Bioinspired carbide-derived carbons with hierarchical pore structure for the adsorptive removal of mercury from aqueous solution.

Biosilica of the diatom species Thalassiosira pseudonana is used as hard template for the synthesis of silicon carbide-derived carbons. The typical species-specific macroporous structure is retained during the nanocasting-chlorine treatment process and the resulting materials exhibit very high specific surface areas up to 2300 m2 g-1. Bioinspired carbons show very high capacities in mercury adsorption from aqueous solution compared to reference materials.

Inflammasome-dependent IL-1ß release depends upon membrane permeabilisation.

Interleukin-1ß (IL-1ß) is a critical regulator of the inflammatory response. IL-1ß is not secreted through the conventional ER-Golgi route of protein secretion, and to date its mechanism of release has been unknown. Crucially, its secretion depends upon the processing of a precursor form following the activation of the multimolecular inflammasome complex. Using a novel and reversible pharmacological inhibitor of the IL-1ß release process, in combination with biochemical, biophysical, and real-time single-cell confocal microscopy with macrophage cells expressing Venus-labelled IL-1ß, we have discovered that the secretion of IL-1ß after inflammasome activation requires membrane permeabilisation, and occurs in parallel with the death of the secreting cell. Thus, in macrophages the release of IL-1ß in response to inflammasome activation appears to be a secretory process independent of nonspecific leakage of proteins during cell death. The mechanism of membrane permeabilisation leading to IL-1ß release is distinct from the unconventional secretory mechanism employed by its structural homologues fibroblast growth factor 2 (FGF2) or IL-1α, a process that involves the formation of membrane pores but does not result in cell death. These discoveries reveal key processes at the initiation of an inflammatory response and deliver new insights into the mechanisms of protein release.

[Care counselling - the client's expectations]. / Pflegeberatung - die Erwartungen der Betroffenen.

The amendment of legal care consultations in the context of the long-term care insurance law (2008) has broadened recent consulting practice within the action range of the nursing care insurance in Germany. The informational needs and consulting requests of the clients were not investigated so far. Our aim was to examine information needs and consulting requests of those in need of care and their informal carers.The consulting requests of visitors of 2 open citizen events were documented by the use of a semi-structured questionnaire. Content analysis following Mayring (2008) was used for data analysis.158 consulting discussions were documented, from which 177 consulting requests were formed. The consulting requests can be divided in 4 main categories: (1) inquiry about the care system [56/32%], (2) inquiry about individual access to care offers [43/24%], (3) inquiry about regional care suppliers [43/24%], (4) situation- and disease-specific inquiries [35/20%].Inquiries about local suppliers of care and situation- and disease-specific inquiries outweigh the number of inquiries about the care system in general. Furthermore, our results show that the informational needs of those in need of care do not only refer to the scope of care insurance law, but to additional social security codes.

[Structures of long-term care facilities: a study in nursing homes in Leipzig]. / Versorgungsstrukturen in stationären Pflegeeinrichtungen: Eine Untersuchung in Leipziger Pflegeheimen.

BACKGROUND: In order to provide guidelines for the organization and design of nursing homes, it is important to evaluate current care facilities. The aim of the study was to describe the structures of nursing homes in the German city of Leipzig. MATERIALS AND METHODS: As part of a cross-sectional design, 47 nursing homes participated in a written survey. RESULTS: The majority of nursing homes focus on caring for residents diagnosed with dementia. Only a few special care units exist. A precise description of the structure of nursing homes, concepts and nursing methods, however, is rare. Established concepts exist in a small number of institutions. Approved service offers are largely available, but do not apply specifically to the large group of nursing home residents with dementia. CONCLUSION: The study showed not only the diversity of the stationary nursing homes in a large city, but also their resources and limitations.

[What information do people needing care and their care-giving relatives need?]. / Welche Informationsbedürfnisse haben pflegebedürftige ältere Menschen und pflegende Angehörige?

