Growth factor induction of Cripto-1 shedding by glycosylphosphatidylinositol-phospholipase D and enhancement of endothelial cell migration

Cripto-1 (CR-1) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that has been shown to play an important role in embryogenesis and cellular transformation. CR-1 is reported to function as a membrane-bound co-receptor and as a soluble ligand. Although a number of studies implicate the role of CR-1 as a soluble ligand in tumor progression, it is unclear how transition from the membrane-bound to the soluble form is physiologically regulated and whether differences in biological activity exist between these forms. Here, we demonstrate that CR-1 protein is secreted from tumor cells into the conditioned medium after treatment with serum, epidermal growth factor, or lysophosphatidic acid, and this soluble form of CR-1 exhibits the ability to promote endothelial cell migration as a paracrine chemoattractant. On the other hand, membrane-bound CR-1 can stimulate endothelial cell sprouting through direct cell-cell interaction. Shedding of CR-1 occurs at the GPI-anchorage site by the activity of GPI-phospholipase D (GPI-PLD), because CR-1 shedding was suppressed by siRNA knockdown of GPI-PLD and enhanced by overexpression of GPI-PLD. These findings describe a novel molecular mechanism of CR-1 shedding, which may contribute to endothelial cell migration and possibly tumor angiogenesis.     

Requirement of glycosylphosphatidylinositol anchor of Cripto-1 for trans activity as a Nodal co-receptor

Cripto-1 (CR-1) has an indispensable role as a Nodal co-receptor for patterning of body axis in embryonic development. CR-1 is reported to have a paracrine activity as a Nodal co-receptor, although CR-1 is primarily produced as a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Regulation of cis and trans function of CR-1 should be important to establish the precise body patterning. However, the mechanism by which GPI-anchored CR-1 can act in trans is not well known. Here we confirmed the paracrine activity of CR-1 by fluorescent cell-labeling and immunofluorescent staining. We generated COOH-terminal-truncated soluble forms of CR-1 based on the attachment site for the GPI moiety (omega-site), which we identified in the present study. GPI-anchored CR-1 has a significantly higher activity than COOH-terminal-truncated soluble forms to induce Nodal signal in trans as well as in cis. Moreover, transmembrane forms of CR-1 partially retained their ability to induce Nodal signaling only when type I receptor Activin-like kinase 4 was overexpressed. NTERA2/D1 cells, which express endogenous CR-1, lost the cell-surface expression of CR-1 after phosphatidylinositol-phospholipase C treatment and became refractory to stimulation of Nodal. These observations suggest that GPI attachment of CR-1 is required for the paracrine activity as a Nodal co-receptor.     

Connexin Channels at the Glio-Vascular Interface: Gatekeepers of the Brain

Neuronal survival, electrical signaling and synaptic activity require a well-balanced micro-environment in the central nervous system. This is achieved by the blood–brain barrier (BBB), an endothelial barrier situated in the brain capillaries, that controls near-to-all passage in and out of the brain. The endothelial barrier function is highly dependent on signaling interactions with surrounding glial, neuronal and vascular cells, together forming the neuro-glio-vascular unit. Within this functional unit, connexin (Cx) channels are of utmost importance for intercellular communication between the different cellular compartments. Connexins are best known as the building blocks of gap junction (GJ) channels that enable direct cell–cell transfer of metabolic, biochemical and electric signals. In addition, beyond their role in direct intercellular communication, Cxs also form unapposed, non-junctional hemichannels in the plasma membrane that allow the passage of several paracrine messengers, complementing direct GJ communication. Within the NGVU, Cxs are expressed in vascular endothelial cells, including those that form the BBB, and are eminent in astrocytes, especially at their endfoot processes that wrap around cerebral vessels. However, despite the density of Cx channels at this so-called gliovascular interface, it remains unclear as to how Cx-based signaling between astrocytes and BBB endothelial cells may converge control over BBB permeability in health and disease. In this review we describe available evidence that supports a role for astroglial as well as endothelial Cxs in the regulation of BBB permeability during development as well as in disease states.     

