Golgi enzymes are enriched in perforated zones of golgi cisternae but are depleted in COPI vesicles

In the most widely accepted version of the cisternal maturation/progression model of intra-Golgi transport, the polarity of the Golgi complex is maintained by retrograde transport of Golgi enzymes in COPI-coated vesicles. By analyzing enzyme localization in relation to the three-dimensional ultrastructure of the Golgi complex, we now observe that Golgi enzymes are depleted in COPI-coated buds and 50- to 60-nm COPI-dependent vesicles in a variety of different cell types. Instead, we find that Golgi enzymes are concentrated in the perforated zones of cisternal rims both in vivo and in a cell-free system. This lateral segregation of Golgi enzymes is detectable in some stacks during steady-state transport, but it was significantly prominent after blocking endoplasmic reticulum-to-Golgi transport. Delivery of transport carriers to the Golgi after the release of a transport block leads to a diminution in Golgi enzyme concentrations in perforated zones of cisternae. The exclusion of Golgi enzymes from COPI vesicles and their transport-dependent accumulation in perforated zones argues against the current vesicle-mediated version of the cisternal maturation/progression model.     

Modulation of SF3B1 in the pre-mRNA spliceosome induces a RIG-I-dependent type I IFN response

Nucleic acid–sensing pathways play critical roles in innate immune activation through the production of type I interferon (IFN-I) and proinflammatory cytokines. These factors are required for effective antitumor immune responses. Pharmacological modulators of the pre-mRNA spliceosome splicing factor 3b subunit 1 (SF3B1) are under clinical investigation as cancer cytotoxic agents. However, potential roles of these agents in aberrant RNA generation and subsequent RNA-sensing pathway activation have not been studied. In this study, we observed that SF3B1 pharmacological modulation using pladienolide B (Plad B) induces production of aberrant RNA species and robust IFN-I responses via engagement of the dsRNA sensor retinoic acid–inducible gene I (RIG-I) and downstream interferon regulatory factor 3. We found that Plad B synergized with canonical RIG-I agonism to induce the IFN-I response. In addition, Plad B induced NF-κB responses and secretion of proinflammatory cytokines and chemokines. Finally, we showed that cancer cells bearing the hotspot SF3B1^K700E mutation, which leads to global aberrant splicing, had enhanced IFN-I response to canonical RIG-I agonism. Together, these results demonstrate that pharmacological modulation of SF3B1 in cancer cells can induce an enhanced IFN-I response dependent on RIG-I expression. The study suggests that spliceosome modulation may not only induce direct cancer cell cytotoxicity but also initiate an innate immune response via activation of RNA-sensing pathways.     

Clinical variation in X-linked adrenoleukodystrophy: fatty acid and lipid metabolism in cultured fibroblasts

To determine whether the clinical phenotype of ALD correlates with the extent of metabolic abnormality, we investigated VLFA metabolism in cultured fibroblasts from patients with the clinically severe childhood from of ALD and the milder AMN variant. No differences were seen in the content of neutral lipids or phospholipids, in incorporation of [1-14C]lignocerate into cellular lipids, or in the fatty acid composition of fibroblasts from patients with childhood ALD or AMN. [1-14C]Lignocerate oxidation was deficient to a similar extent (35-40% of normal) in both intact fibroblasts and cell homogenates from patients with childhood ALD and AMN. With the use of fibroblast homogenates, oxidation of lignocerate was partially inhibited by various long-chain fatty acids, and residual activity in ALD homogenates was more susceptible to inhibition by palmitate than normal. In the presence of competing palmitate, residual lignocerate oxidative activity in fibroblast homogenates was reduced to 20 +/- 4% of normal in childhood ALD and 24 +/- 2% of normal in AMN. These results indicate that residual VLFA oxidative activity, fatty acid composition, VLFA metabolism, and lipid content of cultured fibroblasts do not correlate with the clinical expression of the ALD gene.     

Barley hordothionin accumulates in transgenic oat seeds and purified protein retains anti-fungal properties in vitro

Summary Transgenic anti-fungal gene expression in heterologous species provides a means to test resistance protein combinations across species barriers. This is the first report of transgenic anti-fungal seed storage protein accumulation in oat seed. An anti-fungal barley (Hordeum vulgare L.) hordothionin (Hthl) gene was genetically engineered into oat (Avena sativa L.) to determine the effect of hordothionin on pathogen resistance. The transgene was expressed in both leaf and seed tissue, with transgenic protein accumulation occurring only in the seed. Transgenic oat line HTH-Av5 expressed c. 94 μg HTH/g seed, 19% of native barley seed levels. The anti-fungal activities of HTH fractions from barley cv. Morex and oat (transgenic and control) were tested in an in vitro growth assay against an important small grain pathogen. Fusarium graminearum. The partially purified HTH fractions from control oat seeds did not inhibit fungal growth, while HPLC-purified HTH positive control, as well as partially purified barley and transgenic oat HTH inhibited growth similarly over a range of concentrations. These results indicate hordothionin can be expressed in a heterologous cereal species and still maintain its anti-fungal properties. Future studies with HTH targeted to additional tissues are planned to test for increased fungal resistance. The University of Wisconsin and the USDA neither guarantee nor warrant the standard of the products named herein, and the use of the name by University of Wisconsin or USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Isolation and characterization of Oct-4+/HLA-G+ mesenchymal stem cells from human umbilical cord matrix: differentiation potential and detection of new markers

