Transient Defects Research Articles

Fullerenol C60(OH)24 increases ion permeability of lipid membranes in a pH-dependent manner

Fullerenols are water-soluble analogs of fullerene exhibiting both antioxidant and prooxidant activities in vitro and in vivo. Here we report, for the first time, that fullerenol C60(OH)24 can induce ion permeability of a planar lipid bilayer membrane via the formation of ion pores or conductive defects with a preference for cations over anions. The fullerenol-mediated electrical current displayed non-linear concentration dependence and was reversibly enhanced by alkalinization. Calcium and magnesium ions decreased the fullerenol-induced potassium ion permeability. Voltage dependence of the current was sensitive to membrane composition, with the conductance being well pronounced in fully saturated diphytanoylphosphatidylcholine. Fullerenol did not induce carboxyfluorescein leakage from liposomes, suggesting a small size of fullerenol-induced pores. In contrast to ion permeability, the binding of C60(OH)24 to liposomes increased at acidic pH, as measured by fluorescence quenching of pyrene-labeled lipid. In line with this, the photodynamic action of fullerenol on the peptide gramicidin A also increased at low pH. It is hypothesized that aggregates of fullerenol may stabilize transient conductive lipid defects or pores formed under a variety of stress conditions.

Parkinsonism in a pair of monozygotic CADASIL twins sharing the R1006C mutation: a transcranial sonography study.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common hereditary cerebral small vessel disease, is caused by mutations in the NOTCH3 gene on chromosome 19. Clinical manifestations of CADASIL include recurrent transient ischemic attacks, strokes, cognitive defects, epilepsy, migraine and psychiatric symptoms. Parkinsonian features have variably been reported in CADASIL patients, but only a few patients showed a clear parkinsonian syndrome. We studied two patients, a pair of monozygotic twins, carrying the R1006C mutation of the NOTCH3 gene and affected by a parkinsonian syndrome. For the first time in CADASIL patients, we used transcranial sonography (TCS) to assess basal ganglia abnormalities. TCS showed a bilateral hyperechogenic pattern of substantia nigra in one twin, and a right hyperechogenic pattern in the other. In both patients, lenticular nuclei showed a bilateral hyperechogenic pattern, and the width of the third ventricle was slightly increased. The TCS pattern found in our CADASIL patients is characteristic neither for Parkinson's disease, nor for vascular parkinsonism and seems to be specific and related to the disease-specific pathological features.

Foxn4 promotes gene expression required for the formation of multiple motile cilia.

Multiciliated cell (MCC) differentiation involves extensive organelle biogenesis required to extend hundreds of motile cilia. Key transcriptional regulators known to drive the gene expression required for this organelle biogenesis are activated by the related coiled-coil proteins Multicilin and Gemc1. Here we identify foxn4 as a new downstream target of Multicilin required for MCC differentiation in Xenopus skin. When Foxn4 activity is inhibited in Xenopus embryos, MCCs show transient ciliogenesis defects similar to those seen in mutants of Foxj1, a known key regulator of genes required for motile ciliation. RNAseq analysis indicates that Foxn4 co-activates some Foxj1 target genes strongly and many Foxj1 targets weakly. ChIPseq suggests that whereas Foxn4 and Foxj1 frequently bind to different targets at distal enhancers, they largely bind together at MCC gene promoters. Consistent with this co-regulation, cilia extension by MCCs is more severely compromised in foxn4 and foxj1 double mutants than in single mutants. In contrast to Foxj1, Foxn4 is not required to extend a single motile cilium by cells involved in left-right patterning. These results indicate that Foxn4 complements Foxj1 transcriptionally during MCC differentiation, thereby shaping the levels of gene expression required for the timely and complete biogenesis of multiple motile cilia.

Visual findings as primary manifestations in patients with intracranial tumors.

