Proteasome-Rich PaCS as an Oncofetal UPS Structure Handling Cytosolic Polyubiquitinated Proteins. In Vivo Occurrence, in Vitro Induction, and Biological Role.

In this article, we outline and discuss available information on the cellular site and mechanism of proteasome interaction with cytosolic polyubiquitinated proteins and heat-shock molecules. The particulate cytoplasmic structure (PaCS) formed by barrel-like particles, closely reproducing in vivo the high-resolution structure of 26S proteasome as isolated in vitro, has been detected in a variety of fetal and neoplastic cells, from living tissue or cultured cell lines. Specific trophic factors and interleukins were found to induce PaCS during in vitro differentiation of dendritic, natural killer (NK), or megakaryoblastic cells, apparently through activation of the MAPK-ERK pathway. Direct interaction of CagA bacterial oncoprotein with proteasome was shown inside the PaCSs of a Helicobacter pylori-infected gastric epithelium, a finding suggesting a role for PaCS in CagA-mediated gastric carcinogenesis. PaCS dissolution and autophagy were seen after withdrawal of inducing factors. PaCS-filled cell blebs and ectosomes were found in some cells and may represent a potential intercellular discharge and transport system of polyubiquitinated antigenic proteins. PaCS differs substantially from the inclusion bodies, sequestosomes, and aggresomes reported in proteinopathies like Huntington or Parkinson diseases, which usually lack PaCS. The latter seems more linked to conditions of increased cell proliferation/differentiation, implying an increased functional demand to the ubiquitin⁻proteasome system.

Accumulation of heme oxygenase-1 (HSP32) in Xenopus laevis A6 kidney epithelial cells treated with sodium arsenite, cadmium chloride or proteasomal inhibitors.

The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats.

PaCS is a novel cytoplasmic structure containing functional proteasome and inducible by cytokines/trophic factors.

A variety of ubiquitinated protein-containing cytoplasmic structures has been reported, from aggresomes to aggresome-like induced structures/sequestosomes or particle-rich cytoplasmic structures (PaCSs) that we recently observed in some human diseases. Nevertheless, the morphological and cytochemical patterns of the different structures remain largely unknown thus jeopardizing their univocal identification. Here, we show that PaCSs resulted from proteasome and polyubiquitinated protein accumulation into well-demarcated, membrane-free, cytoskeleton-poor areas enriched in glycogen and glycosaminoglycans. A major requirement for PaCS detection by either electron or confocal microscopy was the addition of osmium to aldehyde fixatives. However, by analyzing living cells, we found that proteasome chymotrypsin-like activity concentrated in well-defined cytoplasmic structures identified as PaCSs by ultrastructural morphology and immunocytochemistry of the same cells. PaCSs differed ultrastructurally and cytochemically from sequestosomes which may coexist with PaCSs. In human dendritic or natural killer cells, PaCSs were induced in vitro by cytokines/trophic factors during differentiation/activation from blood progenitors. Our results provide evidence that PaCS is indeed a novel distinctive cytoplasmic structure which may play a critical role in the ubiquitin-proteasome system response to immune, infectious or proneoplastic stimuli.

Tumour tissue selectivity in the uptake and retention of SN 28049, a new topoisomerase II-directed anticancer agent.

PURPOSE: A variety of anticancer drugs, including doxorubicin and mitoxantrone, have structures in which a DNA-intercalating chromophore is linked to a positively charged side chain. These drugs generally inhibit tumour growth and survival by poisoning the enzyme DNA topoisomerase II. SN 28049, a benzonaphthyridine derivative with these properties, has curative activity against the Colon 38 tumour in mice. Previous pharmacokinetic studies have demonstrated tumour-selective retention with approximately 20-fold higher area under the concentration-time curve (AUC) for tumour tissue as compared to normal tissues. We have investigated here whether such retention is tumour specific. METHODS: Plasma and tissue pharmacokinetics were assessed in the murine Lewis lung (LL3) tumour in C57 BL/6 mice and in xenografts of the NZM4, NZM10 and NZM52 human melanoma lines in Balb/c Rag-1 immunodeficient mice. The in vitro cellular localisation of SN 28049 in murine and human cell lines was studied by confocal fluorescence microscopy. RESULTS: A 260-fold variation, from 8.9 µM h (NZM4) to 2,334 µM h (Colon 38), was found among the different tumours. Only small variations were observed in the corresponding plasma AUC (2.9-5 µM h). Moreover, in vivo activity, as measured by tumour growth delay, varied from 1 day (NZM4) to curative (Colon 38), consistent with the tumour pharmacokinetic data. In cultured cell lines, SN 28049 was found in cytoplasmic bodies, suggesting that drug sequestration could contribute to tumour pharmacokinetics. CONCLUSION: SN 28049 shows dramatic differences in both tumour AUC and antitumour activity against different tumours. These differences point to the presence of a tumour-specific uptake and retention mechanism.

