Secretory Components Research Articles

Mutations affecting the secretory COPII coat component SEC23B cause congenital dyserythropoietic anemia type II

Congenital dyserythropoietic anemias (CDAs) are phenotypically and genotypically heterogeneous diseases. CDA type II (CDAII) is the most frequent CDA. It is characterized by ineffective erythropoiesis and by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance. Other features of the peripheral red blood cells are protein and lipid dysglycosylation and endoplasmic reticulum double-membrane remnants. Development of other hematopoietic lineages is normal. Individuals with CDAII show progressive splenomegaly, gallstones and iron overload potentially with liver cirrhosis or cardiac failure. Here we show that the gene encoding the secretory COPII component SEC23B is mutated in CDAII. Short hairpin RNA (shRNA)-mediated suppression of SEC23B expression recapitulates the cytokinesis defect. Knockdown of zebrafish sec23b also leads to aberrant erythrocyte development. Our results provide in vivo evidence for SEC23B selectivity in erythroid differentiation and show that SEC23A and SEC23B, although highly related paralogous secretory COPII components, are nonredundant in erythrocyte maturation.

Protection by Anti-β-Glucan Antibodies Is Associated with Restricted β-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

Anti-β-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective β-glucan epitope(s) and protection mechanisms, we studied two anti-β-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model.Both mAbs bound to fungal cell surface and to the β1,3-β1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to β1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to β1,6- and β1,4-linked glucose sequences in addition to β1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells.Our study demonstrates that the isotype of anti-β-glucan antibodies may affect details of the β-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize β-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.

Using Transgenic Animal Models in Neuroendocrine Research

Transgenic animals are commonly employed to explore the function of individual proteins. Transgenic animal models include the mouse, the zebrafish, and the South African clawed toad Xenopus laevis. In contrast to mice and zebrafish, with Xenopus transgenesis DNA integration is mostly achieved in the one-cell stage. Moreover, Xenopus (as well as zebrafish) eggs are relatively large, the embryos are transparent, a large offspring is generated, and maintenance of the offspring is easy. In our transgenic studies in Xenopus, we focus on the well-characterized neuroendocrine melanotrope cells of the pituitary pars intermedia that are regulated during the process of adaptation of Xenopus to a changing environment. When the animal is placed on a black background, the melanotrope cells produce and process large amounts of the prohormone proopiomelanocortin (POMC). We apply stable melanotrope-specific transgenesis that is achieved by mixing a Xenopus POMC-promoter/transgene construct with sperm nuclei and injecting this mixture into unfertilized eggs. Since in the melanotrope cells the POMC promoter is much more active in black-adapted animals, the level of transgene expression can be manipulated by placing the animal on either a black or a white background. In this paper we review the possibilities of the Xenopus melanotrope-specific transgenic approach. Following a brief overview of the functioning of Xenopus melanotrope cells, stable melanotrope-specific transgenesis is discussed and our transgenic studies on brain-derived neurotrophic factor and secretory pathway components are described as examples of the transgenic approach in a physiological context and close to the in vivo situation.

Genomic analysis of Campylobacter fetus subspecies: identification of candidate virulence determinants and diagnostic assay targets