To evaluate the visual findings as primary manifestations in patients with intracranial tumors. The medical charts of the patients with intracranial tumors who initially admitted to the Neuro-ophthalmology and Strabismus Department with ocular complaints between August 1999 and December 2012 were reviewed retrospectively. The detailed clinical history and the findings of neuro-ophthalmologic examination were recorded. Ocular symptoms and signs, the types and locations of intracranial tumors, and the duration of symptoms before the diagnosis were evaluated. The mean age of 11 women (61.1%) and 7 men (38.9%) was 42.2±11.0 (range 20-66y) at the time of intracranial tumor diagnosis. Initial symptoms were transient visual obscurations, visual loss or visual field defect in 16 cases (88.9%), and diplopia in 2 cases (11.1%). Neuro-ophthalmologic examination revealed normal optic discs in both eyes of 6 patients (33.3%), paleness, atrophy or edema of optic disc in 12 patients (66.7%), and sixth cranial nerve palsy in 2 patients (11.1%). Visual acuity ranged between normal vision and loss of light perception. Cranial imaging demonstrated craniopharyngioma (n=1), plasmacytoma (n=1), meningioma (n=6; olfactory groove and tuberculum sellae, pontocerebellar angle, anterior cranial fossa, frontal vertex, suprasellar region), and pituitary macroadenoma (n=10). The mean duration between the onset of visual disturbances and the diagnosis of intracranial tumor was 9.8±18mo (range 3d-6y). The ophthalmologist is frequently the first physician to encounter a patient with clinical manifestations of intracranial tumors that may cause neurological and ocular complications. Neuro-ophthalmologic findings should be carefully evaluated to avoid a delay in the diagnosis of intracranial tumors.

Towards a Quantitative Model for BO Regeneration by Means of Charge State Control of Hydrogen

The bulk minority carrier lifetime of p-type Cz-silicon material decreases due to light induced degradation caused by the formation of recombination active BO related defects. Regeneration at elevated temperatures under carrier injection is an adequate method for transformation of these BO-related defects into a non-recombination active complex and thus for long term recovery of the minority carrier lifetime. Next to prolonged temperature treatments and carrier injection, the charge state of hydrogen is assumed to play a major role for regeneration. However, the incorporation of hydrogen and especially correctly charged hydrogen has not been proven. The physical mechanism of the regeneration process is not understood yet and still under discussion. So far no physical model explaining the regeneration process is present.For introducing a model explaining the regeneration process we investigate the influence of the concentration of charged hydrogen on the reaction kinetics on the regeneration process. By combining reaction kinetics with statistical mechanics wewill show a quantitative model for BO-regeneration considering the charge state of hydrogen. Comparing experimental and simulated values a good agreement between the experiment and the model is observed. Based on the model we are able to simulate transient defect concentrations during degradation-regenerations cycles over a wide variety of process parameters: temperature and carrier injection, giving an insight into the sophisticated process control for passivating active BO-complexes. Simulation results confirm the incorporation of fast diffusing non-charged hydrogen in the passivation of BO-related defects in cz-silicon solar cells. We conclude that the dissociation of B-H pairs and subsequent diffusion of non-charged hydrogen is the limiting process for regeneration in the material system investigated within this contribution.

Ultrafast defect dynamics: A new approach to all optical broadband switching employing amorphous selenium thin films

Optical switches offer higher switching speeds than electronics, however, in most cases utilizing the interband transitions of the active medium for switching. As a result, the signal suffers heavy losses. In this article, we demonstrate a simple and yet efficient ultrafast broadband all-optical switching on ps timescale in the sub-bandgap region of the a-Se thin film, where the intrinsic absorption is very weak. The optical switching is attributed to short-lived transient defects that form localized states in the bandgap and possess a large electron-phonon coupling. We model these processes through first principles simulation that are in agreement with the experiments.

Pre-operative fMRI localization of the supplementary motor area and its relationship with postoperative speech deficits.

