Molecular and functional characterization of a novel stefin analogue in large yellow croaker (Pseudosciaena crocea).

In this paper, we report the molecular cloning of a novel stefin analogue from the spleen of large yellow croaker Pseudosciaena crocea (Lycstefin). The open reading frame (ORF) of 297 nucleotides (nt) of Lycstefin encodes a protein of 99 amino acids (aa) with a putative molecular weight of 11kDa, in which no signal peptide and potential N-glycoslation site are predicted. The deduced Lycstefin possesses the structural features of the mammalian stefins, including two conserved motifs known to interact with the active sites of family C1 cysteine peptidases: one glycine in the N-terminal region (G(6)) and Gln-Xaa-Val-Xaa-Gly motif (Q(48)LVAG(52)). It shares 32-47.5% aa sequence identity to the sequences found in mammals and other fish species and is rich in cysteine residues (seven cysteines). Genomic analysis revealed that Lyccys gene, 757 nt long, consisted of three exons and two introns. The Lycstefin gene was constitutively expressed in various tissues examined although at different levels. Upon stimulation with poly(I:C) or inactivated trivalent bacterial vaccine, Lycstefin transcript was significantly up-regulated in spleen and head kidney while down-regulated in blood. Immuno-electron microscopy showed that Lycstefin was mainly localized in the cytoplasm of spleen cells of large yellow croaker, and also in the nucleus. Recombinant Lycstefin protein fused with glutathione S-transferase (rLycstefin) was shown to have strong inhibitory activity against papain with a K(i) of 1.3x10(-13)M. The in vivo experiments revealed that Lycstefin could not modulate the expression levels of large yellow croaker tumor necrosis factor-alpha2 (TNF-alpha2) and interleukin-10 in spleen and head kidney. To our knowledge, this is the first report on the molecular and functional identification of a stefin analogue in bony fish.

Mimicking proteasomal release of polyglutamine peptides initiates aggregation and toxicity.

Several neurodegenerative disorders, including Huntington's disease, are caused by expansion of the polyglutamine (polyQ) tract over 40 glutamines in the disease-related protein. Fragments of these proteins containing the expanded polyQ tract are thought to initiate aggregation and represent the toxic species. Although it is not clear how these toxic fragments are generated, in vitro data suggest that proteasomes are unable to digest polyQ tracts. To examine whether the resulting polyQ peptides could initiate aggregation in living cells, we mimicked proteasomal release of monomeric polyQ peptides. These peptides lack the commonly used starting methionine residue or any additional tag. Only expanded polyQ peptides seem to be peptidase resistant, and their accumulation initiated the aggregation process. As observed in polyQ disorders, these aggregates subsequently sequestered proteasomes, ubiquitin and polyQ proteins, and recruited Hsp70. The generated expanded polyQ peptides were toxic to neuronal cells. Our approach mimics proteasomal release of pure polyQ peptides in living cells, and represents a valuable tool to screen for proteins and compounds that affect aggregation and toxicity.

A new cell culture system for infection with hepatitis B virus that fuses HepG2 cells with primary human hepatocytes.

Hepatitis B virus (HBV) infection exhibits a very narrow host range and shows a strong tropism for liver parenchymal cells, however none of the previously established experimental models can reproduce the natural process of HBV infection. In the present study, primary human hepatocytes were fused with HepG2 cells to establish the hybrid HepCHLine-4 cell line with high susceptibility to HBV. The HepCHLine-4 cells expressed HBV-specific antigen when co-incubated with HBV-positive serum from a hepatitis B patient. Post-infection, HBV relaxed circular DNA and covalently closed circular DNA were detected in HepCHLine-4 cells using a nested polymerase chain reaction, and HBV-specific particles were visualized by electron microscopy of the culture media of HepCHLine-4 cells. HepG2 cells were not susceptible to HBV infection under the same conditions. The HepCHLine-4 cells can be sub-cultured for > 12 months while maintaining susceptibility to HBV and may, therefore, be useful for studying HBV infection and the viral life cycle in human hepatocytes.

Dilated intercellular spaces as a marker of GERD.

Gastroesophageal reflux disease (GERD) is typically heralded by the substernal burning pain of heartburn. On endoscopic examination, about one third of GERD subjects with heartburn have erosive disease, and the remainder have nonerosive reflux disease (NERD). Unlike patients with erosive disease, those with NERD (approximately 50%) often do not respond to therapy with proton pump inhibitors (PPIs), raising the question of whether they have NERD and, if they do, whether the cause of their symptoms is similar to those who respond to PPIs. Recently, biopsies established that subjects with heartburn and PPI-responsive NERD, like those with erosive esophagitis, have lesions within the esophageal epithelium known as dilated intercellular space (DIS). In this article, we discuss the physicochemical basis for DIS in acid-injured esophageal epithelium and its significance in GERD. Although DIS is not pathognomic of GERD, it is a marker of a break in the epithelial (junctional) barrier reflecting an increase in paracellular permeability.