BackgroundCampylobacter fetus subspecies venerealis is the causative agent of bovine genital campylobacteriosis, asymptomatic in bulls the disease is spread to female cattle causing extensive reproductive loss. The microbiological and molecular differentiation of C. fetus subsp. venerealis from C. fetus subsp. fetus is extremely difficult. This study describes the analysis of the available C. fetus subsp. venerealis AZUL-94 strain genome (~75–80%) to identify elements exclusively found in C. fetus subsp. venerealis strains as potential diagnostic targets and the characterisation of subspecies virulence genes.ResultsEighty Kb of genomic sequence (22 contigs) was identified as unique to C. fetus subsp. venerealis AZUL-94 and consisted of type IV secretory pathway components, putative plasmid genes and hypothetical proteins. Of the 9 PCR assays developed to target C. fetus subsp. venerealis type IV secretion system genes, 4 of these were specific for C. fetus subsp. venerealis biovar venerealis and did not detect C. fetus subsp. venerealis biovar intermedius. Two assays were specific for C. fetus subsp. venerealis AZUL-94 strain, with a further single assay specific for the AZUL-94 strain and C. fetus subsp. venerealis biovar intermedius (and not the remaining C. fetus subsp. venerealis biovar venerealis strains tested). C. fetus subsp. fetus and C. fetus subsp. venerealis were found to share most common Campylobacter virulence factors such as SAP, chemotaxis, flagellar biosynthesis, 2-component systems and cytolethal distending toxin subunits (A, B, C). We did not however, identify in C. fetus the full complement of bacterial adherence candidates commonly found in other Campylobacter spp.ConclusionThe comparison of the available C. fetus subsp. venerealis genome sequence with the C. fetus subsp. fetus genome identified 80 kb of unique C. fetus subsp. venerealis AZUL94 sequence, with subsequent PCR confirmation demonstrating inconsistent amplification of these targets in all other C. fetus subsp. venerealis strains and biovars tested. The assays developed here highlight the complexity of targeting strain specific virulence genes for field studies for the molecular identification and epidemiology of C. fetus.

HIF1α Is Essential for Normal Intrauterine Differentiation of Alveolar Epithelium and Surfactant Production in the Newborn Lung of Mice

Neonatal respiratory distress syndrome (RDS) is mainly the result of perturbation in surfactant production and is a common complication seen in premature infants. Normal fetal lung development and alveolar cell differentiation is regulated by a network of transcription factors. Functional loss of any of these factors will alter the developmental program and impact surfactant production and normal gas exchange. During development, the fetus is exposed to varying oxygen concentrations and must be able to quickly adapt to these changes in order to survive. Hypoxia-inducible factor 1alpha (HIF1alpha) is the primary transcription factor that is responsible for regulating the cellular response to changes in oxygen tension and is essential for normal development. Its role in lung maturation is not well defined and to address this knowledge gap, a lung-specific HIF1alpha knock-out model has been developed. Loss of HIF1alpha early in lung development leads to pups that die within hours of parturition, exhibiting symptoms similar to RDS. Lungs from these pups display impaired alveolar epithelial differentiation and an almost complete loss of surfactant protein expression. Ultrastructural analysis of lungs from HIF1alpha deletion pups had high levels of glycogen, aberrant septal development, and decreased expression of several factors necessary for proper lung development, including HIF2alpha, beta-catenin, and vascular endothelial growth factor. These results suggest that HIF1alpha is essential for proper lung maturation and alteration in its normal signaling during premature delivery might explain the pathophysiology of neonatal RDS.

Differential expression of the regulated catecholamine secretory pathway in different hereditary forms of pheochromocytoma.

Pheochromocytomas in patients with von Hippel-Lindau (VHL) syndrome and multiple endocrine neoplasia type 2 (MEN 2) differ in the types and amounts of catecholamines produced and the resulting signs and symptoms. We hypothesized the presence of different processes of catecholamine release reflecting differential expression of components of the regulated secretory pathway among the two types of hereditary tumors. Differences in catecholamine secretion from tumors in patients with VHL syndrome (n = 47) and MEN 2 (n = 32) were examined using measurements of catecholamines in tumor tissue, urine, and plasma, the last of which was under baseline conditions in all subjects and in a subgroup of patients who received intravenous glucagon to provoke catecholamine release. Microarray and proteomics analyses, quantitative PCR, and Western blotting were used to assess expression of tumor tissue secretory pathway components. The rate constant for baseline catecholamine secretion was 20-fold higher in VHL than in MEN 2 tumors (0.359 +/- 0.094 vs. 0.018 +/- 0.009 day(-1)), but catecholamine release was responsive only to glucagon in MEN 2 tumors. Compared with tumors from MEN 2 patients, those from VHL patients were characterized by reduced expression of numerous components of the regulated secretory pathway (e.g., SNAP25, syntaxin, rabphilin 3A, annexin A7, calcium-dependent secretion activator). The mutation-dependent differences in expression of secretory pathway components indicate a more mature regulated secretory pathway in MEN 2 than VHL tumors. These data provide a unique mechanistic link to explain how variations in the molecular machinery governing exocytosis may contribute to clinical differences in the secretion of neurotransmitters or hormones and the subsequent presentation of a disease.