The presence of multipotent cells in several adult and embryo-related tissues opened new paths for their use in regenerative medicine. Extraembryonic tissues such as umbilical cord are considered a promising source of stem cells, potentially useful in therapy. The characterization of cells from the umbilical cord matrix (Wharton’s Jelly) and amniotic membrane revealed the presence of a population of mesenchymal-like cells, sharing a set of core-markers expressed by “mesenchymal stem cells”. Several reports enlightened the differentiation capabilities of these cells, even if at times the lack of an extensive characterization of surface markers and immune co-stimulators expression revealed hidden pitfalls when in vivo transplantation was performed. The present work describes a novel isolation protocol for obtaining mesenchymal stem cells from the umbilical cord matrix. These cells are clonogenic, retain long telomeres, can undergo several population doublings in vitro, and can be differentiated in mature mesenchymal tissues as bone and adipose. We describe for the first time that these cells, besides expressing all of the core-markers for mesenchymal stem cells, feature also the expression, at both protein and mRNA level, of tolerogenic molecules and markers of all the three main lineages, potentially important for both their differentiative potential as well as immunological features. G. La Rocca and R. Anzalone have contributed equally to this work.     

Improved NMR experiments with 13C-isotropic mixing for assignment of aromatic and aliphatic side chains in labeled proteins

Three improved ¹³C-spinlock experiments for side chain assignments of isotope labelled proteins in liquid state are presented. These are based on wide bandwidth spinlock techniques that have become possible with contemporary cryogenic probes. The first application, the H(C^aliC^aro)H-TOCSY, is an HCCH-TOCSY in which all CH_n moieties of a protein are detected in a single experiment, including the aromatic ones. This enables unambiguous assignment of aromatic and aliphatic amino acids in a single, highly sensitive experiment. In the second application, the ¹³C-detected C^all-TOCSY, magnetization transfer comprises all carbons—aliphatic, aromatic as well as the carbonyl carbons—making the complete carbon assignment possible using one spectrum only. Thirdly, the frequently used HC(CCO)NH experiment was redesigned by replacing the long C-carbonyl refocused INEPT transfer step by direct ¹³C–¹³C-TOCSY magnetization transfer from side chain carbons to the backbone carbonyls. The resulting HC(CCO)NH experiment minimizes relaxation losses because it is shorter and represents a more sensitive alternative particularly for larger proteins. The performance of the experiments is demonstrated on isotope labeled proteins up to the size of 43 kDa.     

Cytokine Release Assays as Tests for Exposure to Leishmania,and for Confirming Cure from Leishmaniasis,in Solid Organ Transplant Recipients

Spain has one of the world’s largest pools of organ donors and is a global leader in terms of the number of transplants it performs. The current outbreak of leishmaniasis in Fuenlabrada (in the southwest of the region of Madrid, Spain) has involved 600 clinical cases since late 2009 (prevalence 0.2%). It may therefore be wise to monitor the town’s transplanted population for Leishmania infantum; its members are immunosuppressed and at greater risk of infection and relapse following treatment. The present work examines the use of cytokine release assays to determine the prevalence of Leishmania infection in this population, and to confirm recovery following treatment for visceral leishmaniasis (VL). The humoral and cellular immune responses to L. infantum were characterized in 63 solid organ transplant (SOT) recipients from Fuenlabrada, 57 of whom reported no previous episode of VL (NVL subjects), and six of whom had been cured of VL (CVL subjects). Seventeen subjects (12 NVL and 5 CVL) showed a patent lymphoproliferative response to soluble Leishmania antigen (SLA). Stimulation of peripheral blood mononuclear cell cultures and of whole blood with SLA led to the production of different combinations of cytokines that might serve to confirm Leishmania infection or recovery from VL and help prevent cured patients from relapsing into this serious condition.     

The twin arginine protein transport pathway exports multiple virulence proteins in the plant pathogen Streptomyces scabies

Streptomyces scabies is one of a group of organisms that causes the economically important disease potato scab. Analysis of the S. scabies genome sequence indicates that it is likely to secrete many proteins via the twin arginine protein transport (Tat) pathway, including several proteins whose coding sequences may have been acquired through horizontal gene transfer and share a common ancestor with proteins in other plant pathogens. Inactivation of the S. scabies Tat pathway resulted in pleiotropic phenotypes including slower growth rate and increased permeability of the cell envelope. Comparison of the extracellular proteome of the wild type and ΔtatC strains identified 73 predicted secretory proteins that were present in reduced amounts in the tatC mutant strain, and 47 Tat substrates were verified using a Tat reporter assay. The ΔtatC strain was almost completely avirulent on Arabidopsis seedlings and was delayed in attaching to the root tip relative to the wild‐type strain. Genes encoding 14 candidate Tat substrates were individually inactivated, and seven of these mutants were reduced in virulence compared with the wild‐type strain. We conclude that the Tat pathway secretes multiple proteins that are required for full virulence.     

<Emphasis Type="Italic">Membraniporopsis tubigera</Emphasis>, an invasive bryozoan in sandy beaches of southern Brazil and Uruguay

The massive irruption of the invasive bryozoan Membraniporopsis tubigera (Osburn) in sandy beaches of southern Brazil and Uruguay is reported. The species, originally described from Puerto Rico, Texas and Florida, has also been recorded for Brazilian beaches from 21°S to 26°S as well as for harbours of Australia, New Zealand and the Sea of Japan. The southward spreading rate of this bryozoan along the Brazilian and Uruguayan coasts can be estimated in approximately 183–195 km year⁻¹. The chances that this invasion could proceed southwards in the Southwest Atlantic and the possible impacts that it may be causing are discussed. The case of M. tubigera seems to be qualitatively and quantitatively different from those of other alien bryozoans previously recorded for this region, since it appeared massively in exposed sandy beaches, a habitat regarded to date as apparently free from the pervasive ecological impact of invasion by exotic species in the Southwest Atlantic.