Down-regulation of ZnT8 expression in INS-1 rat pancreatic beta cells reduces insulin content and glucose-inducible insulin secretion.

The SLC30A8 gene codes for a pancreatic beta-cell-expressed zinc transporter, ZnT8. A polymorphism in the SLC30A8 gene is associated with susceptibility to type 2 diabetes, although the molecular mechanism through which this phenotype is manifest is incompletely understood. Such polymorphisms may exert their effect via impacting expression level of the gene product. We used an shRNA-mediated approach to reproducibly downregulate ZnT8 mRNA expression by >90% in the INS-1 pancreatic beta cell line. The ZnT8-downregulated cells exhibited diminished uptake of exogenous zinc, as determined using the zinc-sensitive reporter dye, zinquin. ZnT8-downregulated cells showed reduced insulin content and decreased insulin secretion (expressed as percent of total insulin content) in response to hyperglycemic stimulus, as determined by insulin immunoassay. ZnT8-depleted cells also showed fewer dense-core vesicles via electron microscopy. These data indicate that reduced ZnT8 expression in cultured pancreatic beta cells gives rise to a reduced insulin response to hyperglycemia. In addition, although we provide no direct evidence, these data suggest that an SLC30A8 expression-level polymorphism could affect insulin secretion and the glycemic response in vivo.

Intracellular transport of human immunodeficiency virus type 1 genomic RNA and viral production are dependent on dynein motor function and late endosome positioning.

Our earlier work indicated that the human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) is trafficked to the microtubule-organizing center (MTOC) when heterogeneous nuclear ribonucleoprotein A2/B1 is depleted from cells. Also, Rab7-interacting lysosomal protein promoted dynein motor complex, late endosome and vRNA clustering at the MTOC suggesting that the dynein motor and late endosomes were involved in vRNA trafficking. To investigate the role of the dynein motor in vRNA trafficking, dynein motor function was disrupted by small interference RNA-mediated depletion of the dynein heavy chain or by p50/dynamitin overexpression. These treatments led to a marked relocalization of vRNA and viral structural protein Gag to the cell periphery with late endosomes and a severalfold increase in HIV-1 production. In contrast, rerouting vRNA to the MTOC reduced virus production. vRNA localization depended on Gag membrane association as shown using both myristoylation and Gag nucleocapsid domain proviral mutants. Furthermore, the cytoplasmic localization of vRNA and Gag was not attributable to intracellular or internalized endocytosed virus particles. Our results demonstrate that dynein motor function is important for regulating Gag and vRNA egress on endosomal membranes in the cytoplasm to directly impact on viral production.

Evidence that productive human immunodeficiency virus type 1 assembly can occur in an intracellular compartment.

Human immunodeficiency virus type 1 (HIV-1) assembly occurs predominantly at the plasma membrane of infected cells. The targeting of assembly to intracellular compartments such as multivesicular bodies (MVBs) generally leads to a significant reduction in virus release efficiency, suggesting that MVBs are a nonproductive site for HIV-1 assembly. In the current study, we make use of an HIV-1 Gag-matrix mutant, 29/31KE, that is MVB targeted. We previously showed that this mutant is severely defective for virus particle production in HeLa cells but more modestly affected in primary macrophages. To more broadly examine the consequences of MVB targeting for virus production, we investigated 29/31KE particle production in a range of cell types. Surprisingly, this mutant supported highly efficient assembly and release in T cells despite its striking MVB Gag localization. Manipulation of cellular endocytic pathways revealed that unlike Vpu-defective HIV-1, which demonstrated intracellular Gag localization as a result of Gag endocytosis from the plasma membrane, 29/31KE mutant Gag was targeted directly to an MVB compartment. The 29/31KE mutant was unable to support multiple-round replication; however, this defect could be reversed by truncating the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 and by the acquisition of a 16EK change in matrix. The 16EK/29/31KE matrix mutant replicated efficiently in the MT-4 T-cell line despite maintaining an MVB-targeting phenotype. These results indicate that MVB-targeted Gag can be efficiently released from T cells and primary macrophages, suggesting that under some circumstances, late endosomal compartments can serve as productive sites for HIV-1 assembly in these physiologically relevant cell types.

Platelet structural pathology in a patient with the X-linked GATA-1, R216Q mutation.

