Aspergillus nidulans ArfB plays a role in endocytosis and polarized growth

Filamentous fungi undergo polarized growth throughout most of their life cycles. The Spitzenkörper is an apical organelle composed primarily of vesicles that is unique to filamentous fungi and is likely to act as a vesicle supply center for tip growth. Vesicle assembly and trafficking are therefore important for hyphal growth. ADP ribosylation factors (Arfs), a group of small GTPase proteins, play an important role in nucleating vesicle assembly. Little is known about the role of Arfs in filamentous hyphal growth. We found that Aspergillus nidulans is predicted to encode six Arf family proteins. Analysis of protein sequence alignments suggests that A. nidulans ArfB shares similarity with ARF6 of Homo sapiens and Arf3p of Saccharomyces cerevisiae. An arfB null allele (arfB disrupted by a transposon [arfB::Tn]) was characterized by extended isotropic growth of germinating conidia followed by cell lysis or multiple, random germ tube emergence, consistent with a failure to establish polarity. The mutant germ tubes and hyphae that do form initially meander abnormally off of the axis of polarity and frequently exhibit dichotomous branching at cell apices, consistent with a defect in polarity maintenance. FM4-64 staining of the arfB::Tn strain revealed that another phenotypic characteristic seen for arfB::Tn is a reduction and delay in endocytosis. ArfB is myristoylated at its N terminus. Green fluorescent protein-tagged ArfB (ArfB::GFP) localizes to the plasma membrane and endomembranes and mutation (ArfB^G2A::GFP) of the N-terminal myristoylation motif disperses the protein to the cytoplasm rather than to the membranes. These results demonstrate that ArfB functions in endocytosis to play important roles in polarity establishment during isotropic growth and polarity maintenance during hyphal extension.     