Neurosurgery of the supplementary motor area (SMA) is associated with transient speech defects. We investigated whether SMA laterality correlates with postoperative speech defects. The authors reviewed 17 patients with SMA-area lesion resection and preoperative language fMRI. SMA laterality was calculated by comparison of voxel activation in paired SMAs by hand-drawn regions of interest (ROIs) (drawn by a neuroradiologist), and compared with qualitative assessment by two neuroradiologists. Postoperative speech defects before and after surgery were assessed by chart review. Six patients developed new speech defects that resolved within several months. Two of the patients had a pre-existing speech defect that had developed after prior SMA-area surgery. All these patients had left-sided lesions, while none of the four patients with a right-sided lesion developed a speech defect. Neuroradiologists' assessment of SMA laterality agreed with ROI calculation for the SMAs that were lateralized. However, for the SMAs in the "codominant" range by ROI, the neuroradiologists felt that all but one of the cases clearly lateralized, with the exception deemed indeterminate or codominant. No correlation between laterality of SMA and speech defect was identified. Twelve patients showed lateralization contralateral to the lesion. fMRI lateralization does not correlate with transient speech defects that developed from SMA-area surgery. Qualitative/visual assessment of SMA laterality was superior to ROI calculation because of the close proximity and possible overlap of signal from midline SMA. A majority of patients showed SMA lateralization contralateral to the SMA lesion.

Modulating bilayer mechanical properties to promote the coupled folding and insertion of an integral membrane protein.

Bilayer mechanical properties are not only of crucial importance to the mechanism of action of mechanosensation in lipid membranes but also affect preparative laboratory taskssuch as membrane-protein refolding. We report this for coupled refolding and bilayer insertion of outer membrane phospholipaseA (OmpLA), an integral membrane enzyme that catalyses the hydrolytic cleavage of glycerophospholipids. OmpLA can be refolded into a variety of detergent micelles and unilamellar vesicles composed of short-chain phospholipids but, in the absence of chemical or molecular chaperones, not into thicker membranes. Controlled modulation of bilayer mechanical properties by judicious use of subsolubilising concentrations of detergents induces monolayer curvature strain, acyl chain fluidisation, membrane thinning, and transient aqueous bilayer defects. This enables quantitative and functional refolding of OmpLA even into bilayer membranes composed of long-chain phospholipids to yield enzymatically active proteoliposomes without requiring membrane solubilisation.

Transient but Significant Visual Field Defects after Robot-Assisted Laparoscopic Radical Prostatectomy in Deep Trendelenburg Position

BackgroundRobot-assisted laparoscopic radical prostatectomy (RALP) is a minimally invasive surgical procedure for prostate cancer. During RALP, the patient must be in a steep Trendelenburg (head-down) position, which leads to a significant increase in intraocular pressure (IOP). The association of RALP with visual field sensitivity, however, has not been prospectively studied. The purpose of this study was to evaluate prospectively the visual field, retinal nerve fiber layer (RNFL) thickness, and optic disc morphology in 50 normal eyes of 25 male patients that underwent RALP.MethodsThe subjects were 25 males among 33 consecutive patients who underwent uneventful RALP under general anesthesia in our hospital. Visual field tests using the Humphrey visual field analyzer 30-2 SITA-standard program were performed before, 7 days after, and 1-3 months after RALP. IOP was measured before, during, and after RALP; and ophthalmologic examinations, including slit-lamp, fundus examination, and optical coherence tomography (OCT), were scheduled before and 7 days after surgery.ResultsIOP was significantly increased during RALP up to 29.4 mmHg (P<0.01). Postoperative local visual field defects were detected in 7 eyes of 7 subjects dominantly in the lower hemifield without abnormal findings in the optic nerve head or retina, and the visual field recovered to normal within 3 months after surgery. General factors associated with RALP, IOP, RNFL thickness, or optic disc parameters did not differ significantly between eyes with and without postoperative visual field defects, and parameters of OCT measurements were not altered after surgery.ConclusionTransient but significant unilateral visual field defects were found in 28% of the subjects examined. The probable cause are the increased IOP and altered perfusion during surgery and ophthalmologic examinations are therefore suggested before and after RALP.