The ultrastructural pathology of GATA-1, V205M and G208S macrothrombocytes was discussed in earlier investigations. This study has used the same technology to evaluate macrothrombocytes from a patient with the GATA-1, R216Q mutation. Some of the pathological features observed in macrothrombocytes from patients with the V205M and G208S variations including hypo- and agranular platelets, tubular inclusions and platelets within platelets, as well as platelets within platelets within platelets were identified. However, tubular membrane sheets in megakaryocytes and platelets of the V205M and G208S types and large groups of platelets attached to platelets to form megathrombocytes were not observed. The unique pathology of the megathrombocytes from this patient was the near absence of dense bodies in his giant cells. Storage Pool Deficiency, together with large platelets, defective adhesion and aggregation of his macrocytes under shear stress to vWF and collagen and defective clot retraction may contribute to the pathogenesis of his bleeding disorder.

Salmeterol restores secretory functions in cystic fibrosis airway submucosal gland serous cells.

The activity of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) can be mediated by surface G protein-coupled receptors such as the beta(2)-adrenergic receptor. In this study, we explored the effect of a long-acting beta(2)-adrenergic agonist, salmeterol, on the CFTR-dependent secretory capacity of a human CF tracheal gland serous cell line (CF-KM4), homozygous for the delF508 mutation. We showed that, compared with the untreated CF serous cells, a 24-hour pre-incubation period with 200 nM salmeterol induced an 83% increase in delF508-CFTR-mediated chloride efflux. The restoration of the bioelectric properties is associated with increased apical surface pool of delF508-CFTR. Salmeterol induced a decrease in ion concentration and an increase in the level of hydration of the mucus packaged inside the CF secretory granules. The effects of salmeterol are not associated with a persistent production of cAMP. Western blotting on isolated secretory granules demonstrated immunoreactivity for CFTR and lysozyme. In parallel, we measured by atomic force microscopy an increased size of secretory granules isolated from CF serous cells compared with non-CF serous cells (MM39 cell line) and showed that salmeterol was able to restore a CF cell granule size similar to that of non-CF cells. To demonstrate that the salmeterol effect was a CFTR-dependent mechanism, we showed that the incubation of salmeterol-treated CF serous cells with CFTR-inh172 suppressed the restoration of normal secretory functions. The capacity of salmeterol to restore the secretory capacity of glandular serous cells suggests that it could also improve the airway mucociliary clearance in patients with CF.

Markedly increased intracellular lipid droplets and disruption of intercellular junctions in biopsied myocardium from a patient with arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by the progressive replacement of myocardial cells by fat and fibrous tissue. Here we describe the histopathological features of biopsied myocardium from a patient with ARVC. A large amount of adipose tissue was present in the biopsy specimen, and a group of myocardial cells were isolated as an island-like region in the adipose tissue. Electron microscopic examination of cardiomyocytes revealed a large number of intracellular lipid droplets, including some extremely large droplets. Disruptions of the plasma membrane and dissociation of intercellular junctions were associated with discharge of intracellular lipid droplets into the interstitial space. The high accumulation of intracellular lipid droplets may be involved in the pathogenesis of ARVC and may have played an important role in myocardial cell death and progressive replacement of cardiomyocytes by fatty tissue in the current case.

Toxoplasma gondii Tic20 is essential for apicoplast protein import.

Apicomplexan parasites harbor a secondary plastid that has lost the ability to photosynthesize yet is essential for the parasite to multiply and cause disease. Bioinformatic analyses predict that 5-10% of all proteins encoded in the parasite genome function within this organelle. However, the mechanisms and molecules that mediate import of such large numbers of cargo proteins across the four membranes surrounding the plastid remain elusive. In this work, we identify a highly diverged member of the Tic20 protein family in Apicomplexa. We demonstrate that Tic20 of Toxoplasma gondii is an integral protein of the innermost plastid membrane. We engineer a conditional null-mutant and show that TgTic20 is essential for parasite growth. To characterize this mutant functionally, we develop several independent biochemical import assays to reveal that loss of TgTic20 leads to severe impairment of apicoplast protein import followed by organelle loss and parasite death. TgTic20 is the first experimentally validated protein import factor identified in apicoplasts. Our studies provide experimental evidence for a common evolutionary origin of import mechanisms across the innermost membranes of primary and secondary plastids.

Probing cells with noble metal nanoparticle aggregates.

