Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase.

Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.

Experimental inhibition of porcupine-mediated Wnt O-acylation attenuates kidney fibrosis.

Activated Wnt signaling is critical in the pathogenesis of renal fibrosis, a final common pathway for most forms of chronic kidney disease. Therapeutic intervention by inhibition of individual Wnts or downstream Wnt/β-catenin signaling has been proposed, but these approaches do not interrupt the functions of all Wnts nor block non-canonical Wnt signaling pathways. Alternatively, an orally bioavailable small molecule, Wnt-C59, blocks the catalytic activity of the Wnt-acyl transferase porcupine, and thereby prevents secretion of all Wnt isoforms. We found that inhibiting porcupine dramatically attenuates kidney fibrosis in the murine unilateral ureteral obstruction model. Wnt-C59 treatment similarly blunts collagen mRNA expression in the obstructed kidney. Consistent with its actions to broadly arrest Wnt signaling, porcupine inhibition reduces expression of Wnt target genes and bolsters nuclear exclusion of β-catenin in the kidney following ureteral obstruction. Importantly, prevention of Wnt secretion by Wnt-C59 blunts expression of inflammatory cytokines in the obstructed kidney that otherwise provoke a positive feedback loop of Wnt expression in collagen-producing fibroblasts and epithelial cells. Thus, therapeutic targeting of porcupine abrogates kidney fibrosis not only by overcoming the redundancy of individual Wnt isoforms but also by preventing upstream cytokine-induced Wnt generation. These findings reveal a novel therapeutic maneuver to protect the kidney from fibrosis by interrupting a pathogenic crosstalk loop between locally generated inflammatory cytokines and the Wnt/β-catenin signaling pathway.

Properties of the protein matrix revealed by the free energy of cavity formation.

The classical picture of the hydrophobic stabilization of proteins invokes a resemblance between the protein interior and nonpolar solvents, but the extent to which this is the case has often been questioned. The protein interior is believed to be at least as tightly packed as organic crystals, and was shown to have very low compressibility. There is also evidence that these properties are not uniform throughout the protein, and conflicting views exist on the nature of sidechain packing and on its influence on the properties of the protein.

Stimulation of apolipoprotein D secretion by steroids coincides with inhibition of cell proliferation in human LNCaP prostate cancer cells

Although steroid hormones are known to play a predominant role in the regulation of cell growth in hormone-sensitive cancers, their mechanisms of action, especially their interaction with growth factors and/or growth inhibitors, is poorly understood. We have recently observed that the effects of androgens and estrogens on the expression of the major protein found in human breast gross cystic disease fluid, protein-24, are opposite to their respective action on cell proliferation in human breast cancer cell lines. Somewhat surprisingly, the recent elucidation of the amino acid sequence of this progesterone binding protein reveals that this tumor marker is apolipoprotein D (apo D), a member of a superfamily of lipophilic ligand carrier proteins. The present study was designed to determine whether apo D is secreted by human prostate cancer cells and could thus be a new marker of steroid action in these cancer cells, and whether the sex steroid-induced stimulation of apo D secretion coincides with inhibition of cell proliferation. We took advantage of the biphasic pattern of the effect of steroids on the proliferation of the human prostate cancer LNCaP cell line, which offers the opportunity to discriminate between positive and negative steroid receptor-regulated cell growth processes. A 10-day exposure to low concentrations of dihydrotestosterone and testosterone caused a potent stimulation of LNCaP cell proliferation, whereas incubation with higher concentrations of these androgens led to a progressive decrease in cell proliferation towards basal levels. The biphasic action of androgens was also observed on apo D secretion, the effects on apo D secretion being inversely related to their action on LNCaP cell proliferation. Similar opposite biphasic effects were also observed with 9 other steroids, thus indicating that the stimulation of secretion of this new biochemical marker coincides with inhibition of cell proliferation in LNCaP human prostatic cancer cells.