Mechanism of microtubule array expansion in the cytokinetic phragmoplast.

In land plants, the cell plate partitions the daughter cells at cytokinesis. The cell plate initially forms between daughter nuclei and expands centrifugally until reaching the plasma membrane. The centrifugal development of the cell plate is driven by the centrifugal expansion of the phragmoplast microtubule array, but the molecular mechanism underlying this expansion is unknown. Here, we show that the phragmoplast array comprises stable microtubule bundles and dynamic microtubules. We find that the dynamic microtubules are nucleated by γ-tubulin on stable bundles. The dynamic microtubules elongate at the plus ends and form new bundles preferentially at the leading edge of the phragmoplast. At the same time, they are moved away from the cell plate, maintaining a restricted distribution of minus ends. We propose that cycles of attachment of γ-tubulin complexes onto the microtubule bundles, microtubule nucleation and bundling, accompanied by minus-end-directed motility, drive the centrifugal development of the phragmoplast.

Russell body inducing threshold depends on the variable domain sequences of individual human IgG clones and the cellular protein homeostasis.

Russell bodies are intracellular aggregates of immunoglobulins. Although the mechanism of Russell body biogenesis has been extensively studied by using truncated mutant heavy chains, the importance of the variable domain sequences in this process and in immunoglobulin biosynthesis remains largely unknown. Using a panel of structurally and functionally normal human immunoglobulin Gs, we show that individual immunoglobulin G clones possess distinctive Russell body inducing propensities that can surface differently under normal and abnormal cellular conditions. Russell body inducing predisposition unique to each immunoglobulin G clone was corroborated by the intrinsic physicochemical properties encoded in the heavy chain variable domain/light chain variable domain sequence combinations that define each immunoglobulin G clone. While the sequence based intrinsic factors predispose certain immunoglobulin G clones to be more prone to induce Russell bodies, extrinsic factors such as stressful cell culture conditions also play roles in unmasking Russell body propensity from immunoglobulin G clones that are normally refractory to developing Russell bodies. By taking advantage of heterologous expression systems, we dissected the roles of individual subunit chains in Russell body formation and examined the effect of non-cognate subunit chain pair co-expression on Russell body forming propensity. The results suggest that the properties embedded in the variable domain of individual light chain clones and their compatibility with the partnering heavy chain variable domain sequences underscore the efficiency of immunoglobulin G biosynthesis, the threshold for Russell body induction, and the level of immunoglobulin G secretion. We propose that an interplay between the unique properties encoded in variable domain sequences and the state of protein homeostasis determines whether an immunoglobulin G expressing cell will develop the Russell body phenotype in a dynamic cellular setting.

Sensor specific imaging of proteomic Zn2+ with zinquin and TSQ after cellular exposure to N-ethylmaleimide.

