Rev proteins of human and simian immunodeficiency virus enhance RNA encapsidation

The main function attributed to the Rev proteins of immunodeficiency viruses is the shuttling of viral RNAs containing the Rev responsive element (RRE) via the CRM-1 export pathway from the nucleus to the cytoplasm. This restricts expression of structural proteins to the late phase of the lentiviral replication cycle. Using Rev-independent gag-pol expression plasmids of HIV-1 and simian immunodeficiency virus and lentiviral vector constructs, we have observed that HIV-1 and simian immunodeficiency virus Rev enhanced RNA encapsidation 20- to 70-fold, correlating well with the effect of Rev on vector titers. In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev. Binding of Rev to the RRE or to a heterologous RNA element was required for Rev-mediated enhancement of RNA encapsidation. In addition to specific interactions of nucleocapsid with the packaging signal at the 5' end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA.     

Effectiveness of Antegrade Access in Bladder Tumors With Inaccessible Urethra

Inaccessible urethra with no retrograde endoscopic access due to multiple/diffuse strictures or multiple urethrocutaneous fistulas with acute urinary retention due to posturethral instrumentation (transurethral resection of bladder tumor [TURBT], or TURBT with transurethral resection of the prostate [TURP]), is a rare entity. Management of such a case with a bladder tumor for TURBT/surveillance cystoscopy poses a great challenge. The authors present 12 cases of bladder tumor with inaccessible urethra, 10 cases due to multiple strictures (post-TURBT and/or TURP), and 2 cases due to urethrocutaneous fistulas (post-TURBT), who presented to our emergency department with acute urinary retention. Emergent suprapubic catheterization was used as a temporary treatment method.Key words: Suprapubic cystostomy, Inaccessible urethra, Bladder tumors, Tract seedlingBladder tumors are the most common neoplasm of the lower urinary tract, comprising 6% of all malignancies in men and 2% of those in women.¹ A majority of patients present with gross painless hematuria, usually as the sole presenting symptom.² Bladder carcinoma is unique among human neoplasms in that many of its etiologic factors are known; the urologist should be aware of the possible occupational exposures to urothelial carcinogens.³ Initial symptoms of urothelial carcinoma of the bladder (UCB) include microhematuria, painless macrohematuria, and/or irritative voiding symptoms, and require further investigation. Carcinoma in situ of the bladder causes irritative lower urinary tract symptoms (LUTS) more often than does papillary UCB. Histopathologic evaluation is necessary to assess stage and grade with sufficient certainty after the appearance of bladder tumors.⁴ Bladder tumors spread by implantation in abdominal wounds, denuded epithelium, resected prostatic fossa, or traumatized urethra⁵; implantation occurs most often with high-grade tumors.     

Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag

In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.     

Cardiolipin and mitochondrial phosphatidylethanolamine have overlapping functions in mitochondrial fusion in Saccharomyces cerevisiae

The two non-bilayer forming mitochondrial phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE) play crucial roles in maintaining mitochondrial morphology. We have shown previously that CL and PE have overlapping functions, and the loss of both is synthetically lethal. Because the lack of CL does not lead to defects in the mitochondrial network in Saccharomyces cerevisiae, we hypothesized that PE may compensate for CL in the maintenance of mitochondrial tubular morphology and fusion. To test this hypothesis, we constructed a conditional mutant crd1Δpsd1Δ containing null alleles of CRD1 (CL synthase) and PSD1 (mitochondrial phosphatidylserine decarboxylase), in which the wild type CRD1 gene is expressed on a plasmid under control of the TET(OFF) promoter. In the presence of tetracycline, the mutant exhibited highly fragmented mitochondria, loss of mitochondrial DNA, and reduced membrane potential, characteristic of fusion mutants. Deletion of DNM1, required for mitochondrial fission, restored the tubular mitochondrial morphology. Loss of CL and mitochondrial PE led to reduced levels of small and large isoforms of the fusion protein Mgm1p, possibly accounting for the fusion defect. Taken together, these data demonstrate for the first time in vivo that CL and mitochondrial PE are required to maintain tubular mitochondrial morphology and have overlapping functions in mitochondrial fusion.     