Actin bundling via LIM domains

The LIM domain is defined as a protein-protein interaction module involved in the regulation of diverse cellular processes including gene expression and cytoskeleton organization. We have recently shown that the tobacco WLIM1, a two LIM domain-containing protein, is able to bind to, stabilize and bundle actin filaments, suggesting that it participates to the regulation of actin cytoskeleton structure and dynamics. In the December issue of the Journal of Biological Chemistry we report a domain analysis that specifically ascribes the actin-related activities of WLIM1 to its two LIM domains. Results suggest that LIM domains function synergistically in the full-length protein to achieve optimal activities. Here we briefly summarize relevant data regarding the actin-related properties/functions of two LIM domain-containing proteins in plants and animals. In addition, we provide further evidence of cooperative effects between LIM domains by transiently expressing a chimeric multicopy WLIM1 protein in BY2 cells.Key words: Actin-binding proteins, actin-bundling, cysteine-rich proteins, cytoskeleton, LIM domainThe LIM domain is a ≈55 amino acid peptide domain that was first identified in 1990 as a common cystein-rich sequence found in the three homeodomain proteins LIN-11, Isl1 and MEC-3. It has since been found in a wide variety of eukaryotic proteins of diverse functions. Animals possess several families of LIM proteins, with members containing 1–5 LIM domains occasionally linked to other catalytic or protein-binding domains such as homeodomain, kinase and SH3 domains. In contrast, plants only possess two distinct sets of LIM proteins. One is plant-specific and has not been functionally characterized yet. The other one comprises proteins that exhibit the same overall structure as the animal cystein rich proteins (CRPs), i.e., two very similar LIM domains separated by a ≈50 amino acid-long interLIM domain and a relatively short and variable C-terminal domain (Fig. 1A). The mouse CRP2 protein was the first CRP reported to interact directly with actin filaments (AF) and to stabilize the latter.¹ Identical observations were subsequently described for the chicken CRP1 and tobacco WLIM1 proteins.^2,3 In addition, these two proteins were shown to arrange AF into cables both in vitro and in vivo and thus join the list of actin bundlers.Open in a separate windowFigure 1Domain maps for wild-type WLIM1 (A) and GFP-fused chimeric 3xWLIM1 (B). A. WLIM1 basically comprises a short N-terminal domain (Nt), two LIM domains (LIM1 and LIM2), an interLIM spacer (IL) and a C-terminal domain (Ct). B. 3xWLIM1 consists of three tandem WLIM1 copies. This chimeric protein has been fused in C-terminus to GFP and transiently expressed in tobacco BY2 cells.To identify the peptide domains of WLIM1 responsible for its actin-related properties/activities, we generated domain-deleted and single domain variants and submitted them to a series of in vivo and in vitro assays.⁴ Localization experiments established that both LIM domains are required to efficiently target the actin cytoskeleton in tobacco BY2 cells. High-speed (200,000 g) cosedimentation data confirmed that the actin-binding activity of WLIM1 relies on its LIM domains. Indeed, the deletion of either the first or the second LIM domain respectively resulted in a 5-fold and 10-fold decrease of the protein affinity for AF. Importantly, each single LIM domain was found able to interact with AF in an autonomous manner, although with a reduced affinity compared to the wild-type WLIM1. Low-speed (12,500 g) cosedimentation data and electron microscopy observations revealed that the actin bundling activity of WLIM1 is also triggered by its LIM domains. Surprisingly each single LIM domain was able to bundle AF in an autonomous manner, suggesting that WLIM1 has two discrete actin-bundling sites. However, the bundles induced by the variants containing only one LIM domain, i.e., LIM domain-deleted mutants and single LIM domains, differed from those induced by the full-length WLIM1. They appeared more wavy and loosely packed and formed only at relatively high protein:actin ratios. Together these data suggest that LIM domains are autonomous actin-binding and -bundling modules that function in synergy in wild-type WLIM1 to achieve optimal activities.To further assess the mechanism of cooperation between the LIM domains of plant CRP-related proteins, we generated a chimeric protein composed of three WLIM1 copies in tandem (3 × WLIM1, Fig. 1B), and transiently expressed it as a GFP-fusion in tobacco BY2 cells. We anticipated that such a six LIM domain-containing protein displays an even higher actin-bundling activity. (Fig. 2A) shows the typical actin cytoskeleton pattern in an expanding BY2 cell as visualized using the actin marker GFP-fABD2.⁵ As previously reported by Sheahan et al.,⁵ GFP-fABD2 decorated dense, transversely oriented, cortical networks as well as transvacuolar strands connecting the subcortical-perinuclear region to the cortex. Ectopic expression of WLIM1-GFP (BY2 cells normally do not express the WLIM1 gene) induced moderate but perceptible modifications of the actin cytoskeleton structure (Fig. 2B). Most AF are arranged in bundles thicker than those observed in GFP-fABD2 expressing cells and fine AF arrays are less frequently observed. As expected, this phenotype was significantly enhanced in cells transformed with the 3xWLIM1-GFP protein (Fig. 2C). Indeed, cells were almost devoided of fine AF arrays and exhibited very thick actin cables (Fig. 2C) that, at times (≈30 %), form atypical long looped structures (Fig. 2D). The appearance of such structures may result from the increase of cable stability and thickness induced by the 3xWLIM1-GFP protein, as these parameters are likely to determine, at least partially, the maximal length of actin bundles. Together the present observations support earlier data showing that LIM domains work in concert in LIM proteins to regulate actin bundling in plant cells. Strikingly, vertebrate and plant CRPs invariably contain two LIM domains. The lack, in these organisms, of CRP-related proteins combining more than two LIM domains may be explained by the fact that very thick cables, such as those induced by the artificial 3xWLIM1, may be too stable structures incompatible with the necessary high degree of actin cytoskeleton plasticity. As an exception, a muscle CRP-related protein with five LIM domains (Mlp84B) has been identified in Drosophila.⁶ However, rather than decorating actin filaments in an homogenous manner, this protein has been found to concentrate in a specialized region of the Z-discs where it stabilizes, in concert with D-titin, muscle sarcomeres.⁷Open in a separate windowFigure 2Typical actin cytoskeleton patterns in tobacco BY2 cells that have been transiently transformed, using a particle gun, with GFP-fABD2 (A), WLIM1-GFP (B), and 3xWLIM1-GFP (C and D). For each construct, more than 60 cells were analyzed by confocal microscopy. In the case of 3xWLIM1-GFP, two prevalent patterns have been observed (C and D). Bars = 20 µm.The relatively well conserved spacer length (≈50 amino acids) that separates the two LIM domains in vertebrate CRPs and related plant LIM proteins remains an intriguing feature the importance of which in actin cable organization remains to be established. Using electron microscopy we are currently evaluating the effects of the modification of the interLIM domain length on the structural properties of actin cables.     

