Identification of all FK506-binding proteins from Neurospora crassa

Immunophilins are intracellular receptors of immunosuppressive drugs, carrying peptidyl-prolyl cis-trans isomerase activity, with a general role in protein folding but also involved in specific regulatory mechanisms. Four immunophilins of the FKBP-type (FK506-binding proteins) were identified in the genome of Neurospora crassa. Previously, FKBP22 has been located in the endoplasmic reticulum as part of chaperone/folding complexes and FKBP13 has been found to have a dual location in the cytoplasm and mitochondria. FKBP11 is apparently located exclusively in the cytoplasm. It is not expressed during vegetative development of the fungus although its expression can be induced with calcium and during sexual development. Overexpression of the respective gene appears to confer a growth advantage to the fungus in media containing some divalent ions. FKBP50 is a nuclear protein and its genetic inactivation leads to a temperature-sensitive phenotype. None of these proteins is, alone or in combination, essential for N. crassa, as demonstrated by the isolation of a mutant strain lacking all four FKBPs.     

The evolution of the Ecdysozoa

Ecdysozoa is a clade composed of eight phyla: the arthropods, tardigrades and onychophorans that share segmentation and appendages and the nematodes, nematomorphs, priapulids, kinorhynchs and loriciferans, which are worms with an anterior proboscis or introvert. Ecdysozoa contains the vast majority of animal species and there is a great diversity of body plans among both living and fossil members. The monophyly of the clade has been called into question by some workers based on analyses of whole genome datasets. We review the evidence that now conclusively supports the unique origin of these phyla. Relationships within Ecdysozoa are also controversial and we discuss the molecular and morphological evidence for a number of monophyletic groups within this superphylum.     

The voltage-gated proton channel Hv1 has two pores, each controlled by one voltage sensor

In voltage-gated channels, ions flow through a single pore located at the interface between membrane-spanning pore domains from each of four subunits, and the gates of the pore are controlled by four peripheral voltage-sensing domains. In a striking exception, the newly discovered voltage-gated Hv1 proton channels lack a homologous pore domain, leaving the location of the pore unknown. Also unknown are the number of subunits and the mechanism of gating. We find that Hv1 is a dimer and that each subunit contains its own pore and gate, which is controlled by its own voltage sensor. Our experiments show that the cytosolic domain of the channel is necessary and sufficient for dimerization and that the transmembrane part of the channel is functional also when monomerized. The results suggest a mechanism of gating whereby the voltage sensor and gate are one and the same.     

The first metal complexes bearing ligated 3-iminoisoindolin-1-ones

The reaction between Zn(OAc)₂ · 2H₂O (1) and the 3-iminoisoindolin-1-ones H₂NCNC(O)C₆R¹R²R³R⁴ (R¹-R⁴ = H 2; R¹, R⁴ = H, R², R³ = Cl 3; R¹, R³, R⁴ = H, R² = Me 4) in EtCN at 70 °C for ca. 12 h affords the novel family of complexes [Zn{H₂NCNC(O)C₆R¹R²R³R⁴}₂(OAc)₂] (R¹-R⁴ = H 5; R¹, R⁴ = H, R², R³ = Cl 6; R¹, R³, R⁴ = H, R² = Me 7) in excellent (90% and 93% for 5 and 6, correspondingly) to good (64% for 7) yields. The isolated compounds were characterized by elemental analyses (C, H, N), IR, NMR and ESI⁺-MS. X-ray diffraction data for 2 and 5 indicate that both free (2) and ligated (5) 3-iminoisoindolin-1-ones exist in the zwitterionic form.     

Time- and space-resolved Monte Carlo study of water radiolysis for photon,electron and ion irradiation

Time-dependent yields of the most important products of water radiolysis , ^•OH, H^•, H₃O⁺, H₂, OH⁻ and H₂O₂ have been calculated for ⁶⁰Co-photons, electrons, protons, helium- and carbon-ions incident onto water. G values have been evaluated for the interval from 1 ps to 1 μs after initial energy deposition as a function of time, as well as after 1 ns and at the end of the chemical stage as a function of linear energy transfer (LET), covering an interval from approximately 0.2 up to 750 keV/μm by means of different particle types. In this work, the modules of the biophysical Monte Carlo track structure code PARTRAC dealing with the simulation of prechemical and chemical stages have been improved to extend interaction data sets for heavier ions. Among other newly selected parameter values, the thermalisation distance between the point of generation and hydration of subexcitation electrons has been adopted from recent data in the literature. As far as data from the literature are available, good agreement has been found with the calculated time- and LET-dependent yields in this work, supporting the selection of the revised parameter values.     