Development and validation of an in vitro micronucleus assay platform in TK6 cells

The Organization for Economic Co-operation and Development (OECD) has recently adopted Test Guideline 487 (TG487) for conducting the in vitro micronucleus (MNvit) assay. The purpose of this study is to evaluate and validate treatment conditions for the use of p53 competent TK6 human lymphoblastoid cells in a TG487 compliant MNvit assay. The ten reference compounds suggested in TG487 (mitomycin C, cytosine arabinoside, cyclophosphamide, benzo-a-pyrene, vinblastine sulphate, colchicine, sodium chloride, nalidixic acid and di(2-ethylhexyl)phthalate and pyrene) and noscapine hydrochloride were chosen for this study. In order to optimize the micronucleus response after treatment with some positive substances, we extended the recovery time after pulse treatment from 2 cell cycles recommended in TG487 to 3 cell cycles for untreated cells (40h). Each compound was tested in at least one of four exposure conditions: a 4h exposure followed by a 40h recovery, a 4h exposure followed by a 24h recovery, a 4h exposure in the presence of an exogenous metabolic activation system followed by a 40h recovery period, and a 27h continuous direct treatment. Results show that the direct acting clastogens, clastogens requiring metabolic activation and aneugens caused a robust increase in micronuclei in at least one test condition whereas the negative compounds did not induce micronuclei. The negative control cultures exhibited reproducibly low and consistent micronucleus frequencies ranging from 0.4 to 1.8% (0.8±0.3% average and standard deviation). Furthermore, extending the recovery period from 24h to 40h produced a 2-fold higher micronucleus frequency after a 4h pulse treatment with mitomycin C. In summary, the protocol described in this study in TK6 cells produced the expected result with model compounds and should be suitable for performing the MNvit assay in accordance with guideline TG487.     

Members of RTP and REEP gene families influence functional bitter taste receptor expression

Functional characterization of chemosensory receptors is usually achieved by heterologous expression in mammalian cell lines. However, many chemoreceptor genes, including bitter taste receptors (TAS2Rs), show only marginal cell surface expression. Usually, these problems are circumvented by using chimeric receptors consisting of "export tags" and the receptor sequence itself. It seems likely that chemoreceptor cells express factors for cell surface targeting of native receptor molecules in vivo. For TAS2Rs, however, such factors are still unknown. The present study investigates the influence of RTP and REEP proteins on the functional expression of human TAS2Rs in heterologous cells. We expressed hTAS2Rs in HEK 293T cells and observed dramatic differences in responsiveness to agonist stimulation. By immunocytochemistry we show accumulation of the bitter beta-glucopyranoside receptor hTAS2R16 in the Golgi compartment. Coexpression of RTP and REEP proteins changed the responses of some hTAS2Rs upon agonist stimulation, which is likely due to efficient cell surface localization as demonstrated by cell surface biotinylation experiments. The coimmunoprecipitation of hTAS2R16 and RTP3 or RTP4 suggests that the mechanism by which these cofactors influence hTAS2R16 function might involve direct protein-protein interaction. Finally, expression analyses demonstrate RTP and REEP gene expression in human circumvallate papillae and testis, both of which are sites of TAS2R gene expression.     

Real-time measurement of PMA-induced cellular alterations by microelectrode array-based impedance spectroscopy

For a feasible and cost-effective impedance measurement of cellular alterations in real-time, we combined commercially available microelectrode arrays (MEAs), consisting of 60 microelectrodes, with a conventional impedance analyzer. For proof of principle, a breast carcinoma cell line (MCF-7) was cultured on MEAs, and cellular alterations were measured by impedance spectroscopy at a frequency ranging from 10 Hz to 1 MHz. Cells were stimulated with phorbol 12-myristate 13-acetate (PMA) at different concentrations to activate protein kinase C (PKC)-mediated extra- and intracellular changes. By addition of 0.03 microM PMA, an increase of the relative impedance (Z(rel)) was observed after 10 min with a maximum at 1 kHz. Moreover a gradual elevation of the impedance was measured 60 min after stimulation with PMA. If 0.3 microM PMA was applied, the maximal amplitude of the relative impedance after 60 min shifted from 1 kHz (0.03 microM PMA) to 150 Hz. Subsequently, the impedance was further increased up to 90 min after PMA application, after which the impedance reduced after 240 min. Since we could use MEAs for at least 10 times without affecting the sensitivity, our study revealed that commercially available MEAs comprising nanocolumnar titanium nitrite electrodes are suitable microstructures for a highly reproducible and cost-effective multisite measurement of intracellular processes by impedance spectroscopy.     