A study on the relationship between intraglandular arterial distribution and thyroid lobe shape: Implications for biotechnology of a bioartificial thyroid

We have recently hypothesized that structural and secretory components of the adult human thyroid gland maintain constant reciprocal and geometrical relationships, even if changes occur in the three-dimensional (3D) architecture of any of these elements. This means that thyroid morphology could be studied from the point of view of a 3D topology. As a consequence, we have investigated anatomical aspects that could support this assumption. In the present study, we show that the presence of a constant relationship can be demonstrated between the vascular arrangement of the gland, including the extension of intraglandular arterial fields, arterial anastomoses and arterial calibers, and the shape as well as volume of the thyroid. Specifically, a statistically significant difference has been found between the network amplitudes of the superior and inferior thyroid arteries in relation to either a conic or an ellipsoidal geometry of the thyroid lobe. In addition, a direct relationship has been implicated between the distribution of arterial anastomoses and the behavior of the lobe as a single hemodynamic unit. Finally, a statistically significant correlation has been observed between average arterial caliber of the inferior thyroid artery and thyroid volume. On the basis of these results, we propose a model of architectural assembly between stromal and parenchymal elements of the adult thyroid that might prove useful in designing a bioartificial gland ex situ. Potential clinical applications of this principle in regenerative medicine of other endocrine organs are highlighted.

Coupling Secretion with Transcription

Bacterial virulence factor secretion systems, which contribute to pathogenicity, are assembled when bacteria encounter host cells. Mycobacterium tuberculosis , which causes tuberculosis, produces a type VII secretion system (also called the ESX-1 protein secretion system). In a screen for ESX-1 secretion mutants, Raghavan et al . identified Rv3849 , which encodes a secreted protein that they named EspR for ESX-1 secreted protein regulator. In wild-type bacteria, EspR was secreted by the secretion system. However, mutants defective in EspR function exhibited not only lack of EspR secretion but also diminished secretion of other factors, such as ESAT-6. Thus, EspR was not only a substrate of the secretion system but also required for proper function of the ESX-1 system. Secondary structure modeling suggested that EspR had a helix-turn-helix DNA binding domain and in the Rv3849 − cells three operons were activated and two operons were less active, including one Rv3616c - Rv3612c that encodes proteins that are necessary for the ESX-1 system. After phagocytosis by macrophages, Rv3849 − cells exhibited decreased induction of the Rv3616c - Rv3612c operon. Expression of M. tuberculosis EspR in a nonpathogenic relative that also expressed a Rv3616c promoter reporter showed that EspR stimulated transcription, and electrophoretic mobility shift assays confirmed that EspR bound the Rv3616c promoter. Forced expression of Rv3616c - Rv3612c using a constitutive promoter restored ESX-1 function in EspR-deficient bacteria, suggesting that the transcriptional activity is the relevant mechanism by which EspR contributes to ESX-1 secretion. Addition of an epitope tag to EspR blocked its secretion without compromising its transcriptional activity, and expression of the tagged EspR resulted in enhanced repression of EspR-inhibited genes and enhanced expression of EspR-activated genes. Thus, secretion of EspR appears to serve as a homeostatic mechanism to couple secretion with transcription of the genes encoding secretory system components. S. Raghavan, P. Manzanillo, K. Chan, C. Dovey, J. S. Cox, Secreted transcription factor controls Mycobacterium tuberculosis virulence. Nature 454 , 717-721 (2008). [PubMed]

Large dense-core vesicles in rat adrenal after reserpine: levels of mRNAs of soluble and membrane-bound constituents in chromaffin and ganglion cells indicate a biosynthesis of vesicles with higher secretory quanta.