This review focuses on the integration of noble metal nanoparticle aggregates as tags and transport vessels in cellular applications. The natural tendency of nanoparticles to aggregate can be reduced through surface modification; however, this stabilization is often compromised in the cellular environment. The degree of nanoparticle aggregation has both positive and negative consequences. Nanoparticle aggregates are more efficiently removed by the organism compared with single nanoparticles, preventing delivery to their cellular target. In addition, these aggregates are recognized by cells in different ways versus isolated nanoparticles. Despite these negatives, aggregates exhibit enhancement for many detection and treatment techniques in comparison with single nanoparticles. In coming years, the role of aggregates and better control over the degree of aggregation in cellular studies will be required for the realization of medical applications.

Addressing variability in a Xenopus laevis melanophore cell line.

Xenopus laevis melanophores can be used in high-throughput screens for guanine nucleotide binding protein coupled receptor ligands and have potential as biosensors. Inherent in this immortal cell line is a substantial variability, which macroscopic evaluations disregard. Here we demonstrate a systematic way to incorporate this natural variability in the evaluations. Clusters of similar cells from a sparsely seeded cell culture are examined by imaging changes in cell appearance, pigment motility, and cumulative displacements. The time evolution of the image intensity distributions of clusters upon a pigment-dispersing stimulus is used as a signature of the cell clusters, and their behaviors are classified by multivariate analysis. Conventional image subtraction procedures are used to highlight cumulative and transitory changes in the pigment dynamics, enabling characterization of multiple aspects of the cell response from a single experiment. Additionally, a simple way to accomplish standard optical density changes at the single-cell group level is shown. The present results also provide evidence that natural cell variability arising from a cell culture can enrich the diversity of responses from pigment-containing cells assays and underscore that in conventional macroscopic evaluations these aspects are overlooked and can lead to spurious results.

Intracellular distribution of phototropin 1 protein in the short-day plant Ipomoea nil.

Phototropin 1 (phot1) is a blue-light Ser/Thr receptor kinase that contains two LOV domains. It is a plasma membrane-associated protein that mediates phototropism, blue-light induced chloroplast movement, and stomatal opening. The aim of the present work was to analyze the intracellular localization of phot1 protein in Ipomoea nil seedlings. In cotyledon and hypocotyl cells of etiolated seedlings, phot1 was specifically localized in the plasma membrane regions, whereas in light-treated seedlings, it was homogeneously distributed throughout the whole cytoplasm, excluding cell nuclei and vacuoles. Phot1 was also localized in cotyledon epidermal and guard cells. Such a localization pattern suggests a light-dependent intracellular distribution of phot1 in Ipomoea nil. On the basis of the spatial distribution, the possible role of phot1 is also discussed.

Morphological changes in intracellular lipid droplets induced by different hepatitis C virus genotype core sequences and relationship with steatosis.

UNLABELLED: Hepatocellular steatosis is common in patients with chronic hepatitis C. Steatosis can be considered as a true cytopathic lesion induced by hepatitis C virus (HCV) genotype 3, suggesting that one or more viral proteins produced during genotype 3 infection are involved in the steatogenic process, while the same proteins produced during infection by other genotypes are not. We examined in vitro interactions between lipid droplets and full-length core protein isolated from patients with HCV genotype 3a infection, with and without steatosis, and from steatosis-free patients infected by HCV genotype 1b. We also examined morphological changes in the lipid droplets according to the HCV genotype and the presence of steatosis in vivo. Core protein processing by signal peptide peptidase was not affected by sequence differences between the variants. We showed that the core protein of both HCV genotypes 1b and 3a binds tightly to the surface of intracellular lipid droplets. However, cells transfected with genotype 3a contain more neutral lipids in lipid droplets, and more large lipid droplets, than cells transfected with genotype 1b sequences. This suggests that HCV core protein-lipid droplet interaction could play a role in virus-induced steatosis. Importantly, we found no genetic or functional differences between genotype 3a core proteins from patients with and without HCV-induced steatosis. CONCLUSION: This suggests that other viral proteins and/or host factors are involved in the development of hepatocellular steatosis in patients infected by HCV genotype 3a.

The role of chronic infection in children with otitis media with effusion: evidence for intracellular persistence of bacteria.

OBJECTIVE: Demonstrate mucosal bacterial infection in children with otitis media with effusion (OME). STUDY DESIGN AND SETTING: Middle ear mucosal biopsies from 11 children with OME were examined for bacteria utilizing transmission electron microscopy. This was correlated with standard culture and polymerase chain reaction (PCR) of middle ear effusions. RESULTS: Gram-positive coccal bacteria were demonstrated in middle ear mucosal epithelial cells of 4 of 11 (36%) children. Morphological appearance of bacteria and detection of pneumolysin DNA by PCR in middle ear fluid suggests a role for persistent intracellular infection with Streptococcus pneumoniae and other gram-positive cocci in some cases of OME. CONCLUSION: Intracellular bacterial infection of middle ear mucosal epithelial cells in children with OME may be an important mechanism for bacterial persistence, and contribute to inflammation and mucus production in the pathogenesis of this condition. SIGNIFICANCE: Persistent intracellular infection is a novel paradigm for OME pathogenesis in children and may influence antibiotic effectiveness in treatment of this condition.