Bi-directional exchange of membrane components occurs during co-culture of mesenchymal stem cells and nucleus pulposus cells

Mesenchymal stem cell (MSC)-based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. We have previously demonstrated that when MSCs are co-cultured with nucleus pulposus (NP) cells with direct cell-cell contact, they differentiate along the NP lineage and simultaneously stimulate the degenerate NP cell population to regain a normal (non-degenerate) phenotype, an effect which requires cell-cell communication. However, the mechanisms by which NP cells and MSCs interact in this system are currently unclear. Thus, in this study we investigated a range of potential mechanisms for exchange of cellular components or information that may direct these changes, including cell fusion, gap-junctional communication and exchange of membrane components by direct transfer or via microvesicle formation. Flow cytometry of fluorescently labeled MSCs and NP cells revealed evidence of some cell fusion and formation of gapjunctions, although at the three timepoints studied these phenomena were detectable only in a small proportion of cells. While these mechanisms may play a role in cell-cell communication, the data suggests they are not the predominant mechanism of interaction. However, flow cytometry of fluorescently dual-labeled cells showed that extensive bi-directional transfer of membrane components is operational during direct co-culture of MSCs and NP cells. Furthermore, there was also evidence for secretion and internalization of membrane-bound microvesicles by both cell types. Thus, this study highlights bi-directional intercellular transfer of membrane components as a possible mechanism of cellular communication between MSC and NP cells.     

Fractalkine, a CX3C-chemokine, functions predominantly as an adhesion molecule in monocytic cell line THP-1

A newly identified CX3C-chemokine, fractalkine, expressed on activated endothelial cells plays an important role in leucocyte adhesion and migration. Co-immobilized fractalkine with fibronectin or intercellular adhesion molecule-1 enhanced adhesion of THP-1 cells, which express the fractalkine receptor (CX3CR1), compared with that observed for each alone. That adherence was fractalkine-dependent and was confirmed in blocking studies. However, soluble fractalkine induced little chemotaxis in THP-1 cells in comparison to monocyte chemotactic protein-1 (MCP-1), which induced a strong chemotactic response. Moreover, the membrane form of fractalkine expressed on ECV304 cells reduced MCP-1 mediated chemotaxis of THP-1 cells. These results indicate that fractalkine may function as an adhesion molecule between monocytes and endothelial cells rather than as a chemotactic factor.     

Soluble and membrane-bound forms of dopamine beta-hydroxylase are encoded by the same mRNA.

A full length cDNA clone for bovine dopamine beta-hydroxylase was expressed in rat pheochromocytoma PC12 cells by stable transformation of this cell line with a plasmid expression vector. The recombinant protein exhibited dopamine beta-hydroxylase enzyme activity and was found in both the soluble and membrane fractions of the secretory vesicle. Immunoprecipitation of cell extracts from recombinant cell lines with dopamine beta-hydroxylase antisera followed by fractionation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two subunits, which migrated to relative molecular masses of 76 and 78 kDa. The recombinant protein co-fractionated with neurotransmitter when subcellular structures were separated by sucrose gradient density centrifugation, suggesting that the protein was routed to the secretory vesicles. Dopamine beta-hydroxylase immunoreactivity in those sucrose gradient fractions presumed to contain secretory vesicles was resistant to treatment with trypsin unless the nonionic detergent Triton X-100 was also present to disrupt membrane structure. The 76- and 78-kDa isoform were each found in both the membrane and soluble fractions of the secretory vesicle. Treatment of cultured cells with nerve growth factor or 8-(4-chlorophenylthio)-cyclic AMP alters the relative distribution of the subunits such that the 76-kDa form predominates. The subcellular distribution of a dopamine beta-hydroxylase cDNA clone lacking the first 16 nucleotide residues was also determined. The predicted amino acid sequence of the protein encoded by this cDNA would be deleted of the first 13 residues of the signal sequence, which were reported to be present in the membrane-bound form, but not the soluble form, of native dopamine beta-hydroxylase (Taljanidisz, J., Stewart, L., Smith, A. J., and Klinman, J. P. (1989) Biochemistry 28, 10054-10061). Immunoprecipitable dopamine beta-hydroxylase derived from expression of the deleted cDNA was found in both the membrane-bound and soluble fractions of the secretory vesicle. These experiments demonstrate that the membrane-bound and soluble forms of dopamine beta-hydroxylase are derived from one primary translation product, which is also sufficient to produce enzyme activity. In addition, the amino-terminal amino acids encoding residues 1-13, which compose the hydrophilic region of the signal sequence, are not necessary for the biogenesis of membrane-bound dopamine beta-hydroxylase.     