Intracellular Serotonin Modulates Insulin Secretion from Pancreatic β-Cells by Protein Serotonylation

While serotonin (5-HT) co-localization with insulin in granules of pancreatic β-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1−/−) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1−/− β-cells, which clearly showed that the secretory defect is downstream of Ca²⁺-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in β-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic β-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.     

An Unusual ERAD-Like Complex Is Targeted to the Apicoplast of Plasmodium falciparum

Many apicomplexan parasites, including Plasmodium falciparum, harbor a so-called apicoplast, a complex plastid of red algal origin which was gained by a secondary endosymbiotic event. The exact molecular mechanisms directing the transport of nuclear-encoded proteins to the apicoplast of P. falciparum are not well understood. Recently, in silico analyses revealed a second copy of proteins homologous to components of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) system in organisms with secondary plastids, including the malaria parasite P. falciparum. These proteins are predicted to be endowed with an apicoplast targeting signal and are suggested to play a role in the transport of nuclear-encoded proteins to the apicoplast. Here, we have studied components of this ERAD-derived putative preprotein translocon complex in malaria parasites. Using transfection technology coupled with fluorescence imaging techniques we can demonstrate that the N terminus of several ERAD-derived components targets green fluorescent protein to the apicoplast. Furthermore, we confirm that full-length PfsDer1-1 and PfsUba1 (homologues of yeast ERAD components) localize to the apicoplast, where PfsDer1-1 tightly associates with membranes. Conversely, PfhDer1-1 (a host-specific copy of the Der1-1 protein) localizes to the ER. Our data suggest that ERAD components have been “rewired” to provide a conduit for protein transport to the apicoplast. Our results are discussed in relation to the nature of the apicoplast protein transport machinery.The apicomplexan parasite Plasmodium falciparum is the etiological agent of malaria tropica, the most severe form of human malaria, responsible for over 250 million infections and 1 million deaths annually (61). Many apicomplexan parasites, including P. falciparum, harbor a so-called apicoplast, a complex plastid of red algal origin which was gained by a secondary endosymbiotic event (27, 58). Although during the course of evolution this plastid organelle has lost the ability to carry out photosynthesis, it is still the site of several important biochemical pathways, including isoprenoid and heme biosynthesis, and as such is essential for parasite survival (60). As in other plastids, the vast majority of genes originally encoded on the plastid genome have been transferred to the nucleus of the host. As a result, their gene products (predicted to constitute up to 10% of all nucleus-encoded proteins) must be imported back into the apicoplast (12). The apicoplast is surrounded by four membranes (55), and this protein import process thus represents a major cell biological challenge and has attracted much research interest, not least due to the importance of P. falciparum as a human pathogen (16, 50).The signals directing transport of nucleus-encoded proteins to complex plastids, including the apicomplexan apicoplast, have been studied in great detail in recent years, and reveal that such proteins are endowed with specific N-terminal targeting sequences, referred to as a bipartite topogenic signals (BTS), that direct their transport to this compartment (50). BTS are composed of an N-terminal endoplasmic reticulum (ER)-type signal sequence, which initially allows proteins to enter the secretory system via the Sec61 complex (59). Following this, proteins are carried via a Golgi complex-independent transport step to the second outermost membrane, from where they are then translocated across the remaining three apicoplast membranes, directed by the second part of the BTS, the transit peptide (51). Based on evolutionary considerations, it has long been suggested that transport across the inner two apicoplast membranes occurs via a Toc/Tic-like (where Toc and Tic are translocons of the outer and inner chloroplast envelopes, respectively) protein translocase machinery, and this is supported by a recent publication that provides evidence for an essential role of a Toxoplasma gondii Tic20 homologue in this transport process (50, 57). Despite this progress, it is still unclear how proteins travel across the second and third outer apicoplast membranes. Several models have been discussed to account for this transport step, including vesicular shuttle and translocon-based mechanisms (recently reviewed in reference 19), but until recently no actual molecular equipment had been found which could account for these membrane translocation events. To address this question, Sommer et al. screened the nucleomorph genome of the chromalveolate cryptophyte Guillardia theta (which, similar to P. falciparum, contains a four-membrane-bound plastid organelle) for genes encoding potential translocon-related proteins (49). Surprisingly, the authors identified genes encoding proteins usually involved in the ER-associated protein degradation pathway (ERAD), which recognizes incorrectly folded protein substrates and retrotranslocates them to the cell cytosol for degradation by the ubiquitin (Ub)-proteasome system (35, 44). As such, the ERAD system functions as a translocation complex, capable of transporting proteins across a biological membrane. Further characterization of one of these proteins (G. theta Der1-1, a homologue of yeast Der1p, a component of the ERAD system) provided strong evidence for a plastid localization. These data suggested an attractive solution to the mechanistic problem of transport across the second and third outermost membrane of complex plastids by hypothesizing a role for an ERAD-derived protein translocon complex. Intriguingly, this study also identified several members of this ERAD-derived translocon complex (apicoplast ERAD [apERAD]) in the nuclear genome of P. falciparum endowed with an N-terminal BTS (49). The BTS derived from one of these proteins, P. falciparum sDer1-1 [PfsDer1-1], was sufficient to direct transport of green fluorescent protein (GFP) to the apicoplast of P. falciparum, suggesting that this ERAD-like machinery is ubiquitous among chromalveolates with four membrane-bound plastids (49). In this current report we extend our study of the P. falciparum apERAD complex.     

