Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G).

Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed Ca2+-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower Ca2+-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. Ca2+-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower Ca2+-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the Ca2+-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm.

Integration of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) into Cellular Structures.

The human mutant cardiac α-actins p.A295S or p.R312H and p.E361G, correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by the baculovirus/Sf21 insect cell system and purified to homogeneity. The purified cardiac actins maintained their native state but showed differences in Ca2+-sensitivity to stimulate the myosin-subfragment1 ATPase. Here we analyzed the interactions of these c-actins with actin-binding and -modifying proteins implicated in cardiomyocyte differentiation. We demonstrate that Arp2/3 complex and the formin mDia3 stimulated the polymerization rate and extent of the c-actins, albeit to different degrees. In addition, we tested the effect of the MICAL-1 monooxygenase, which modifies the supramolecular actin organization during development and adaptive processes. MICAL-1 oxidized these c-actin variants and induced their de-polymerization, albeit at different rates. Transfection experiments using MDCK cells demonstrated the preferable incorporation of wild type and p.A295S c-actins into their microfilament system but of p.R312H and p.E361G actins into the submembranous actin network. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding HA-tagged c-actin variants showed their incorporation into microfilaments after one day in culture and thereafter into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferentially incorporated at the minus ends.

The formin Drosophila homologue of Diaphanous2 (Diaph2) controls microtubule dynamics in colorectal cancer cells independent of its FH2-domain.

In this study, we analyzed the functional role of the formin Drosophila Homologue of Diaphanous2 (Diaph2) in colorectal cancer cells. We show that stable down-regulation of Diaph2 expression in HT29 cells decreased chromosome alignment and the velocity of chromosome movement during M-phase, thus reducing the proliferation rate and colony formation. In interphase cells, Diaph2 was diffusely distributed in the cytosol, while in metaphase cells the protein was located to spindle microtubules (MTs). Diaph2-depletion increased the concentration of stable spindle MTs, showing that the formin is required to control spindle MT-dynamics. Our cellular data indicate that Diaph2-controls spindle MT-dynamics independent of Cdc42 activity and our in vitro results reveal that bacterially produced full-length (FL) Diaph2 strongly altered MT-dynamics in absence of Cdc42, where its actin-nucleating activity is auto-inhibited. FL-Diaph2 mediates a 10-fold increase in MT-polymerization compared to the Diaph2-FH2-domain. Interestingly, a Diaph2-mutant lacking the FH2-domain (ΔFH2) increased MT-polymerization to a similar extent as the FH2-domain, indicating the existence of a second MT-binding domain. However, in contrast to FL-Diaph2 and the FH2-domain, ΔFH2 did not alter the density of taxol-stabilized MTs. Thus, the FH2-domain and the second Diaph2-binding domain appear to control MT-dynamics by different mechanisms. In summary, our data indicate that Diaph2 controls M-phase progression under basal conditions by regulating spindle MT-dynamics. In addition, a region outside of the canonical MT-regulating FH2-domain is involved in Diaph2-mediated control of MT-dynamics.

BCAS1 expression defines a population of early myelinating oligodendrocytes in multiple sclerosis lesions.

Investigations into brain function and disease depend on the precise classification of neural cell types. Cells of the oligodendrocyte lineage differ greatly in their morphology, but accurate identification has thus far only been possible for oligodendrocyte progenitor cells and mature oligodendrocytes in humans. We find that breast carcinoma amplified sequence 1 (BCAS1) expression identifies an oligodendroglial subpopulation in the mouse and human brain. These cells are newly formed, myelinating oligodendrocytes that segregate from oligodendrocyte progenitor cells and mature oligodendrocytes and mark regions of active myelin formation in development and in the adult. We find that BCAS1+ oligodendrocytes are restricted to the fetal and early postnatal human white matter but remain in the cortical gray matter until old age. BCAS1+ oligodendrocytes are reformed after experimental demyelination and found in a proportion of chronic white matter lesions of patients with multiple sclerosis (MS) even in a subset of patients with advanced disease. Our work identifies a means to map ongoing myelin formation in health and disease and presents a potential cellular target for remyelination therapies in MS.