Three-dimensional co-culture model employing silica nonwoven fabrics to enhance cell-to-cell communication of paracrine signaling between hepatocytes and fibroblasts

The realization that soluble factors secreted by heterotypic cells play an importanta role in paracrine signaling, which facilitates intercellular communication, enabled the development of physiologically relevant co-culture models for drug screening and the engineering of tissues, such as hepatic tissues. The most crucial issues confronting the use of conventional membrane inserts in segregated co-culture models that are used to study paracrine signaling between heterotypic cells have been identified as long-term viability and retention of cell-specific functions, especially when isolated primary cells are used. Herein, we present an in vitro segregated co-culture model consisting of a well plate incubated with rat primary hepatocytes and normal human dermal fibroblasts which were segregated using a membrane insert with silica nonwoven fabric (SNF) on it. SNF, which mimics a physiological environment much more effectively than a two-dimensional (2D) one, promotes cell differentiation and resultant paracrine signaling in a manner that is not possible in a conventional 2D culture, owing to high mechanical strength generated by its inorganic materials and interconnected network structure. In segregated co-cultures, SNF clearly enhanced the functions of hepatocytes and fibroblasts, thereby showing its potential as a measure of paracrine signaling. These results may advance the understanding of the role played by paracrine signaling in cell-to-cell communication and provide novel insights into the applications of drug metabolism, tissue repair, and regeneration.     

Posttranslational Processing of Carboxypeptidase E, a Neuropeptide-Processing Enzyme, in AtT-20 Cells and Bovine Pituitary Secretory Granules

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of peptide hormones and neurotransmitters. Like other peptide processing enzymes, CPE is present in secretory granules in soluble and membrane-associated forms that arise from posttranslational processing of a single precursor, “proCPE.” To identify the intracellular site of proCPE processing, the biosynthesis and posttranslational processing were investigated in the mouse anterior pituitary-derived cell line, AtT-20. Following a 15-min pulse with [³⁵S]Met, both soluble and membrane-bound forms of CPE were identified, indicating that the posttranslational processing event that generates these forms of CPE occurs in the endoplasmic reticulum or early Golgi apparatus. The relative proportion of soluble and membrane-bound forms of CPE changed when cells were chased for 2 h at 37°C but was unaffected when cells were chased at either 20 or 15°C, suggesting that further processing of membrane forms to the soluble form occurs in a post-Golgi compartment. Treatment of the cells with chloroquine did not alter the relative distribution of soluble and membrane forms, suggesting that an acidic compartment is not required for this processing event. Overexpression of CPE did not influence the distribution of soluble and membrane forms of CPE, indicating that the CPE-processing enzymes are not rate-limiting. To examine directly CPE-processing enzymes, bovine anterior pituitary secretory vesicles were isolated. An enzyme activity that releases the membrane-bound form of CPE was detected in the purified secretory vesicle membranes. This enzyme, which removes the C-terminal region of CPE, is partially inhibited by EDTA and phenylmethylsulfonyl fluoride and is activated by CaCI₂. Together, the data indicate that posttranslational processing of CPE occurs in secretory granules and that this activity may be mediated by a prohormone convertase-like enzyme.     

Hodgkin's cell lectin, a lymphocyte adhesion molecule and mitogen

We have previously shown a novel galactose/N-acetylgalactosamine specific lectin activity (Hodgkin's disease (HD) lectin) on the surface of cultured HD cells (lines L428, its variants, and line L540) to mediate lymphocyte adhesion. We here demonstrate that both surface membrane-bound and secreted HD lectin activities participate in the activation of agglutinated lymphocytes. Among known adhesion molecules expressed by the HD cells, only the intercellular adhesion molecule-1 (ICAM-1) contributed to this activation as an alternative PBL binding site. As yet we have not identified the cellular ligand(s) for the HD lectin on the lymphocyte surface. Pretreatment of lymphocytes with mAb to the accessory molecules CD2, CD3, CD4, CD8, CD11b, or CD11c did not interfere with their response to HD cells. mAb to CD11a (LFA-1), the alleged ligand of ICAM-1, inhibited the ICAM-1 but not the HD lectin-mediated lymphocyte stimulation. Although lymphocyte binding could proceed via either pathway, lymphocyte activation always depended upon factors secreted by the HD cells, one of which we identified as a soluble form of the HD lectin based on its shared properties with the membrane-bound form including immunologic cross-recognition and carbohydrate-binding specificity. Although HD cell-conditioned medium alone stimulated lymphocytes, HD cell plasma membranes could compensate for low concentrations of this medium. In addition, resting lymphocytes, normally unresponsive, were triggered into DNA synthesis by growth medium when cocultured with HD cell membranes. The unique functions of the surface-expressed HD lectin and its soluble counterpart as lymphocyte adhesion molecule and mitogen might be physiologically relevant to the severe immunodeficiencies occurring in patients with HD.     