The impact of the thiol binding reagent N-ethylmaleimide (NEM) on proteomic Zn(2+) availability was investigated in rat glioma cells. Zinquin (ZQ) or TSQ, two related fluorescent sensors, were used to observe reactive Zn(2+). Control cells contained proteomic Zn(2+) but no detectable low molecular weight (LMW) Zn(2+). With either sensor, basal cellular fluorescence emission centered near 470 nm, indicative of sensor-Zn-proteins. ZQ sequestered 13% of proteomic Zn(2+) as Zn(ZQ)(2); TSQ reacted only with the Zn-proteome. NEM (100 µM) abolished LMW thiols, including glutathione (GSH) and lowered proteomic sulfhydryl content about 30%. In ZQ-treated cells, NEM exposure enhanced fluorescent intensity and the formation of Zn(ZQ)(2) (λ(MAX), 492 nm). Cells incubated with TSQ and NEM also displayed increased fluorescence without a spectral shift in wavelength maximum, consistent with increased formation of TSQ-Zn-protein adducts but not Zn(TSQ)(2). In neither experiment was Zn(2+) lost from cells. NEM altered Zn(2+) accessibility to sensors in membrane-nuclear and cytosolic fractions, but Zn(ZQ)(2) was only generated in the cytosol. Similar results were obtained when cell supernatant replaced cells. In contrast, when isolated proteome was reacted with ZQ and 100 µM NEM in the absence of GSH, 70% of the proteomic thiols underwent reaction. As a consequence, most of the ZQ-Zn-protein adducts were converted to Zn(ZQ)(2). Substituting TSQ for ZQ, only increased TSQ-Zn-proteins were observed. Evidently, the results of imaging cells with Zn(2+) sensors are dependent upon the specific chemical properties of the sensors and can only be understood after detailed chemical analysis.

Dark cell change of the cerebellar Purkinje cells induced by terbutaline under transient disruption of the blood-brain barrier in adult rats: morphological evaluation.

This study aimed to establish a cerebellar degeneration animal model and to characterize the dark cell change of Purkinje cells. We hypothesized that terbutaline, a ß2-adrenoceptor agonist, induces cerebellar degeneration not only in neonatal rats, but also in adult rats. Nine-week-old adult male Sprague-Dawley rats were anesthetized and infused with 25% mannitol via the left common carotid artery. Thirty seconds later, terbutaline was infused via the same artery. Dark-stained Purkinje cells were observed in the entire cerebellum on day 3. Prominent Bergmann glial cells accompanied by swelling of the glial processes were present, and were closely associated with the dark-stained Purkinje cells. These findings were found continuously throughout day 30. Ultrastructurally, dilated Golgi vesicles and/or endoplasmic reticulum and large lamella bodies were present in both severely changed and slightly changed Purkinje cells. Bergmann glial cells in the area of synaptic contacts of the severely changed Purkinje cells showed swelling. The Bergmann glial process in close contact with the slightly changed Purkinje cell dendrite in molecular layer showed slight swelling, and large lamella bodies in the dendrite were observed close to the dendritic spines. These findings may suggest that terbutaline induced a failure of Bergmann glial cell and resulted in dark cell degeneration of the Purkinje cells due to glutamate excitotoxicity.

Microtubule disruption targets HIF-1alpha mRNA to cytoplasmic P-bodies for translational repression.

The hypoxia inducible factor 1α (HIF-1α) is overexpressed in solid tumors, driving tumor angiogenesis and survival. However, the mechanisms regulating HIF-1α expression in solid tumors are not fully understood. In this study, we find that microtubule integrity and dynamics are intricately involved in orchestrating HIF-1α translation. HIF-1α messenger RNA (mRNA) traffics on dynamic microtubules when it is actively translated. Microtubule perturbation by taxol (TX) and other microtubule-targeting drugs stalls HIF-1α mRNA transport and releases it from polysomes, suppressing its translation. Immunoprecipitation of the P-body component Argonaute 2 (Ago2) after microtubule disruption shows significant enrichment of HIF-1α mRNAs and HIF-targeting microRNAs (miRNAs). Inhibition of HIF-repressing miRNAs or Ago2 knockdown abrogates TX's ability to suppress HIF-1α translation. Interestingly, microtubule repolymerization after nocodazole washout allows HIF-1α mRNA to reenter active translation, suggesting that microtubule dynamics exert tight yet reversible control over HIF-1α translation. Collectively, we provide evidence for a new mechanism of microtubule-dependent HIF-1α translation with important implications for cell biology.

Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells.

BACKGROUND: Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined. METHODOLOGY/PRINCIPAL FINDINGS: Distinct cytoplasmic rods (∼3-10 µm in length) and rings (∼2-5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin. CONCLUSIONS/SIGNIFICANCE: RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.

Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella.