Rats were injected with a large dose of reserpine known to stimulate the adrenal medulla. Various times after drug treatment the mRNA levels of several constituents of large dense-core vesicles were determined by northern blot analysis and in situ hybridization. The latter method allowed detection of changes in mRNA levels not only in chromaffin cells, but also in the ganglion cells found in adrenal medulla. Levels of the mRNAs of secretory components of large dense-core vesicles (chromogranins A and B, secretogranin II, VGF, and neuropeptide Y) increased in chromaffin cells by 215-857% after 1-3 days of drug treatment. For partly membrane-bound components (dopamine beta-hydroxylase, prohormone convertase 2, carboxypeptidase H, and peptidylglycine alpha-amidating monooxygenase) the changes ranged from 182 to 315%, whereas for glycoprotein III and for intrinsic membrane proteins (cytochrome b561 and vesicle monoamine transporter 2) no change occurred. In ganglion cells the mRNAs that could be detected for VGF, neuropeptide Y, secretogranin II, carboxypeptidase H, and vesicle monoamine transporter 1 showed an analogous pattern of change, with significant increases for the secretory proteins and no change for the membrane components. From these and previous results we suggest the following concept: Long-lasting stimulation of chromaffin cells or neurons does not induce the biosynthesis of a larger number of vesicles but rather leads to the formation of vesicles containing higher secretory quanta of chromogranins and neuropeptides.

The Immunology of Immediate and Delayed Hypersensitivity Reaction to Gluten

The immunology of gluten hypersensitivity and celiac disease has been pursued with significant interest in the past 20 years. For the prevention of systemic diseases, most pathogens that gain entry into our bodies must be met with an effective immune response, yet in the gastrointestinal tract it is equally important that commensal bacteria and a diverse collection of dietary proteins and peptides be recognized without eliciting an active immune response or constant activation of the inflammatory pathway. This phenomenon of hyporesponsiveness to food antigens is known as oral tolerance. This oral tolerance to dietary antigens is maintained by three different mechanisms: anergy, cell deletion and immune suppression. However, in the presence of mechanical/chemical stressors and infections, this tolerance may break down, and gut associated lymphoid tissues (GALT) will react to different luminal antigens. The reaction of GALT to these antigens may lead to the production of pro-inflammatory cytokines, opening of tight junctions, entry of undigested antigens into the circulation, and the subsequent production of IgA, IgG, IgM and IgE antibodies in blood and secretory components. Like any other food hypersensitivity reaction, gluten sensitivity can be divided into immediate and delayed hypersensitivities. In this review an attempt is made first to differentiate immediate hypersensitivity to gliadin, mediated by IgE, from delayed hypersensitivity, which is mediated by IgA and IgG. Furthermore, we attempt to differentiate between gluten hypersensitivity with enteropathy (celiac disease) and gluten hypersensitivity without enteropathy.

A flax fibre proteome: identification of proteins enriched in bast fibres.