BACKGROUND: The recent reform of the German Care Insurance Law (2008) was expanded to include independent consultancy for care issues. The goal of this study was to explore the informational needs of people in need of care and their informal caregivers. METHOD: A semi-structured questionnaire was used to document 89 consultation conversations. The data were analyzed using qualitative content analysis. RESULTS: The findings identified that information was needed about (1) the German health care insurance system, (2) access to care, (3) local care services, and (4) situation and disease-specific concerns. CONCLUSION: Consultancy services for people in need of care and informal caregivers require detailed knowledge about local care services and, therefore, should be integrated into the neighborhoods of the users.

Recruitment to Golgi membranes of ADP-ribosylation factor 1 is mediated by the cytoplasmic domain of p23.

Binding to Golgi membranes of ADP ribosylation factor 1 (ARF1) is the first event in the initiation of COPI coat assembly. Based on binding studies, a proteinaceous receptor has been proposed to be critical for this process. We now report that p23, a member of the p24 family of Golgi-resident transmembrane proteins, is involved in ARF1 binding to membranes. Using a cross-link approach based on a photolabile peptide corresponding to the cytoplasmic domain of p23, the GDP form of ARF1 (ARF1-GDP) is shown to interact with p23 whereas ARF1-GTP has no detectable affinity to p23. The p23 binding is shown to localize specifically to a 22 amino acid C-terminal fragment of ARF1. While a monomeric form of a non-photolabile p23 peptide does not significantly inhibit formation of the cross-link product, the corresponding dimeric form does compete efficiently for this interaction. Consistently, the dimeric p23 peptide strongly inhibits ARF1 binding to native Golgi membranes suggesting that an oligomeric form of p23 acts as a receptor for ARF1 before nucleotide exchange takes place.

Evidence for segregation of sphingomyelin and cholesterol during formation of COPI-coated vesicles.

In higher eukaryotes, phospholipid and cholesterol synthesis occurs mainly in the endoplasmic reticulum, whereas sphingomyelin and higher glycosphingolipids are synthesized in the Golgi apparatus. Lipids like cholesterol and sphingomyelin are gradually enriched along the secretory pathway, with their highest concentration at the plasma membrane. How a cell succeeds in maintaining organelle-specific lipid compositions, despite a steady flow of incoming and outgoing transport carriers along the secretory pathway, is not yet clear. Transport and sorting along the secretory pathway of both proteins and most lipids are thought to be mediated by vesicular transport, with coat protein I (COPI) vesicles operating in the early secretory pathway. Although the protein constituents of these transport intermediates are characterized in great detail, much less is known about their lipid content. Using nano-electrospray ionization tandem mass spectrometry for quantitative lipid analysis of COPI-coated vesicles and their parental Golgi membranes, we find only low amounts of sphingomyelin and cholesterol in COPI-coated vesicles compared with their donor Golgi membranes, providing evidence for a significant segregation from COPI vesicles of these lipids. In addition, our data indicate a sorting of individual sphingomyelin molecular species. The possible molecular mechanisms underlying this segregation, as well as implications on COPI function, are discussed.

Functional architecture of an intracellular membrane t-SNARE.

Lipid bilayer fusion is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) located on the vesicle membrane (v-SNAREs) and the target membrane (t-SNAREs). The assembled v-SNARE/t-SNARE complex consists of a bundle of four helices, of which one is supplied by the v-SNARE and the other three by the t-SNARE. For t-SNAREs on the plasma membrane, the protein syntaxin supplies one helix and a SNAP-25 protein contributes the other two. Although there are numerous homologues of syntaxin on intracellular membranes, there are only two SNAP-25-related proteins in yeast, Sec9 and Spo20, both of which are localized to the plasma membrane and function in secretion and sporulation, respectively. What replaces SNAP-25 in t-SNAREs of intracellular membranes? Here we show that an intracellular t-SNARE is built from a 'heavy chain' homologous to syntaxin and two separate non-syntaxin 'light chains'. SNAP-25 may thus be the exception rather than the rule, having been derived from genes that encoded separate light chains that fused during evolution to produce a single gene encoding one protein with two helices.

Allosteric activation of acid alpha-glucosidase by the human papillomavirus E7 protein.