TRPC3 Facilitates Adaptation of Collecting Duct Cells to Changes in Osmolality

Mechanical stress induced by changes in tubular flow rate and osmotic gradients is viewed to be an important regulator of the water and electrolytes transport in the collecting duct (CD). Our recent experimental effort identified that pharmacological inhibition or genetic ablation of TRPV4 precluded [Ca2+]i responses to elevated flow but not to hypotonicity suggesting involvement of discrete signaling determinants. Here we combined direct measurements of [Ca2+]i dynamics in freshly isolated split‐opened CDs with genetic tools to unravel molecular components of cellular responses to decreased osmolality. [Ca2+]i elevations elicited by hypotonicity in CD cells were abolished upon removal of extracellular Ca2+, but not by depletion of the endoplasmic reticulum stores, indicating a critical role of direct Ca2+ entry via the plasma membrane. Inhibition of Ca2+‐permeable TRPC3 channel markedly diminishes the amplitude of [Ca2+]i signal induced by hypotonic stimuli in the CD. Interestingly, genetic deletion of TRPC3 significantly increases the activation threshold for [Ca2+]i responses to changes in extracellular osmolality. Water deprivation (18 hours) greatly augmented sensitivity of CD cells to extracellular osmolality in wild‐type, but only mildly in TRPC3‐/‐ mice. At the systemic level, TRPC3 ablation results in a transient defect in urinary concentration ability in response to water deprivation. Overall, our findings demonstrate that elevated flow and osmotic gradients recruit distinct signaling cascades to increase [Ca2+]i in CD cells. Genetic ablation of TRPC3 disturbs sensitivity of CD cells to hypotonic stress contributing to delayed adaptation of the kidney to restricted water intake.

Super-Resolution Mapping of the Dynamics of Periodic Structural Defects in Collagen Fibrils

Single-molecule tracking of matrix metalloproteinases (MMPs) moving on fibrillar collagen reveals a regular binding pattern with a 1.1 µm periodicity. The binding sites exhibit collective motion that preserves the distribution but not the phase of the binding pattern. Over short timescales (∼1 s), the motion of individual binding sites is consistent with diffusion in a harmonic potential. At longer timescales (∼20 s), the potential wells slowly migrate, bifurcate, and merge. The dynamic nature of the binding sites suggests that they correspond to transient local defects in the collagen fibril structure. However, the long-range order of their pattern, exceeding any known structural scale of the fibril, indicates a collective defect formation process. We propose a model in which internal strain energy in fibrillar collagen is relieved by the formation of defects that are distributed along its length. This model falls into the general class of mechanical instabilities that generate long-range spatial patterning in physical systems ranging from mud cracking to skin wrinkling. However, unlike cracks and wrinkles that are stable structures, the microscopic fibril features thermally excited structural dynamics and self-healing of defect states. One physiological consequence of the proposed model is that external tension opposing the internal strain in the fibril can suppress defect formation and exposure of the MMP binding sites. Experiments showing that external loading attenuates the enzymatic degradation of fibrillar collagen are consistent with this prediction of the model. More generally, many aspects of collagen degradation, including cleavage initiation, processivity, and kinetics, may largely be a consequence of a previously unrecognized structural heterogeneity in the underlying fibrillar substrate. Thus, mapping the periodic array of defects in the molecular architecture of collagen elucidates a key feature regulating enzymatic activity and remodeling of the extracellular matrix.

A Case of Transient Color Anomaly and Persistent Visual Field Defect after Sildenafil Citrate Overdose

A Case of Transient Color Anomaly and Persistent Visual Field Defect after Sildenafil Citrate Overdose

80.: Cardiac effects of high-dose fingolimod ingestion

Fingolimod is associated with first dose transient atrioventricular conduction defects mandating cardiac monitoring at initiation. This side effect is not seen with chronic use. Theoretically, overdose of fingolimod should not lead to cardiac side effects, but there are very few reports. We present a case and review the literature of fingolimod overdoses. We report an intentional overdose of 21 mg of fingolimod in a patient with relapsing remitting multiple sclerosis and depression. We retrospectively reviewed the clinical notes of our patient and searched for any similar reports in the literature. At the time of initial assessment and during the subsequent observation period of approximately 4 h, no cardiovascular symptoms, signs or atrioventricular conduction delay were noted. Though there has been a recent report of cardiac side effects noticed after intentional fingolimod overdose, in our patient no such side effects were observed. This highlights the relative safety of fingolimod and that the adverse effects can be variable. We expect further reports of effects of overdoses of this drug with more widespread use.