BackgroundBast fibres from the phloem tissues of flax are scientifically interesting and economically useful due in part to a dynamic system of secondary cell wall deposition. To better understand the molecular mechanisms underlying the process of cell wall development in flax, we extracted proteins from individually dissected phloem fibres (i.e. individual cells) at an early stage of secondary cell wall development, and compared these extracts to protein extracts from surrounding, non-fibre cells of the cortex, using fluorescent (DiGE) labels and 2D-gel electrophoresis, with identities assigned to some proteins by mass spectrometry.ResultsThe abundance of many proteins in fibres was notably different from the surrounding non-fibre cells of the cortex, with approximately 13% of the 1,850 detectable spots being significantly (> 1.5 fold, p ≤ 0.05) enriched in fibres. Following mass spectrometry, we assigned identity to 114 spots, of which 51 were significantly enriched in fibres. We observed that a K+ channel subunit, annexins, porins, secretory pathway components, β-amylase, β-galactosidase and pectin and galactan biosynthetic enzymes were among the most highly enriched proteins detected in developing flax fibres, with many of these proteins showing electrophoretic patterns consistent with post-translational modifications.ConclusionThe fibre-enriched proteins we identified are consistent with the dynamic process of secondary wall deposition previously suggested by histological and biochemical analyses, and particularly the importance of galactans and the secretory pathway in this process. The apparent abundance of β-amylase suggests that starch may be an unappreciated source of materials for cell wall biogenesis in flax bast fibres. Furthermore, our observations confirm previous reports that correlate accumulation proteins such as annexins, and specific heat shock proteins with secondary cell wall deposition.

Transient potential receptor channel 4 controls thrombospondin‐1 secretion and angiogenesis in renal cell carcinoma

Angiogenic switch in renal cell carcinoma (RCC) is attributed to the inactivation of the von Hippel-Lindau tumor suppressor, stabilization of hypoxia inducible factor-1 transcription factor and increased vascular endothelial growth factor. To evaluate the role of an angiogenesis inhibitor, thrombopsondin-1 (TSP1), we compared TSP1 production in human RCC and normal tissue and secretion by the normal renal epithelium (human normal kidney, HNK) and RCC cells. Normal and RCC tissues stained positive for TSP1, and the levels of TSP1 mRNA and total protein were similar in RCC and HNK cells. However, HNK cells secreted high TSP1, which rendered them nonangiogenic, whereas RCC cells secreted little TSP1 and were angiogenic. Western blot and immunostaining revealed TSP1 in the cytoplasm of RCC cells on serum withdrawal, whereas, in HNK cells, it was rapidly exported. Seeking mechanisms of defective TSP1 secretion, we discovered impaired calcium uptake by RCC in response to vascular endothelial growth factor. In HNK cells, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, a calcium chelator, simulated TSP1 retention, mimicking the RCC phenotype. Further analysis revealed a profound decrease in transient receptor potential canonical ion channel 4 (TRPC4) Ca(2+) channel expression in RCC cells. TRPC4 silencing in HNK cells caused TSP1 retention and impaired secretion. Double labeling of the secretory system components revealed TSP1 colocalization with coatomer protein II (COPII) anterograde vesicles in HNK cells. In contrast, in RCC cells, TSP1 colocalized with COPI vesicles, pointing to the retrograde transport to the endoplasmic reticulum caused by misfolding. Our study indicates that TRPC4 loss in RCC leads to impaired Ca(2+) intake, misfolding, retrograde transport and diminished secretion of antiangiogenic TSP1, thus enabling angiogenic switch during RCC progression.

Rab6 and the secretory pathway affect oocyte polarity inDrosophila

The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. Here, we describe a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. We found that Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. We also provide evidence that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGFalpha homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. Our results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport.

Linking division and growth

Aprotein that pushes forward the cell cycle coordinates division with the ensuing growth of the cell surface, according to work from Derek McCusker, Douglas Kellogg (University of California, Santa Cruz, CA), and colleagues. Figure 1 Vesicles delivering new surface materials in the growing bud are missing (right) when Cdk1 activity is blocked. It's well accepted that progress through the cell cycle depends on cell growth. In contrast, says Kellogg, “general thinking held that growth is continuous and independent of the cell cycle.” But McCusker et al. now show that Cdk1, which triggers the G1 to S phase transition in budding yeast, also directly promotes the bud's growth. Bud growth quickly stalled upon blocking Cdk1 activity. The stall stemmed from a loss of polarized secretion. Normal cells placed secretory components such as vesicle tethering proteins and a myosin motor at the growing bud tip, but these proteins were rapidly delocalized when Cdk1 was inhibited. The group then sought targets of Cdk1 that regulated cell surface growth. Several proteins that depended on Cdk1 for phosphorylation during bud growth were found in a complex that included a guanine nucleotide exchange factor and an activating protein for the Cdc42 GTPase, which is required for polarized cell growth. How phosphorylation of proteins in this complex leads to new bud membrane addition is not yet clear. The complex might orient the actin cytoskeleton in the right direction for secretion, although at least one affected secretory protein did not require actin for its polarization. Reference: McCusker, D., et al. 2007. Nat. Cell Biol. doi: 10.1038/ncb1568 [PubMed] [Cross Ref]