[Chronic acromioclavicular joint injury of Rockwood V type with concomitant chronic anterior sternoclavicular instability]. / Chronische Acromioclaviculargelenkverletzung Typ Rockwood V mit gleichzeitiger chronischer anteriorer sternoclavicularer Instabilität.

Bipolar dislocation of the clavicle is rare. In the literature, every reported bipolar dislocation of the clavicle is caused by a traumatic injury with loss of function of the affected shoulder. Currently, there is no recommendation to treat. A conservative treatment can be tried first to achieve adequate shoulder function. If this cannot be achieved, surgical treatment will be indicated. In the literature, many options for surgical treatment are described. This article presents a case of a chronic Rockwood V injury with chronic anterior sternoclavicular joint instability. The special feature of this case was the arthroscopically assisted stabilization of the acromioclavicular joint (ACJ) with the ipsilateral semitendinosus tendon graft and the open stabilization of the sternoclavicular joint (SCJ) with the ipsilateral gracilis tendon graft. A lateral fracture of the clavicle in the course of the postoperative treatment was treated with a plate osteosynthesis. At follow-up after six months, the postoperative shoulder function was restored. The ACJ and the SCJ were stable in clinical and radiographic examination. This case report shows the first surgical treatment using two tendon grafts for combined stabilization of the ACJ and SCJ.

Effect directed analysis and mixture effects of estrogenic compounds in a sediment of the river Elbe.

INTRODUCTION: Endocrine disrupting chemicals (EDCs) are present in the environment and can have serious effects on humans and wildlife. For the establishment of environmental quality guidelines and regulation of EDCs, a better understanding and knowledge of the occurrence and the behavior of environmental EDCs is necessary. The aim of the present study was to comprehensively identify substances that are responsible for the estrogenic effect of an environmental sediment sample taken from the river Elbe/Germany. DISCUSSION: The estrogenic effect of the organic sediment extract was determined using the yeast-estrogen-screen (YES). The sample was fractionated by liquid chromatography (LC) for effect directed analysis. The composition of estrogen-active fractions was further investigated by gas chromatography-mass spectrometry and high-resolution LC-MS analysis. The composition of the environmental sample was rebuilt with pure compounds in order to assess the partition of estrogenic activity caused by the identified compounds. The organic sediment extract showed an estrogenic potential of 1.9 ± 0.4 ng/g ethinylestradiol equivalents in the sediment. The most prominent contaminants with an estrogenic potential were 17ß-estradiol, estrone, and 4-iso-nonylphenols, but other xenoestrogens like bisphenol A and stigmasterol could be found as well. A rebuild of the sample was measured in the YES in order to investigate mixture effects. About 67 % of the observed estrogenic effect in the sediment extract could be explained by a mixture which contained all identified compounds. Chlorophene (o-benzyl-p-chlorophenol)-a widely used antiseptic that was also identified in the sediment extract-has xenoestrogenic properties in the YES that are in the range of other xenoestrogens like 4-n-nonylphenol. This is the first report on chlorophene acting as a xenoestrogen.

Thiol-based redox switches in eukaryotic proteins.

For many years, oxidative thiol modifications in cytosolic proteins were largely disregarded as in vitro artifacts, and considered unlikely to play significant roles within the reducing environment of the cell. Recent developments in in vivo thiol trapping technology combined with mass spectrometric analysis have now provided convincing evidence that thiol-based redox switches are used as molecular tools in many proteins to regulate their activity in response to reactive oxygen and nitrogen species. Reversible oxidative thiol modifications have been found to modulate the function of proteins involved in many different pathways, starting from gene transcription, translation and protein folding, to metabolism, signal transduction, and ultimately apoptosis. This review will focus on three well-characterized eukaryotic proteins that use thiol-based redox switches to influence gene transcription, metabolism, and signal transduction. The transcription factor Yap1p is a good illustration of how oxidative modifications affect the function of a protein without changing its activity. We use glyeraldehyde-3-phosphate dehydrogenase to demonstrate how thiol modification of an active site cysteine re-routes metabolic pathways and converts a metabolic enzyme into a pro-apoptotic factor. Finally, we introduce the redox-sensitive protein tyrosine phosphatase PTP1B to illustrate that reversibility is one of the fundamental aspects of redox-regulation.