The pathogenesis of macrophage activation syndrome

The term macrophage activation syndrome (MAS) identifies a severe and potentially fatal complication of s-JIA, and, more rarely of other rheumatic diseases. MAS share similarities in clinical features and laboratory abnormalities with primary and secondary heamophagocytic lymphohystiocytosis (HLH). Indeed it is currently classified among secondary HLH and the term rheuma-HLH has been used to indicate this condition. The clinical and laboratory similarities with primary genetic- caused HLH led to the hypothesis that pathogenic mechanisms leading to the typical features of MAS/rheuma-HLH are similar to those involved in primary HLH. We will review the evidence supporting this hypothesis; particularly the role of hyper-responses to TLR activation, of subclinical variants of genes involved in the cytotoxic pathways, and of the transient NK cytotoxicity defect induced by inflammatory cytokines. We will also present evidence on the role of IL-6, IL-1 and IFN-g in this syndrome and discuss the potential benefits of therapies targeted to these cytokines.

The importance of membrane defects-lessons from simulations.

The defects and pores within lipid membranes are scientifically interesting and have a number of biological applications. Although lipid bilayers are extremely thin hydrophobic barriers, just ∼3 nm thick, they include diverse chemistry and have complex structures. Bilayers are soft and dynamic, and as a result, they can bend and deform in response to different stimuli by means of structural changes in their component lipids. Though defects occur within these structures, their transience and small size have made it difficult to characterize them. However, with recent advances in computer power and computational modeling techniques, researchers can now use simulations as a powerful tool to probe the mechanism and energies of defect and pore formation in a number of situations. In this Account, we present results from our detailed molecular dynamics computer simulations of hydrophilic pores and related defects in lipid bilayers at an atomistic level. Electroporation can be used to increase the permeability of cellular membranes, with potential therapeutic applications. Atomistic simulations of electroporation have illustrated the molecular details of this process, including the importance of water dipole interactions at the water-membrane interface. Characterization of the lipid-protein interactions provides an important tool for understanding transmembrane protein structure and thermodynamic stability. Atomistic simulations give a detailed picture of the free energies of model peptides and side chains in lipid membranes; the energetic cost of defect formation strongly influences the energies of interactions between lipids and polar and charged residues. Many antimicrobial peptides form hydrophilic pores in lipid membranes, killing bacteria or cancer cells. On the basis of simulation data, at least some of these peptides form defects and pores near the center of the bilayer, with a common disordered structure where hydrated headgroups form an approximately toroidal shape. The localization and trafficking of lipids supports general membrane structure and a number of important signaling cascades, such as those involving ceramide, diacylglycerol, and cholesterol. Atomistic simulations have determined the rates and free energies of lipid flip-flop. During the flip-flop of most phosphatidylcholine lipids, a hydrophilic pore forms when the headgroup moves near the center of the bilayer. Simulations have provided novel insight into many features of defects and pores in lipid membranes. Simulation data from very different systems and models show how water penetration and defect formation can determine the free energies of many membrane processes. Bilayers can deform and allow transient defects and pores when exposed to a diverse range of stimuli. Future work will explore many aspects of membrane defects with increased resolution and scope, including the study of more complex lipid mixtures, membrane domains, and large-scale membrane remodeling. Such studies will examine processes including vesicle budding and fusion, non-bilayer lipid phases, and interactions between lipid bilayers and other biomolecules. Simulations provide information that complements experimental studies, allowing microscopic insight into experimental observations and suggesting novel hypotheses and experiments. These studies should enable a deeper understanding of the role of lipid bilayers in cellular biology and support the development of future lipid-based biotechnology.

Telomerase Reverse Transcriptase Has an Extratelomeric Function in Somatic Cell Reprogramming