Proteomics of Neuroendocrine Secretory Vesicles Reveal Distinct Functional Systems for Biosynthesis and Exocytosis of Peptide Hormones and Neurotransmitters

Regulated secretory vesicles produce, store, and secrete active peptide hormones and neurotransmitters that function in cell-cell communication. To gain knowledge of the protein systems involved in such secretory vesicle functions, we analyzed proteins in the soluble and membrane fractions of dense core secretory vesicles purified from neuroendocrine chromaffin cells. Soluble and membrane fractions of these vesicles were subjected to SDS-PAGE separation, and proteins from systematically sectioned gel lanes were identified by microcapillary LC-MS/MS (microLC-MS/MS) of tryptic peptides. The identified proteins revealed functional categories of prohormones, proteases, catecholamine neurotransmitter metabolism, protein folding, redox regulation, ATPases, calcium regulation, signaling components, exocytotic mechanisms, and related functions. Several novel secretory vesicle components involved in proteolysis were identified consisting of cathepsin B, cathepsin D, cystatin C, ubiquitin, and TIMP, as well carboxypeptidase E/H and proprotein convertases that are known to participate in prohormone processing. Significantly, the membrane fraction exclusively contained an extensive number of GTP nucleotide-binding proteins related to Rab, Rho, and Ras signaling molecules, together with SNARE-related proteins and annexins that are involved in trafficking and exocytosis of secretory vesicle components. Membranes also preferentially contained ATPases that regulate proton translocation. These results implicate membrane-specific functions for signaling and exocytosis that allow these secretory vesicles to produce, store, and secrete active peptide hormones and neurotransmitters released from adrenal medulla for the control of physiological functions in health and disease. In summary, this proteomic study illustrates secretory vesicle protein systems utilized for the production and secretion of regulatory factors that control neuroendocrine functions.

Heat labile secretory proteins released by Naegleria fowleri induces apoptotic cell death of Jurkat T cells (51.13)

Abstract Naegleria fowleri, a free-living amoeba of soil, ponds, andfresh water, causes primary amoebic meningoencephalitis in humansand other mammals. Upon invasion in vivo, N. fowleri is capable of destructing or killing host cells by cytolysis. However, it is unknown on the mechanism by which N. fowleri induces host cell death. In this study, we investigated the mechanism of host cell death induced by N. fowleri. Incubation of Jurkat T cells with live trophozoites of N. fowleri resulted in cell death, as measured by LDH release assay or propidium iodide staining. In addition, N. fowleri induced DNA fragmentation in Jurkat cells in a time and dose-dependent manner. N. fowleri caused cleavage of calpain, caspase-3, and cytoskeletal proteins such as vinculin and paxillin. Pretreatment of cells with NADPH oxidase inhibitor DPI inhibited N. foweleri-induced apoptosis, whereas calpain inhibitor calpeptin or pan-caspase inhibitor z-VAD-fmk did not. When we incubated Jurkat cells with N. fowleri in a Transwell chamber (pore size 0.4 μm), comparable level of cell death rate was found compared to control without chamber, suggesting an important role of secretory factors in N. foweleri-induced cell death. Moreover, secretroy components released by N. fowleri induced host cell death, and heat treatment abolished host cell death by secretory components. These results suggest that heat labile proteins secreted by N. fowleri induce apoptotic cell death in Jurkat T cells.