Changes in the cellular carbohydrate metabolism are a hallmark of malignant transformation and represent one of the earliest discernible events in tumorigenesis. In the early stages of certain epithelial cancers, a metabolic switch is regularly observed, in which slowly growing glycogenotic cells are converted to highly proliferating basophilic cells. This step is accompanied by a rapid depletion of the intracellular glycogen stores, which in liver carcinogenesis results from the activation of the enzyme acid alpha-glucosidase by an as yet unknown mechanism. We show here that acid alpha-glucosidase is a target for the E7 protein encoded by human papillomavirus type 16, a human tumor virus that plays a key role in the genesis of cervical carcinoma. We show that expression of E7 induces the catalytic activity of acid alpha-glucosidase in vivo and wild type E7, but not transformation-deficient mutants bind directly to acid alpha-glucosidase and increase the catalytic activity of the enzyme in vitro. The data suggest that the E7 protein encoded by human papillomavirus type 16 can act as an allosteric activator of acid alpha-glucosidase.

Putative fusogenic activity of NSF is restricted to a lipid mixture whose coalescence is also triggered by other factors.

It has recently been reported that N-ethylmaleimide-sensitive fusion ATPase (NSF) can fuse protein-free liposomes containing substantial amounts of 1,2-dioleoylphosphatidylserine (DOPS) and 1, 2-dioleoyl-phosphatidyl-ethanolamine (DOPE) (Otter-Nilsson et al., 1999). The authors impart physiological significance to this observation and propose to re-conceptualize the general role of NSF in fusion processes. We can confirm that isolated NSF can fuse liposomes of the specified composition. However, this activity of NSF is resistant to inactivation by N-ethylmaleimide and does not depend on the presence of alpha-SNAP (soluble NSF-attachment protein). Moreover, under the same conditions, either alpha-SNAP, other proteins apparently unrelated to vesicular transport (glyceraldehyde-3-phosphate dehydrogenase or lactic dehydrogenase) or even 3 mM magnesium ions can also cause lipid mixing. In contrast, neither NSF nor the other proteins nor magnesium had any significant fusogenic activity with liposomes composed of a biologically occurring mixture of lipids. A straightforward explanation is that the lipid composition chosen as optimal for NSF favors non-specific fusion because it is physically unstable when formed into liposomes. A variety of minor perturbations could then trigger coalescence.

Content mixing and membrane integrity during membrane fusion driven by pairing of isolated v-SNAREs and t-SNAREs.

Membrane bilayer fusion has been shown to be mediated by v- and t-SNAREs initially present in separate populations of liposomes and to occur with high efficiency at a physiologically meaningful rate. Lipid mixing was demonstrated to involve both the inner and the outer leaflets of the membrane bilayer. Here, we use a fusion assay that relies on duplex formation of oligonucleotides introduced in separate liposome populations and report that SNARE proteins suffice to mediate complete membrane fusion accompanied by mixing of luminal content. We also find that SNARE-mediated membrane fusion does not compromise the integrity of liposomes.

p24 and p23, the major transmembrane proteins of COPI-coated transport vesicles, form hetero-oligomeric complexes and cycle between the organelles of the early secretory pathway.

COPI-coated vesicles that bud off the Golgi complex contain two major transmembrane proteins, p23 and p24. We have localized the protein at the Golgi complex and at COPI-coated vesicles. Transport from the intermediate compartment (IC) to the Golgi can be blocked at 15 degrees C, and under these conditions p24 accumulates in peripheral punctated structures identified as IC. Release from the temperature block leads to a redistribution of p24 to the Golgi, showing that p24, similar to p23, cycles between the IC and Golgi complex. Immunoprecipitations of p24 from cell lysates and from detergent-solubilized Golgi membranes and COPI-coated vesicles show that p24 and p23 interact with each other to form a complex. Transient transfection of p23 in HeLa cells shows that p23 and p24 colocalize in structures induced by the overexpression of p23. Taken together p24 interacts with p23 and constitutively cycles between the organelles of the early secretory pathway.

Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors.