Semen CD4+ T Cells and Macrophages Are Productively Infected at All Stages of SIV infection in Macaques

The mucosal events of HIV transmission have been extensively studied, but the role of infected cells present in the genital and rectal secretions, and in the semen, in particular, remains a matter of debate. As a prerequisite to a thorough in vivo investigation of the early transmission events through infected cells, we characterized in detail by multi-parameter flow cytometry the changes in macaque seminal leukocytes during SIVmac251 infection, focusing on T cells, macrophages and dendritic cells. Using immunocytofluorescence targeting SIV proteins and real-time quantitative PCR targeting SIV DNA, we investigated the nature of the infected cells on sorted semen leukocytes from macaques at different stages of infection. Finally, we cocultured semen CD4⁺ T cells and macrophages with a cell line permissive to SIV infection to assess their infectivity in vitro. We found that primary infection induced strong local inflammation, which was associated with an increase in the number of leukocytes in semen, both factors having the potential to favor cell-associated virus transmission. Semen CD4⁺ T cells and macrophages were productively infected at all stages of infection and were infectious in vitro. Lymphocytes had a mucosal phenotype and expressed activation (CD69 & HLA-DR) and migration (CCR5, CXCR4, LFA-1) markers. CD69 expression was increased in semen T cells by SIV infection, at all stages of infection. Macrophages predominated at all stages and expressed CD4, CCR5, MAC-1 and LFA-1. Altogether, we demonstrated that semen contains the two major SIV-target cells (CD4+ T cells and macrophages). Both cell types can be productively infected at all stages of SIV infection and are endowed with markers that may facilitate transmission of infection during sexual exposure.     

From Molecular to Process Simulation: Novel Approaches to the Prediction of Phase Equilibria and PVT Behavior Based on Molecular/Quantum Mechanics and Molecular Dynamics Simulations

Abstract

Actually, in modern process simulators, more than 75% of the code implemented is dedicated to physical properties estimation, calculation and predictions. Data banks storing pure component parameters and binary interaction parameters for phase equilibrium calculations are extensively used and continuously implemented in actual process simulators. This gives an idea of the important role physical properties availability plays in process simulation.

In this paper we propose a new way for coupling molecular and process simulation. The basic machinery is to resort to molecular/quantum mechanics and molecular dynamics simulation techniques for generating the parameters of some equations of state that will subsequently be used for the prediction of phase equilibria and PVT behavior of small and polymeric molecules as well. This information, in turn, will be used as input in the process simulator, thus creating a final and well-defined bridge between molecular and process simulations in chemical engineering.     

Inhibition of protein kinase CK2 with the clinical-grade small ATP-competitive compound CX-4945 or by RNA interference unveils its role in acute myeloid leukemia cell survival,p53-dependent apoptosis and daunorubicin-induced cytotoxicity

Background

The involvement of protein kinase CK2 in sustaining cancer cell survival could have implications also in the resistance to conventional and unconventional therapies. Moreover, CK2 role in blood tumors is rapidly emerging and this kinase has been recognized as a potential therapeutic target. Phase I clinical trials with the oral small ATP-competitive CK2 inhibitor CX-4945 are currently ongoing in solid tumors and multiple myeloma.

Methods

We have analyzed the expression of CK2 in acute myeloid leukemia and its function in cell growth and in the response to the chemotherapeutic agent daunorubicin We employed acute myeloid leukemia cell lines and primary blasts from patients grouped according to the European LeukemiaNet risk classification. Cell survival, apoptosis and sensitivity to daunorubicin were assessed by different means. p53-dependent CK2-inhibition-induced apoptosis was investigated in p53 wild-type and mutant cells.

Results

CK2α was found highly expressed in the majority of samples across the different acute myeloid leukemia prognostic subgroups as compared to normal CD34⁺ hematopoietic and bone marrow cells. Inhibition of CK2 with CX-4945, K27 or siRNAs caused a p53-dependent acute myeloid leukemia cell apoptosis. CK2 inhibition was associated with a synergistic increase of the cytotoxic effects of daunorubicin. Baseline and daunorubicin-induced STAT3 activation was hampered upon CK2 blockade.

Conclusions

These results suggest that CK2 is over expressed across the different acute myeloid leukemia subsets and acts as an important regulator of acute myeloid leukemia cell survival. CK2 negative regulation of the protein levels of tumor suppressor p53 and activation of the STAT3 anti-apoptotic pathway might antagonize apoptosis and could be involved in acute myeloid leukemia cell resistance to daunorubicin.