Trichothecene-induced cytotoxicity on human cell lines

Trichothecene cytotoxicity of type A (T-2 toxin and HT-2 toxin), type B (deoxynivalenol, DON, and nivalenol, NIV), and type D (satratoxins G and H) compounds was determined comparatively by using eight permanent human cell lines (Hep-G2, A549, CaCo-2, HEp-2, A204, U937, RPMI 8226, and Jurkat). Viability of cells was measured by a water-soluble tetrazolium (WST-1) reagent cell proliferation assay assessing mitochondrial metabolic activity. Toxicity was expressed as the toxin concentration inhibiting 50% of cell viability (IC₅₀). Depending on the chemotype of the tested trichothecenes, relative cytotoxic activity differed by a factor of 100–1,000, and the corresponding IC₅₀ values were in the range from 2.2 nmol/l (satratoxin H on Jurkat and U937 cells) to 4,900 nmol/l (deoxynivalenol on HEp-2 cells). In contrast, the specific toxicity of each individual mycotoxin towards different cell lines was within remarkable close limits, and between-cell line differences were much smaller than previously reported. For the cell lines tested, IC₅₀ values were 4.4–10.8 nmol/l for T-2 toxin, 7.5–55.8 mol/l for HT-2 toxin, 600–4,900 nmol/l for DON, 300–2,600 nmol/l for NIV, and 2.2–18.3 nmol/l for satratoxins G/H. In addition, for the first time, the toxic activity of trichothecenes on primary cell culture of human endothelial cells (HUVEC) was tested. The susceptibility of this cell line was comparable to the other cell lines tested, with IC₅₀ values ranging from 16.5 nmol/l (T-2 toxin) to 4,500 nmol/l (DON). The results suggest that the current focus of cytotoxicological studies on trichothecenes on lymphoid cell lines may lead to an underestimate of their potential on other target cell systems.     

Functional dissection of XDppa2/4 structural domains in Xenopus development

The maintenance of pluripotency in mammalian embryonic stem cells depends upon the expression of regulatory genes like Oct3/4 and Sox2. While homologues of these genes are also characterized in non-mammalian vertebrates, like birds, amphibians and fish, existence and function of developmental pluripotency associated genes (Dppa) in lower vertebrates have not yet been reported. Here we describe a Dppa2/4-like gene, XDppa2/4, in Xenopus. The protein contains a SAP domain and a conserved C-terminal region. Overexpression of XDppa2/4, murine Dppa2 or Dppa4 produces similar phenotypes (defects in blastopore closure), while injection of XDppa2/4 morpholino generates a loss of blastopore closure and neural fold formation. Embryos die up to tailbud stage. mDppa2 (but not mDppa4) rescues blastopore closure and neurulation defects caused by XDppaMO, but does not prevent subsequent death of embryos. Although XDppa2/4 exhibits a Dppa-like expression pattern and is indispensable for embryogenesis, analyses of various marker genes make its role as a pluripotency factor rather unlikely. Both the gain and loss of function effects until the end of neurulation are caused by the conserved C-terminal region but not by the SAP domain. The SAP domain is required for association of XDppa2/4 to chromatin and for embryonic survival at later stages of development suggesting epigenetic programming events.     

UBXD1 binds p97 through two independent binding sites

The chaperone-related p97 protein plays a central role in various cellular processes involving the ubiquitin-proteasome system. The diverse functions of p97 are controlled by a large number of cofactors that recruit specific substrates or influence their ubiquitylation state. Many cofactors bind through a UBX or PUB domain, two major p97 binding modules. However, the recently identified UBXD1 cofactor possesses both domains. To elucidate the molecular basis underlying the interaction between UBXD1 and p97, we analyzed the contribution of both domains to p97 binding biochemically and in living cells. The PUB domain mediated robust binding to the carboxy-terminus of p97, while the UBX domain did not contribute to p97 binding. Importantly, we identified an additional p97 binding site in UBXD1 that competed with the p47 cofactor for binding to the N domain of p97. This novel, bipartite binding mode suggests that UBXD1 could be an efficient regulator of p97 cofactor interactions.     

Mutations in Smooth Muscle Alpha-Actin (ACTA2) Cause Coronary Artery Disease, Stroke, and Moyamoya Disease, Along with Thoracic Aortic Disease

The vascular smooth muscle cell (SMC)-specific isoform of α-actin (ACTA2) is a major component of the contractile apparatus in SMCs located throughout the arterial system. Heterozygous ACTA2 mutations cause familial thoracic aortic aneurysms and dissections (TAAD), but only half of mutation carriers have aortic disease. Linkage analysis and association studies of individuals in 20 families with ACTA2 mutations indicate that mutation carriers can have a diversity of vascular diseases, including premature onset of coronary artery disease (CAD) and premature ischemic strokes (including Moyamoya disease [MMD]), as well as previously defined TAAD. Sequencing of DNA from patients with nonfamilial TAAD and from premature-onset CAD patients independently identified ACTA2 mutations in these patients and premature onset strokes in family members with ACTA2 mutations. Vascular pathology and analysis of explanted SMCs and myofibroblasts from patients harboring ACTA2 suggested that increased proliferation of SMCs contributed to occlusive diseases. These results indicate that heterozygous ACTA2 mutations predispose patients to a variety of diffuse and diverse vascular diseases, including TAAD, premature CAD, ischemic strokes, and MMD. These data demonstrate that diffuse vascular diseases resulting from either occluded or enlarged arteries can be caused by mutations in a single gene and have direct implications for clinical management and research on familial vascular diseases.