Reactivation of the endogenous telomerase reverse transcriptase (TERT) catalytic subunit and telomere elongation occur during the reprogramming of somatic cells to induced pluripotent stem (iPS) cells. However, the role of TERT in the reprogramming process is unclear. To clarify its function, the reprogramming process was examined in TERT-KO somatic cells. To exclude the effect of telomere elongation, tail-tip fibroblasts (TTFs) from first generation TERT-KO mice were used. Although iPS cells were successfully generated from TERT-KO TTFs, the efficiency of reprogramming these cells was markedly lower than that of WT TTFs. The gene expression profiles of iPS cells induced from TERT-KO TTFs were similar to those of WT iPS cells and ES cells, and TERT-KO iPS cells formed teratomas that differentiated into all three germ layers. These data indicate that TERT plays an extratelomeric role in the reprogramming process, but its function is dispensable. However, TERT-KO iPS cells showed transient defects in growth and teratoma formation during continuous growth. In addition, TERT-KO iPS cells developed chromosome fusions that accumulated with increasing passage numbers, consistent with the fact that TERT is essential for the maintenance of genome structure and stability in iPS cells. In a rescue experiment, an enzymatically inactive mutant of TERT (D702A) had a positive effect on somatic cell reprogramming of TERT-KO TTFs, which confirmed the extratelomeric role of TERT in this process.

Cytosolic peroxidases protect the lysosome of bloodstream African trypanosomes from iron-mediated membrane damage.

African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px)-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I–II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I–II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective stage of T. brucei. The respective knockout of the cytosolic px I–II in the procyclic insect form resulted in cells that were fully viable in Trolox-free medium.

Deletion of Vitamin C biosynthesis enzyme, Arabino‐1, 4‐lactone oxidase in Leishmania donovani results in increased pro‐inflammatory responses from host immune cells

Recently, we reported molecular characterization, localization and functional importance of Arabino-1, 4-lactone oxidase (ALO) enzyme from Leishmania donovani that catalyses the last step in ascorbate biosynthesis pathway. Vitamin C (l-ascorbic acid) is implicated in several crucial physiological processes. To elucidate the biological role of d-arabinono-γ-lactone oxidase in Leishmania, we made L. donovani ALO null mutant (ΔALO) by double targeted gene replacement. This mutant lacked ALO activity, showed transient growth defect and reduced ascorbate levels. ΔALO grown in ascorbate depleted media further enhanced growth defect with no detectable levels of ascorbate, implying that parasites have the ability to scavenge ascorbate. ΔALO mutants showed reduced survival in mouse macrophages and are impaired in their infectivity in vivo. Furthermore, the ΔALO mutant induced production of pro-inflammatory cytokines gamma interferon (IFN-gamma), interleukin-12 (IL-12) and tumour necrosis factor-α (TNF-α) by infected mouse macrophages. These mutants were susceptible to oxidative stresses in vitro as revealed by the decreased survival inside macrophages by increased production of reactive oxygen or nitrogen species. Complementation of the ΔALO mutants restored the phenotypic effects in these parasites. Our description of ALO null mutant parasite that triggers pro-inflammatory host responses provides a novel platform for targeting ALO in anti-parasitic strategies.

The spindle and kinetochore–associated (Ska) complex enhances binding of the anaphase-promoting complex/cyclosome (APC/C) to chromosomes and promotes mitotic exit

The spindle and kinetochore-associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA-mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore-microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit.

Mice deficient for the epidermal dermokine β and γ isoforms display transient cornification defects.

Expression of the human dermokine gene (DMKN) leads to the production of four dermokine isoform families. The secreted α, β and γ isoforms have an epidermis-restricted expression pattern, with Dmkn β and γ being specifically expressed by the granular keratinocytes. The δ isoforms are intracellular and ubiquitous. Here, we performed an in-depth characterization of Dmkn expression in mouse skin and found an expression pattern that was less complex than in humans. In particular, mRNA coding for the δ family were absent. Homozygous mice null for the Dmkn β and γ isoforms had no obvious phenotype but only a temporary scaly skin during the first week of life. The pups null for the Dmkn β and γ isoforms had smaller keratohyalin granules and their cornified envelopes were more sensitive to mechanical stress. At the molecular level, amounts of profilaggrin and filaggrin monomers were reduced whereas amino acid components of the natural moisturizing factor were increased. In addition, the electrophoretic mobility of involucrin was modified, suggesting post-translational modifications. Finally, the mice null for the Dmkn β and γ isoforms strongly overexpressed Dmkn α. These data are evocative of compensatory mechanisms relevant to the temporary phenotype. Overall, we improved the knowledge of Dmkn expression in mouse and highlighted a role for Dmkn β and γ in cornification.