Intracellular pathogens and endogenous danger signals in the cytosol engage NOD-like receptors (NLRs), which assemble inflammasome complexes to activate caspase-1 and promote the release of proinflammatory cytokines IL-1beta and IL-18. However, the NLRs that respond to microbial pathogens in vivo are poorly defined. We show that the NLRs NLRP3 and NLRC4 both activate caspase-1 in response to Salmonella typhimurium. Responding to distinct bacterial triggers, NLRP3 and NLRC4 recruited ASC and caspase-1 into a single cytoplasmic focus, which served as the site of pro-IL-1beta processing. Consistent with an important role for both NLRP3 and NLRC4 in innate immune defense against S. typhimurium, mice lacking both NLRs were markedly more susceptible to infection. These results reveal unexpected redundancy among NLRs in host defense against intracellular pathogens in vivo.

Defects in the secretory pathway and high Ca2+ induce multiple P-bodies.

mRNA is sequestered and turned over in cytoplasmic processing bodies (PBs), which are induced by various cellular stresses. Unexpectedly, in Saccharomyces cerevisiae, mutants of the small GTPase Arf1 and various secretory pathway mutants induced a significant increase in PB number, compared with PB induction by starvation or oxidative stress. Exposure of wild-type cells to osmotic stress or high extracellular Ca(2+) mimicked this increase in PB number. Conversely, intracellular Ca(2+)-depletion strongly reduced PB formation in the secretory mutants. In contrast to PB induction through starvation or osmotic stress, PB formation in secretory mutants and by Ca(2+) required the PB components Pat1 and Scd6, and calmodulin, indicating that different stressors act through distinct pathways. Consistent with this hypothesis, when stresses were combined, PB number did not correlate with the strength of the translational block, but rather with the type of stress encountered. Interestingly, independent of the stressor, PBs appear as spheres of approximately 40-100 nm connected to the endoplasmic reticulum (ER), consistent with the idea that translation and silencing/degradation occur in a spatially coordinated manner at the ER. We propose that PB assembly in response to stress occurs at the ER and depends on intracellular signals that regulate PB number.

Distribution of activator of G-protein signaling 3 within the aggresomal pathway: role of specific residues in the tetratricopeptide repeat domain and differential regulation by the AGS3 binding partners Gi(alpha) and mammalian inscuteable.

AGS3, a receptor-independent activator of G-protein signaling, is involved in unexpected functional diversity for G-protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G-protein regulatory (GPR) motifs that serve as docking sites for Gialpha-GDP. The positioning of AGS3 within the cell and the intramolecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G-protein signaling. We report that AGS3 enters into the aggresome pathway and that distribution of the protein is regulated by the AGS3 binding partners Gialpha and mammalian Inscuteable (mInsc). Gialpha rescues AGS3 from the aggresome, whereas mInsc augments the aggresome-like distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is accelerated by disruption of the TPR organizational structure or introduction of a nonsynonymous single-nucleotide polymorphism. These data present AGS3, G-proteins, and mInsc as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies.

Formation of GW/P bodies as marker for microRNA-mediated regulation of innate immune signaling in THP-1 cells.

GW bodies (GWB or P bodies) are cytoplasmic foci thought to result from microRNA (miRNA) regulation of messenger RNA (mRNA) targets and subsequent mRNA degradation. The purpose of this study is to examine the effects of lipopolysaccharide (LPS) stimulation of human monocytes on GWB formation, miRNA induction, miRNA target regulation and downstream cytokine and chemokine expression. In response to LPS stimulation, the number of GWB consistently increased by twofold at 8 h after stimulation and this increase was abolished when the miRNA-effector proteins Rck/p54 or argonaute 2 were depleted. As the level of miR-146a increased from 19-fold up to 100-fold during LPS stimulation, the transfection of a miR-146a mimic into THP-1 cells was examined to determine whether miR-146a alone can induce similar changes in GWB. The results showed transfected miR-146a could produce a comparable increase in the number of GWB and this was accompanied by a reduction in major cytokines/chemokines induced by LPS. These data show that the increase in size and number of GWB may serve as a biomarker for miRNA-mediated gene regulation, and miR-146a has a significant role in the regulation of LPS-induced cytokine production in THP-1 cells.

The virion-packaged endoribonuclease of herpes simplex virus 1 cleaves mRNA in polyribosomes.