Transcriptional regulation by protein kinase A in Cryptococcus neoformans.

A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5′-monophosphate (cAMP)–dependent protein kinase A (PKA) is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential targets for anti-cryptococcal therapy.

Secretory Antibody Formation: Conserved Binding Interactions between J Chain and Polymeric Ig Receptor from Humans and Amphibians

Abs of the secretory Ig (SIg) system reinforce numerous innate defense mechanisms to protect the mucosal surfaces against microbial penetration. SIgs are generated by a unique cooperation between two distinct cell types: plasma cells that produce polymers of IgA or IgM (collectively called pIgs) and polymeric Ig receptor (pIgR)-expressing secretory epithelial cells that mediate export of the pIgs to the lumen. Apical delivery of SIgs occurs by cleavage of the pIgR to release its extracellular part as a pIg-bound secretory component, whereas free secretory components are derived from an unoccupied receptor. The joining chain (J chain) is crucial in pIg/SIg formation because it serves to polymerize Igs and endows them with a binding site for the pIgR. In this study, we show that the J chain from divergent tetrapods including mammals, birds, and amphibians efficiently induced polymerization of human IgA, whereas the J chain from nurse shark (a lower vertebrate) did not. Correctly assembled polymers showed high affinity to human pIgR. Sequence analysis of the J chain identified two regions, conserved only in tetrapods, which by mutational analysis were found essential for pIgA-pIgR complexing. Furthermore, we isolated and characterized pIgR from the amphibian Xenopus laevis and demonstrated that its pIg binding domain showed high affinity to human pIgA. These results showed that the functional site of interaction between pIgR, J chain and Ig H chains is conserved in these species and suggests that SIgs originated in an ancestor common to tetrapods.

Phyllodes breast tumour showing invasive squamous-cell carcinoma with invasive ductal, clear-cell, secretory, and squamous components

A 69-year-old woman was found to have a complex carcinoma arising from a phyllodes tumour. Histological examination showed parts of the mass consisting of benign phyllodes tumour (yellow arrow), with most of the tissue consisting of an invasive squamous carcinoma (dark blue arrow) as well as a complex component of invasive ductal carcinoma with clear-cell (red arrow), secretory (green arrow), and squamous (light blue arrow) components. The term phyllodes is derived from the Greek word phyllon (meaning leaf), and refers to rapidly growing, large breast tumours that can cause breast distortion and pressure necrosis of overlying skin and seem leaf-like in cross-section.

A role for serglycin proteoglycan in granular retention and processing of mast cell secretory granule components

In the absence of serglycin proteoglycans, connective tissue-type mast cells fail to assemble mature metachromatic secretory granules, and this is accompanied by a markedly reduced ability to store neutral proteases. However, the mechanisms behind these phenomena are not known. In this study, we addressed these issues by studying the functionality and morphology of secretory granules as well as the fate of the secretory granule proteases in bone marrow-derived mast cells from serglycin(+/+) and serglycin(-/-) mice. We show that functional secretory vesicles are formed in both the presence and absence of serglycin, but that dense core formation is defective in serglycin(-/-) mast cell granules. The low levels of mast cell proteases present in serglycin(-/-) cells had a granular location, as judged by immunohistochemistry, and were released following exposure to calcium ionophore, indicating that they were correctly targeted into secretory granules even in the absence of serglycin. In the absence of serglycin, the fates of the serglycin-dependent proteases differed, including preferential degradation, exocytosis or defective intracellular processing. In contrast, beta-hexosaminidase storage and release was not dependent on serglycin. Together, these findings indicate that the reduced amounts of neutral proteases in the absence of serglycin is not caused by missorting into the constitutive pathway of secretion, but rather that serglycin may be involved in the retention of the proteases after their entry into secretory vesicles.