Distribution and biosynthesis of aminopeptidase N and dipeptidyl aminopeptidase IV in rat small intestine

The regional, cellular and subcellular distribution patterns of aminopeptidase N and dipeptidyl aminopeptidase IV were examined in rat small intestine. Aminopeptidase N of brush border membrane had maximal activity in the upper and middle intestine, while dipeptidyl aminopeptidase IV had a more uniform distribution profile with relatively high activity in the ileum. Along the villus and crypt cell gradient, the activity of both enzymes was maximally expressed in the mid-villus cells. However there was substantial dipeptidyl aminopeptidase IV activity in the crypt cells. Both enzymes were primarily associated with brush border membranes in all segments, however, in the proximal intestine, a significant amount of dipeptidyl aminopeptidase IV activity was associated with the cytosol fraction. The cytosol and brush border membrane forms of dipeptidyl aminopeptidase IV were immunologically identical and had the same electrophoretic mobility on disc gels. In contrast, the soluble and brush border membrane-bound forms of aminopeptidase N were immunologically distinct. When the total amount of aminopeptidase N and dipeptidyl aminopeptidase IV was determined by competitive radioimmunoassay, there were no regional or cellular differences in specific activity (enzyme activity/mg of enzyme protein) of either enzyme in brush border membrane and homogenate. The specific activity of both enzymes in a purified Golgi membrane fraction as measured by radioimmunoassay was about half that of the brush border membrane fraction. These results suggest that (1) aminopeptidase N and dipeptidyl aminopeptidase IV have different regional, cellular and subcellular distribution patterns; (2) there are enzymatically inactive forms of both enzymes present in a constant proportion to active molecules and that (3) a two-fold activation of precursor enzyme forms occurs during transfer from the Golgi membranes to the brush border membranes.     

A new anti-mesothelin antibody targets selectively the membrane-associated form

Mesothelin is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that shows promise as a target for antibody-directed cancer therapy. High levels of soluble forms of the antigen represent a barrier to directing therapy to cellular targets. The ability to develop antibodies that can selectively discriminate between membrane-bound and soluble conformations of a specific protein, and thus target only the membrane-associated antigen, is a substantive issue. We show that use of a tolerance protocol provides a route to such discrimination. Mice were tolerized with soluble mesothelin and a second round of immunizations was performed using mesothelin transfected P815 cells. RNA extracted from splenocytes was used in phage display to obtain mesothelin-specific antigen-binding fragments (Fabs) that were subsequently screened by flow cytometry and ELISA. This approach generated 147 different Fabs in 34 VH-CDR3 families. Utilizing competition assays with soluble protein and mesothelin-containing serum obtained from metastatic cancer patients, 10 of these 34 VH-CDR3 families were found to bind exclusively to the membrane-associated form of mesothelin. Epitope mapping performed for the 1H7 clone showed that it does not recognize GPI anchor. VH-CDR3 sequence analysis of all Fabs showed significant differences between Fabs selective for the membrane-associated form of the antigen and those that recognize both membrane bound and soluble forms. This work demonstrates the potential to generate an antibody specific to the membrane-bound form of mesothelin. 1H7 offers potential for therapeutic application against mesothelin-bearing tumors, which would be largely unaffected by the presence of the soluble antigen.     

Interleukin-6 and the network of several cytokines in multiple myeloma: an overview of clinical and experimental data.

