coupling between Ran binding and substrate dissociation, the two ligand sites on Kapbeta2 are spatially distinct. The uncoupling of Ran binding and substrate dissociation in the TL-Kapbeta2 mutant is further evident in significant loss of Ran-mediated nuclear uptake of fluorescent substrate in digitonin-permeabilized HeLa cells. These results support our previously proposed GTPase-mediated Kapbeta2-substrate dissociation mechanism where the acidic loop of Kapbeta2 physically couples distinct Ran and substrate binding sites.