The virion host shutoff protein product of the U(L)41 gene of herpes simplex virus 1 is an endoribonuclease that selectively degrades mRNAs during the first hours after infection. Specifically, in contrast to the events in uninfected cells or cells infected with a mutant lacking the RNase, in wild-type virus-infected cells mRNA of housekeeping genes exemplified by GAPDH is degraded rapidly, whereas mRNAs containing AU elements are cleaved and the 5' cleavage product of these RNAs persists for many hours. We report that in wild-type virus-infected cells there was a rapid increase in the number and size of processing bodies (P-bodies). These P-bodies were also preset in cycloheximide (CHX)-treated cells but not in either treated or untreated uninfected cells or cells infected with the RNase minus mutant. Additional studies revealed that polyribosomes extracted from cytoplasm of wild-type virus-infected cells treated with CHX and displayed in sucrose gradients contained ribosome-loaded, truncated AU-rich mRNAs lacking the 3' UTR and poly(A) tails. The results suggest that the virion RNase is bound to polyribosomes by virtue of the reported association with translation machinery and cleaves the RNAs 5' to the AU elements. In contrast to the slow degradation of the of the residual 5' domain, the 3' UTR of the AU-rich mRNA and the GAPDH mRNA are rapidly degraded in wild-type virus-infected cells.

Accelerated proteasomal activity induced by Pb2+, Ga3+, or Cu2+ exposure does not induce degradation of alpha-synuclein.

The involvement of environmental heavy metals in Parkinson's disease (PD) has been suggested by epidemiologic studies; however, the mechanism of this effect is unknown. PD is characterized by the aggregation of alpha-synuclein in Lewy bodies. We previously showed that Pb2+ accelerates proteasomal activity. Therefore, we examined the effect of Pb2+, Ga3+, and Cu2+ on alpha-synuclein in human SH-SY5Y cells. The heavy metals induced an increase in heme-oxygenase-1 levels without significant cell death or ROS generation. The metals inhibited ALA-dehydratase, which is the inhibitory subunit of the proteasome, thereby accelerating proteasomal activity and decreasing protein levels of CDK-1 and PBGD. However, alpha-synuclein protein levels increased after exposure to metals, similar to the effect obtained with the proteasome inhibitor, hemin, suggesting that alpha-synuclein is inaccessible to proteasomal degradation. Indeed, electron microscopy revealed the formation of aggresomes in Pb2+- or hemin-treated cells. Thus, although heavy metals enhance proteasomal activity, alpha-synuclein is protected from degradation, and its protein levels and aggregation are increased.

Cardioprotective effect of EGb 761 on myocardial ultrastructure of young and old rat heart and antioxidant status during acute hypoxia.

BACKGROUND AND AIMS: Acute hypoxia is a threatening clinical case of emergency and may result in ultrastructural damage, with complete loss of cellular and organ functions. However, little is known about the differences in hypoxia tolerance between young and old myocardia and the protective effects of radical scavenging agents in acute hypoxic stress situations. METHODS: We investigated the age-dependent differences of the myocardial ultrastructure and antioxidative status (superoxide-dismutase (SOD) activity and malondialdehyde (MDA) content) of young (6 months) and old (22-24 months) Wistar rats (Crl (Wi)Br) after acute respiratory hypoxia of 20 min at 5% v/v O2 in N2O mixture, and the protective effect of Ginkgo biloba extract (EGb 761). RESULTS: Ultrastructural-morphometric and biochemical age analysis only revealed a decrease in the sarcoplasma volume fraction, an increase in homogeneous intramitochondrial areas, significant higher SOD activity and lower MDA levels in the group of old rats. Pretreatment with EGb 761 led to a significant decrease in MDA content in both control groups. Acute hypoxic stress increased the volume fractions of sarcoplasmatic reticulum, t-tubules, vacuoles, and lipid droplets, and caused mitochondrial swelling, with a more significant increase in degenerated and homogeneous intramitochondrial areas in the old group. SOD activity decreased only in the old hypoxic group; MDA content fell in both. Pretreatment with EGb 761 reduced ultrastructural-morphometric hypoxic damage in both groups, significantly below the levels of control. Young rat myocardium showed significantly higher SOD activity after hypoxia than untreated or older specimens. CONCLUSIONS: Better hypoxia tolerance is demonstrated by the young myocardium, and an obvious hypoxia-protective effect of EGb 761 in both age groups.

Neuropathological effect of carbamate molluscicides on the land snail, Eobania vermiculata.