Expression of AMIDASE1 (AMI1) is suppressed during the first two days after germination

The regulation of cellular auxin levels is a critical factor in determining plant growth and architecture, as indole-3-acetic acid (IAA) gradients along the plant axis and local IAA maxima are known to initiate numerous plant growth responses. The regulation of auxin homeostasis is mediated in part by transport, conjugation and deconjugation, as well as by de novo biosynthesis. However, the pathways of IAA biosynthesis are yet not entirely characterized at the molecular and biochemical level. It is suggested that several biosynthetic routes for the formation of IAA have evolved. One such pathway proceeds via the intermediate indole-3-acetamide (IAM), which is converted into IAA by the activity of specific IAM hydrolases, such as Arabidopsis AMIDASE1 (AMI1). In this article we present evidence to support the argument that AMI1-dependent IAA synthesis is likely not to be used during the first two days of seedling development.Key words: Arabidopsis thaliana, auxin biosynthesis, AMIDASE1, indole-3-acetic acid, indole-3-acetamide, LEAFY COTYLEDON1, seed developmentAuxins are versatile plant hormones that play diverse roles in regulating many aspects of plant growth and development.¹ To enable auxins to develop their activity, a tight spatiotemporal control of cellular indole-3-acetic acid (IAA) contents is absolutely necessary since it is well-documented that auxin action is dose dependent, and that high IAA levels can have inhibitory effects on plant growth.² To achieve this goal, plants have evolved a set of different mechanisms to control cellular hormone levels. On the one hand, plants possess several pathways that contribute to the de novo synthesis of IAA. This multiplicity of biosynthetic routes presumably facilitates fine-tuning of the IAA production. On the other hand, plants are equipped with a variety of enzymes that are used to conjugate free auxin to either sugars, amino acids or peptides and small proteins, respectively, or on the contrary, that act as IAA-conjugate hydrolases, releasing free IAA from corresponding conjugates. IAA-conjugates serve as a physiologically inactive storage form of IAA from which the active hormone can be quickly released on demand. Alternatively, conjugation of IAA can mark the first step of IAA catabolism. In general, conjugation and deconjugation of free IAA are ways to positively or negatively affect active hormone levels, which adds another level of complexity to the system. Additionally, IAA can be transported from cell to cell in a polar manner, which is dependent on the action of several transport proteins. All together, these means are used to form auxin gradients and local maxima that are essential to initiate plant growth processes, such as root or leaf primordia formation.³