Multiscale modeling of the onset of freckle formation during vacuum arc remelting

A multiscale model for the Vacuum Arc Remelting process (VAR) was developed to simulate dendritic microstructures during solidification and investigate the onset of freckle formation. On the macroscale, a 3D multi-physics model of VAR was used to study complex physical phenomena, including liquid metal flow with turbulence, heat transfer, and magnetohydrodynamics. The results showed that unsteady fluid flow in the liquid pool caused significant thermal perturbation at the solidification front. These results were coupled into a micromodel to simulate dendritic growth controlled by solute diffusion, including local remelting. The changes in Rayleigh number as the microstructure remelts was quantified to provide an indicator of when fluid flow channels (i.e. freckles) will initiate in the mushy zone. By examining the simulated microstructures, it was found that the Rayleigh number increased more than 300 times during remelting, which suggests that thermal perturbation could be responsible for the onset of freckle formation.

Inhibition of prandial and waterspray-induced rat grooming by 8-OH-DPAT

In addition to its hyperphagic effect in rats, 8-OH-DPAT also reduces grooming, but it is uncertain whether the inhibition of grooming is a specific effect or a consequence of response competition from eating. The present experiments explored the effects of 8-OH-DPAT on periprandial grooming and grooming elicited by spraying rats with water. Momentary time sampling over 30 or 60 min, with behaviour scored in one of 6 or 7 (depending on food availability) mutually exclusive categories (feeding, active, scratching, face grooming, body grooming, genital grooming and resting) at 15s intervals, was used for data collection. Non-deprived rats were tested in the presence and absence of food and baseline grooming levels were manipulated by spraying the dorsal surface of the back with water. Data were submitted to ANOVA. The first experiment confirmed that 8-OH-DPAT increased food intake and that this was associated with a parallel increase in feeding observations; active observations were also increased, but resting and total grooming observations were reduced: scratching was reduced even at 0.003mg/kg, face- and body-grooming were reduced at doses > 0.03mg/kg and genital-grooming was least sensitive, only being reduced at 0.1mg/kg. The second experiment revealed that spraying with water had no effect on food intake, feeding or resting observations, but increased total grooming (largely due to increased body-grooming) and reduced activity observations. In rats sprayed with water, 8-OH-DPAT increased food intake (0.1mg/kg) and observations of feeding (0.003 & 0.1mg/kg), but total grooming was dose-dependently inhibited, with genital-grooming most sensitive(> 0.003mg/kg), followed by face-grooming (> 0.01mg/kg) and body-grooming (>0.03mg/kg), whilst low levels of scratching were unaffected. The final experiment tested water-sprayed rats in the absence of food: 8-OH-DPAT increased resting and reduced total grooming, mostly as a consequence of reductions in face- and body-grooming, but there were also modest reductions in scratching. These results confirm that 8-OH-DPAT has a suppressant effect on all aspects of grooming, except where there are probable floor effects, and that this is independent of response competition from increased eating.

Inhibition of prandial and waterspray-induced rat grooming by 8-OH-DPAT

The effects of 8-OH-DPAT treatment on rat grooming behaviour, elicited either prandially or in response to spraying with water were investigated. Dose (≤0.1 mg/kg s.c.) response studies employed momentary time sampling over 30 or 60 min with behaviour being scored in one of 6 or 7 (depending on food availability) mutually exclusive categories (feeding, active, scratching, face-grooming, body grooming, genital-grooming and resting) at 15 s intervals. In non-deprived rats, tested with wet mash available, feeding and activity frequencies were increased, but resting and total grooming were inhibited by 8-OH-DPAT. Face-, body- and genital-grooming occurred at higher levels than scratching, but all categories were reduced with reductions in scratching occurring at a lower dose (0.01 mg/kg). Misting rats with a fine water spray selectively increased body grooming and decreased activity without altering feeding, while 8-OH-DPAT increased feeding and reduced face-, body- and genital-grooming, without affecting already low levels of scratching. In misted rats, tested without food, 8-OH-DPAT reduced face-, body- and genital-grooming and increased resting. These results confirm i) that the water spray technique is a useful method for increasing grooming and ii) that 8-OH-DPAT has a suppressant effect on grooming independent of response competition from enhanced feeding.