Study of the network of cytokines has helped identify cell growth factors in multiple myeloma. Plasma cells themselves may produce autocrine interleukin 6 (IL-6) while IL-6 production by bone marrow stromal cells may operate a paracrine mechanism. Involvement of IL-6 in multiple myeloma is indicated by its ability to induce the differentiation of myeloma plasmablasts into mature malignant plasma cells. Differential diagnosis between multiple myeloma and monoclonal gammopathies of undetermined significance (MGUS) is generally based on clinical and laboratory parameters. Nevertheless, evaluation of the serum level of IL-6, C reactive protein, soluble IL-6 receptor, soluble IL-2 receptor together with the activity exerted by IL-3 and IL-4 on some cellular subsets constitutes an additional element in the differential diagnosis of border-line cases. Serum levels of IL-6, soluble IL-6 receptor (sIL-6R), soluble interleukin-2 receptor (sIL-2R) and the expression of membrane-bound IL-2 receptors, both on bone marrow plasma cells and on peripheral blood mononuclear cells are correlated with disease activity and disease stage. In addition, IL-6 and sIL-6R serum levels correlate with the duration of survival, as high values at the time of diagnosis correlate with short duration of survival.     

Distinct Cellular Localization of Membrane-Bound and Soluble Forms of Catechol-O-Methyltransferase in Brain

Abstract: The cellular localization of the two forms of catechol- O -methyltransferase (COMT) was investigated by measuring their activities in rat striatum following unilateral stereotaxic injection of kainic acid, which causes degeneration of striatal neurons followed by proliferation of astroglial cells. Membrane-bound COMT activity was decreased in the lesioned striatum, while soluble COMT activity was increased. There was a statistically significant correlation between the ratio of lesioned to control activity for membrane-bound COMT and the neuronal marker enzyme glutamate decarboxylase. Similarly the increase in soluble COMT activity paralleled that of the astroglial marker enzyme, glutamine synthetase. These results indicate that the K _m membrane-bound catechol- O -methyltransferase may be localized predominantly in neurons, whereas the high-K_m soluble enzyme is found in glial cells.     

THP-1 macrophage membrane-bound plasmin activity is up-regulated by transforming growth factor-β1 via increased expression of urokinase and the urokinase receptor

Receptors for urokinase (uPA) and plasminogen provide a mechanism to direct the cellular activation of plasminogen. The regulation of these receptors is important for several macrophage functions. In these studies, the effect of transforming growth factor-b?1 (TGF-b?1) on uPA, uPA receptor, and plasminogen receptor expression by human THP-1 macrophage was examined. TGF-b?1 induction of uPA expression by THP-1 cells was differentiation dependent. Suspension and adherent cultures expressed similar constitutive levels of uPA. Exposure of adherent cells to TGF-b?1 led to a dose- and time-dependent increase in uPA activity which was paralleled by an increase in uPA antigen and uPA mRNA. In contrast, uPA expression by suspension cultures was unresponsive to TGF-b?1. The differential response exhibited by suspension and adherent THP-1 cells may reflect differences in their expression of TGF-b?1 receptors, since when assayed by crosslinking techniques, suspension cells primarily expressed a 65 kDa receptor; whereas, the adherent cells expressed 65 and 100 kDa receptors. TGF-b?1-induced alterations in uPA receptor expression by adherent THP-1 cells were examined by quantitating membrane-bound uPA activity. Membrane-bound uPA activity increased three-fold when cells were incubated with TGF-b?1. The increase in membrane-uPA activity expressed by TGF-b?1-treated cells was not due to increased uPA receptor occupancy since incubation of either control or TGF-b?1 primed cells with exogenous uPA did not lead to an increase in membrane-bound uPA activity. Furthermore, immunoreactive uPA receptor was increased in TGF-b?1-treated cells. Following incubation with plasminogen, membrane-bound plasmin activity increased three-fold in TGF-b?1-treated cells. However, no change in immunoreactive membrane-bound plasmin(ogen) was observed. In addition, binding of ¹²⁵I-Lys-plasminogen to THP-1 cells was not affected by TGF-b?1 treatment. We conclude that TGF-b?1 stimulates membrane-bound plasmin activity, without affecting plasminogen receptor expression, through the up-regulation of uPA and the uPA receptor expression. © 1995 Wiley-Liss, Inc.     