COPI-coated vesicle budding from lipid bilayers whose composition resembles mammalian Golgi membranes requires coatomer, ARF, GTP, and cytoplasmic tails of putative cargo receptors (p24 family proteins) or membrane cargo proteins (containing the KKXX retrieval signal) emanating from the bilayer surface. Liposome-derived COPI-coated vesicles are similar to their native counterparts with respect to diameter, buoyant density, morphology, and the requirement for an elevated temperature for budding. These results suggest that a bivalent interaction of coatomer with membrane-bound ARF[GTP] and with the cytoplasmic tails of cargo or putative cargo receptors is the molecular basis of COPI coat assembly and provide a simple mechanism to couple uptake of cargo to transport vesicle formation.

A role for ADP ribosylation factor in the control of cargo uptake during COPI-coated vesicle biogenesis.

ARF-mediated hydrolysis of GTP has been demonstrated to regulate coat disassembly of Golgi-derived COPI transport vesicles (Tanigawa, G., Orci, L., Amherdt, M., Ravazzola, M., Helms, J.B. and Rothman, J.E. (1993) J. Cell Biol. 123, 1365-1371). In addition, a requirement for GTP hydrolysis at an early stage of COPI vesicle biogenesis has been established since cargo uptake is impaired in the presence of GTPgammaS (Nickel, W., Malsam, J., Gorgas, K., Ravazzola, M., Jenne, N., Helms, J.B. and Wieland, F.T. (1998) J. Cell Sci. 111, 3081-3090), a non-hydrolyzable analogue of GTP. We now demonstrate that the GTPase involved in the regulation of cargo uptake is ARF, revealing a multi-functional role of this GTPase in COPI-mediated vesicular transport. The molecular mechanism of cargo uptake as well as the functional implications of these findings on the overall process of COPI vesicle biogenesis are discussed.

Protein and lipid sorting between the endoplasmic reticulum and the Golgi complex.

Vesicular traffic within the early secretory pathway is mediated by COPI- and COPII-coated vesicles. While COPII-coated vesicles appear to be involved exclusively in the export of secretory proteins and lipids from the endoplasmic reticulum (ER), COPI-coated vesicles seem to function in both anterograde and retrograde transport between the ER-Golgi intermediate compartment (IC) and the Golgi as well as in intra-Golgi transport. Here, we focus on (i) the mechanisms how these transport carriers are formed from a given donor membrane; and (ii) the possible mechanisms involved in sorting of proteins and lipids into such transport vesicles.

Uptake by COPI-coated vesicles of both anterograde and retrograde cargo is inhibited by GTPgammaS in vitro.

On the basis of the cell surface protein CD8 we have constructed reporter molecules for both anterograde and retrograde transport from the Golgi complex. The cytoplasmic tail of CD8 was exchanged by a construct comprising a hemagglutinin (HA) epitope, the C-terminal sequence of the viral protein E19 (containing a KKXX retrieval signal) followed by a myc epitope (CD8-LT). Due to this masking of the KKXX retrieval signal CD8-LT is transported to the cell surface. Since the KKXX motif is joined to the myc epitope via a thrombin cleavage site, CD8-LT in isolated Golgi membranes can be proteolytically converted into an unmasked reporter molecule for retrograde transport (CD8-ST) in vitro. A CHO cell line stably expressing CD8-LT was generated and used for the isolation of Golgi membranes. These membranes were shown to contain CD8-LT en route to the cell surface. By addition of thrombin, CD8-LT could be efficiently converted into CD8-ST, and this allows us to study the sorting into coat protein COPI-coated vesicles of these different kinds of cargo on a comparative basis. COPI-coated vesicles were generated in vitro from Golgi membranes containing either CD8-LT or CD8-ST. When the incubation was performed in the presence of GTP, both CD8-LT and CD8-ST were packaged into COPI-coated vesicles. However, COPI-coated vesicles generated in the presence of the slowly hydrolyzable analogue of GTP, GTP(&ggr ;)S contained strikingly lower amounts of CD8-LT and CD8-ST. While COPI-coated vesicles accumulated about 12-fold in the presence of GTPgammaS these vesicles together contained only one fifth of cargo compared to the few vesicles generated in the absence of GTPgammaS. These data indicate that cargo packaging into COPI-coated vesicles requires hydrolysis of GTP.