     

Effete,a Drosophila Chromatin-Associated Ubiquitin-Conjugating Enzyme That Affects Telomeric and Heterochromatic Position Effect Variegation

Drosophila telomeres are elongated by the transposition of telomere-specific retrotransposons rather than telomerase activity. Proximal to the terminal transposon array, Drosophila chromosomes contain several kilobases of a complex satellite DNA termed telomere-associated sequences (TASs). Reporter genes inserted into or next to the TAS are silenced through a mechanism called telomere position effect (TPE). TPE is reminiscent of the position effect variegation (PEV) induced by Drosophila constitutive heterochromatin. However, most genes that modulate PEV have no effect on TPE, and systematic searches for TPE modifiers have so far identified only a few dominant suppressors. Surprisingly, only a few of the genes required to prevent telomere fusion have been tested for their effect on TPE. Here, we show that with the exception of the effete (eff; also called UbcD1) mutant alleles, none of the tested mutations at the other telomere fusion genes affects TPE. We also found that mutations in eff, which encodes a class I ubiquitin-conjugating enzyme, act as suppressors of PEV. Thus, eff is one of the rare genes that can modulate both TPE and PEV. Immunolocalization experiments showed that Eff is a major constituent of polytene chromosomes. Eff is enriched at several euchromatic bands and interbands, the TAS regions, and the chromocenter. Our results suggest that Eff associates with different types of chromatin affecting their abilities to regulate gene expression.     

Evaluation of the Antioxidant Properties of Propofol and its Nitrosoderivative. Comparison with Homologue Substituted Phenols

Propofol (2,6-diisopropylphenol), some substituted phenols (2,6-dimethylphenol and 2,6-ditertbutylphenol) and their 4-nitrosoderivatives have been compared for their scavenging ability towards 1,1-diphenyl-2-picrylhydrazyl and for their inhibitory action on lipid peroxidation. These products were also compared to the classical antioxidants butylated hydroxytoluene and butylated hydroxyanisole. When measuring the reactivity of the various phenolic derivatives with 1,1-diphenyl-2-picrylhydrazyl the following order of effectiveness was observed: butylated hydroxyanisole>propofol>2,6-dimethylphenol>2,6-di-tertbutylphenol?>?butylated hydroxytoluene. In cumene hydroperoxide-dependent microsomal lipid peroxidation, propofol acts as the most effective antioxidant, while butylated hydroxyanisole, 2,6-di-tertbutylphenol and butylated hydroxytoluene exhibit a rather similar effect, although lower than propofol. In the iron/ascorbate-dependent lipid peroxidation propofol, at concentrations higher than 10?μM, exhibits antioxidant properties comparable to those of butylated hydroxytoluene and butylated hydroxyanisole. 2,6-Dimethylphenol is scarcely effective in both lipoperoxidative systems. The antioxidant properties of the various molecules depend on their hydrophobic characteristics and on the steric and electronic effects of their substituents. However, the introduction of the nitroso group in the 4-position almost completely removes the antioxidant properties of the examined compounds. The nitrosation of the aromatic ring of antioxidant molecules and the consequent loss of antioxidant capacity can be considered a condition potentially occurring in vivo since nitric oxide and its derivatives are continuously formed in biological systems.     

Will an Unsupervised Self-Testing Strategy for HIV Work in Health Care Workers of South Africa? A Cross Sectional Pilot Feasibility Study

Background

In South Africa, stigma, discrimination, social visibility and fear of loss of confidentiality impede health facility-based HIV testing. With 50% of adults having ever tested for HIV in their lifetime, private, alternative testing options are urgently needed. Non-invasive, oral self-tests offer a potential for a confidential, unsupervised HIV self-testing option, but global data are limited.

Methods

A pilot cross-sectional study was conducted from January to June 2012 in health care workers based at the University of Cape Town, South Africa. An innovative, unsupervised, self-testing strategy was evaluated for feasibility; defined as completion of self-testing process (i.e., self test conduct, interpretation and linkage). An oral point-of-care HIV test, an Internet and paper-based self-test HIV applications, and mobile phones were synergized to create an unsupervised strategy. Self-tests were additionally confirmed with rapid tests on site and laboratory tests. Of 270 health care workers (18 years and above, of unknown HIV status approached), 251 consented for participation.

Findings

Overall, about 91% participants rated a positive experience with the strategy. Of 251 participants, 126 evaluated the Internet and 125 the paper-based application successfully; completion rate of 99.2%. All sero-positives were linked to treatment (completion rate:100% (95% CI, 66.0–100). About half of sero-negatives were offered counselling on mobile phones; completion rate: 44.6% (95% CI, 38.0–51.0). A majority of participants (78.1%) were females, aged 18–24 years (61.4%). Nine participants were found sero-positive after confirmatory tests (prevalence 3.6% 95% CI, 1.8–6.9). Six of nine positive self-tests were accurately interpreted; sensitivity: 66.7% (95% CI, 30.9–91.0); specificity:100% (95% CI, 98.1–100).

Interpretation

Our unsupervised self-testing strategy was feasible to operationalize in health care workers in South Africa. Linkages were successfully operationalized with mobile phones in all sero-positives and about half of the sero-negatives sought post-test counselling. Controlled trials and implementation research studies are needed before a scale-up is considered.