Electron tomography and immunonanogold electron microscopy for investigating intracellular trafficking and secretion in human eosinophils.

Electron tomography (ET) has increasingly been used to understand the complexity of membrane systems and protein-trafficking events. By ET and immunonanogold electron microscopy, we recently defined a route for vesicular transport and release of granule-stored products from within activated human eosinophils, cells specialized in the secretion of numerous cytokines and other proteins during inflammatory responses. Here, we highlight these techniques as important tools to unveil a distinct eosinophil vesicular system and secretory pathway.

A reliable method for cell phenotype image classification.

OBJECTIVE: Image-based approaches have proven to be of great utility in the automated cell phenotype classification, it is very important to develop a method that efficiently quantifies, distinguishes and classifies sub-cellular images. METHODS AND MATERIALS: In this work, the invariant locally binary patterns (LBP) are applied, for the first time, to the classification of protein sub-cellular localization images. They are tested on three image datasets (available for download), in conjunction with support vector machines (SVMs) and random subspace ensembles of neural networks. Our method based on invariant LBP provides higher accuracy than other well-known methods for feature extraction; moreover, our method does not require to (direct) crop the cells for the classification. RESULTS AND CONCLUSION: The experimental results show that the random subspace ensemble of neural networks outperforms the SVM in this problem. The proposed approach based on the solely LBP features gives accuracies of 85%, 93.9% and 88.4% on the 2D HeLa dataset, LOCATE endogenous and transfected datasets, respectively, and in combination with other state-of-the-art methods for the cell phenotype image classification we obtain a classification accuracy of 94.2%, 98.4% and 96.5%.

Intracellular organelle dynamics at nm resolution.

In the past 15 years, the dynamics of intracellular membrane-bound secretory vesicles ranging in size from 200 to 1200 nm in pancreatic acinar cells to 30-50 nm in neurons, have been extensively studied, providing for the first time the molecular process involved in vesicular discharge during cell secretion. Live pancreatic acinar cells in near physiological buffer, when imaged using the atomic force microscope (AFM), reveal at nanometer resolution the size of secretory vesicles called zymogen granules (ZGs) lying immediately below the surface of the apical plasma membrane. Within 2.5 min of exposure to a secretory stimulus, majority of ZGs within cells swell, followed by a decrease in ZG size, and a concomitant release of secretory products. These studies directly demonstrated intracellular swelling of secretory vesicles following stimulation of cell secretion in live cells, and vesicle deflation following partial discharge of vesicular contents. Furthermore, a direct estimation of vesicle size dynamics at nm resolution under various experimental conditions, have enabled the determination of the molecular mechanism of secretory vesicle swelling. Atomic force microscopy and photon correlation spectroscopy have been major players in these studies.

Active migration into the subcellular space precedes Campylobacter jejuni invasion of epithelial cells.

The bacterial pathogen Campylobacter jejuni invades mucosal cells via largely undefined and rather inefficient (0.01-2 bacteria per cell) mechanisms. Here we report a novel, highly efficient C. jejuni infection pathway resulting in 10-15 intracellular bacteria per cell within 3 h of infection. Electron microscopy, pulse-chase infection assays and time-lapse multiphoton laser confocal microscopy demonstrated that the mechanism involved active and rapid migration of the pathogen into the subcellular space (termed 'subvasion'), followed by bacterial entry ('invasion') at the cell basis. Efficient subvasion was maximal after repeated rounds of selection for the subvasive phenotype. Targeted mutagenesis indicated that the CadF, JlpA or PEB1 adhesins were not required. Dissection of the selected and parental phenotypes by SDS-PAGE yielded comparable capsule polysaccharide and lipooligosaccharide profiles. Proteomics revealed reduced amounts of the chemotaxis protein CheW for the subvasive phenotype. Swarming assays confirmed that the selected phenotype exhibited altered migration behaviour. Introduction of a plasmid carrying chemotaxis genes into the subvasive strain yielded wild-type subvasion levels and migration behaviour. These results indicate that alterations in the bacterial migration machinery enable C. jejuni to actively penetrate the subcellular space and gain access to the cell interior with unprecedented efficiency.