Biosynthetic protein transport through the early secretory pathway.

Newly synthesized proteins destined for delivery to the cell surface are inserted cotranslationally into the endoplasmic reticulum (ER) and, after their correct folding, are transported out of the ER. During their transport to the cell surface, cargo proteins pass through the various cisternae of the Golgi apparatus and, in the trans-most cisternae of the stack, are sorted into constitutive secretory vesicles that fuse with the plasma membrane. Simultaneously with anterograde protein transport, retrograde protein transport occurs within the Golgi complex as well as from the Golgi back to the ER. Vesicular transport within the early secretory pathway is mediated by two types of non-clathrin coated vesicles: COPI- and COPII-coated vesicles. The formation of these carrier vesicles depends on the recruitment of cytosolic coat proteins that are thought to act as a mechanical device to shape a flattened donor membrane into a spherical vesicle. A general molecular machinery that mediates targeting and fusion of carrier vesicles has been identified as well. Beside a general overview of the various coat structures known today, we will discuss issues specifically related to the biogenesis of COPI-coated vesicles: (1) a possible role of phospholipase D in the formation of COPI-coated vesicles; (2) a functional role of a novel family of transmembrane proteins, the p24 family, in the initiation of COPI assembly; and (3) the direction COPI-coated vesicles may take within the early secretory pathway. Moreover, we will consider two alternative mechanisms of protein transport through the Golgi stack: vesicular transport versus cisternal maturation.

A putative heterotrimeric G protein inhibits the fusion of COPI-coated vesicles. Segregation of heterotrimeric G proteins from COPI-coated vesicles.

Heterotrimeric G proteins have been implicated in the regulation of intracellular protein transport, but their mechanism of action remains unclear. In vivo, secretion of chromogranin B, tagged with the green fluorescent protein, was inhibited by the addition of a general activator of trimeric G proteins (AlF4-) to stably transfected Vero cells and resulted in an accumulation of the tagged protein in the Golgi apparatus. In an in vitro assay that reconstitutes intra-Golgi protein transport, we find that a membrane-bound and AlF4--sensitive factor is involved in the fusion reaction. To determine whether this effect is mediated by a heterotrimeric G protein localized to COPI-coated transport vesicles, we determined the presence of G proteins on these vesicles and found that they were segregated relative to the donor membranes. Because G proteins do not have an obvious sorting, retention, or retrieval signal, we considered the possibility that other interactions might be responsible for this segregation. In agreement with this, we found that trimeric G proteins from isolated Golgi membranes were partially insoluble in Triton X-100. Identification of the proteins that interact with the heterotrimeric G proteins in the Golgi-derived detergent-insoluble complex might help to reveal the regulation of protein secretion mediated by heterotrimeric G proteins.

p23, a major COPI-vesicle membrane protein, constitutively cycles through the early secretory pathway.

A novel type I transmembrane protein of COPI-coated vesicles, p23, has been demonstrated to be localized mainly to the Golgi complex. This protein and p24, another member of the p24 family, have been shown to bind coatomer via their short cytoplasmic tails. Here we demonstrate that p23 continuously cycles through the early secretory pathway. The cytoplasmic tail of p23 is shown to act as a functional retrieval signal as it confers endoplasmic reticulum (ER) residence to a CD8-p23 fusion protein. This ER localization is, at least in part, a result of retrieval from post-ER compartments because CD8-p23 fusion proteins receive post-ER modifications. In contrast, the cytoplasmic tail of p24 has been shown not to retrieve a CD8-p24 fusion protein. The coatomer binding motifs FF and KK in the cytoplasmic tail of p23 are reported to influence the steady-state localization of the CD8-p23 fusion protein within the ER-Golgi recycling pathway. It appears that the steady-state Golgi localization of endogenous p23 is maintained by its lumenal domain, as a fusion protein with the lumenal domain of CD8, and the membrane span as well as the cytoplasmic tail of p23 is no longer detected in the Golgi.