The present study was designed to investigate the neuropathological effect of the two carbamate pesticides: methomyl and methiocarb on the neurons of the buccal ganglia in the land snail Eobania vermiculata using topical application and baiting technique. Their in vivo effects on acetylcholinesterase (AChE, EC 3.1.1.7) activity were also investigated. Sublethal dose and concentration (1/4 LD(50) and 1/4 LC(50)) of both pesticides were used, and the experiment lasted for 14 days. Histopathological and ultrastuctural alterations in the buccal ganglia were more obvious after the baiting technique treatment than after the topical application method, and methomyl was found to be more toxic than methiocarb. These alterations included shrinkage of the perikarya of neurons, increased cytoplasmic basophilia, and extreme indentation of the plasma membrane. In addition, the nuclei appeared karyolitic, eccentric, and highly shrunken with an irregular nuclear envelope. The most outstanding symptom observed after topical application of methiocarb was a highly vacuolated cytoplasm with a peripheral increase in electron density associated with dense accumulations of free ribosomes. On the other hand, an increased number of lysosomes and autophagosomes were observed after topical application of methomyl. Mitochondrial damage, increased number of lipid droplets, and myelin figures were frequently observed in ganglia treated with either methomyl or methiocarb. Moreover, it was noticed that both compounds induced reductions in AChE activity. However, methomyl exhibited more potency in reducing AChE activity than methiocarb.

Characterization of unusual families of ATG8-like proteins and ATG12 in the protozoan parasite Leishmania major.

Leishmania major possesses, apparently uniquely, four families of ATG8-like genes, designated ATG8, ATG8A, ATG8B and ATG8C, and 25 genes in total. L. major ATG8 and examples from the ATG8A, ATG8B and ATG8C families are able to complement a Saccharomyces cerevisiae ATG8-deficient strain, indicating functional conservation. Whereas ATG8 has been shown to form putative autophagosomes during differentiation and starvation of L. major, ATG8A primarily form puncta in response to starvation-suggesting a role for ATG8A in starvation-induced autophagy. Recombinant ATG8A was processed at the scissile glycine by recombinant ATG4.2 but not ATG4.1 cysteine peptidases of L. major and, consistent with this, ATG4.2-deficient L. major mutants were unable to process ATG8A and were less able to withstand starvation than wild-type cells. GFP-ATG8-containing puncta were less abundant in ATG4.2 overexpression lines, in which unlipidated ATG8 predominated, which is consistent with ATG4.2 being an ATG8-deconjugating enzyme as well as an ATG8A-processing enzyme. In contrast, recombinant ATG8, ATG8B and ATG8C were all processed by ATG4.1, but not by ATG4.2. ATG8B and ATG8C both have a distinct subcellular location close to the flagellar pocket, but the occurrence of the GFP-labeled puncta suggest that they do not have a role in autophagy. L. major genes encoding possible ATG5, ATG10 and ATG12 homologues were found to complement their respective S. cerevisiae mutants, and ATG12 localized in part to ATG8-containing puncta, suggestive of a functional ATG5-ATG12 conjugation pathway in the parasite. L. major ATG12 is unusual as it requires C-terminal processing by an as yet unidentified peptidase.

Zinc as a translation regulator in neurons: implications for P-body aggregation.

Post-transcriptional mechanisms of gene expression in neuronal cells include mRNA transport and local protein synthesis, which play a vital role in the control of polarity, synaptic plasticity and growth cone motility. RNA-binding proteins, which form the transported ribonucleoparticle (RNP), control mRNA stability and local translation. Recently, the existence of processing bodies (P-bodies), in which mRNA decapping and degradation take place, was revealed in neurons. It was suggested that P-bodies serve as a transient storage compartment for mRNAs, which can be released and, upon stimulation, resume translation. In this study, we focused on the localization of the Dcp1a protein, which serves as a P-body marker, in PC12 growth cones and P19 neuronal cells and its association with the tau mRNA-binding protein HuD. We found that stimulation of neurons by zinc, which is stored and released from synaptic vesicles, caused a disruption of polysomes into monosomes, whereas HuD protein distribution in sucrose gradient fractions remained unaffected. In addition, zinc application caused an aggregation of Dcp1a protein in an RNA-dependent manner. These findings suggest a role for zinc in translation regulation via disruption of polysomes, aggregation of P-bodies in neurons and impairment of the RNP-polysome interaction.