Activation of cellular Arf GTPases by poliovirus protein 3CD correlates with virus replication

We have previously shown that synthesis of poliovirus protein 3CD in uninfected HeLa cell extracts induces an increased association with membranes of the cellular Arf GTPases, which are key players in cellular membrane traffic. Arfs cycle between an inactive, cytoplasmic, GDP-bound form and an active, membrane-associated, GTP-bound form. 3CD promotes binding of Arf to membranes by initiating recruitment to membranes of guanine nucleotide exchange factors (GEFs), BIG1 and BIG2. GEFs activate Arf by replacing GDP with GTP. In poliovirus-infected cells, there is a dramatic redistribution of cellular Arf pools that coincides with the reorganization of membranes used to form viral RNA replication complexes. Here we demonstrate that Arf translocation in vitro can be induced by purified recombinant 3CD protein; thus, concurrent translation of viral RNA is not required. Coexpression of 3C and 3D proteins was not sufficient to target Arf to membranes. 3CD expressed in HeLa cells was retained after treatment of the cells with digitonin, indicating that it may interact with a membrane-bound host factor. A F441S mutant of 3CD was shown previously to have lost Arf translocation activity and was also defective in attracting the corresponding GEFs to membranes. A series of other mutations were introduced at 3CD residue F441. Mutations that retained Arf translocation activity of 3CD also supported efficient growth of virus, regardless of their effects on 3D polymerase elongation activity. Those that abrogated Arf activation by 3CD generated quasi-infectious RNAs that produced some plaques from which revertants that always restored the Arf activation property of 3CD were rescued.     

Intracellular translocation of phosphatidate phosphatase in maturing safflower seeds: a possible mechanism of feedforward control of triacylglycerol synthesis by fatty acids

Phosphatidate phosphatase activity was found both in the cytosol and in the microsomal membrane of maturing safflower seeds. The combined and relative activities of these two forms varied with seed maturation. During the period of rapid triacylglycerol accumulation in the cell, most of the phosphatidate phosphatase activity was membrane-bound; at the initial and last stages of seed development when triacylglycerol synthesis was at an insignificant level, the majority of the activity was soluble. The potassium salts of palmitic, stearic and oleic acids, which are the fatty acid products of proplastids, caused the translocation of the cytosolic phosphatidate phosphatase to the microsomal membrane, while laurate and linoleate, which are not products of proplastids, showed no effect. Oleoyl-CoA did not convert the soluble form of the enzyme into the membrane-bound form. The translocation induced by oleate was reversible. The cytosolic phosphatidate phosphatase of safflower seeds was not transferred to the microsomal membranes prepared from soybean, a plant species of Leguminosae, and from rapeseed, a species of Cruciferae, but was transferred to that from sunflower, which belongs to the same family as safflower, Compositae. These observations suggest that in maturing oil seeds the rate of fatty acid synthesis in proplastids may regulate the species-specific translocation of phosphatidate phosphatase between the cytosol and the endoplasmic reticulum membrane where triacylglycerol synthesis occurs and that in turn the translocation of this ambiquitous enzyme could control the rate of triacylglycerol synthesis in the cell.     

Location of anaerobic respiratory enzyme trimethylamine N-oxide reductase in the cytoplasmic membrane ofEscherichia coli strain K10

Trimethylamine N-oxide (TMAO) reductase, which is anaerobically induced by TMAO, is a terminal enzyme in anaerobic electron transport inEscherichia coli. When the organism was anaerobically grown with TMAO, a marked increase in the specific activity of TMAO reductase was observed mainly in a cell membrane fraction and stopped after exhausting TMAO. On the other hand, activity was moderately increased in a soluble fraction of the cell even after exhaustion of TMAO. Immunoblot analysis with an antiserum against the TMAO reductase purified from the soluble fractions showed that the cells growing with TMAO contained only a membrane-bound enzyme, which has a molecular mass of 94 kDa, while a soluble enzyme with 92 kDa appeared in the stationary growth phase lacking TMAO. Experiments with right-side-out and inside-out vesicles of cytoplasmic membrane indicated that the membrane-bound enzyme faces the cytoplasm. The soluble enzyme was mainly found in the cytoplasm of the cell, but also at a negligible amount in the periplasm. The membrane-bound form of TMAO reductase functioning in anaerobic electron transport seems to be cleaved and released into the cytoplasm as soluble enzyme after exhaustion of TMAO.     

Cutting edge: Membrane nanotubes in vivo: a feature of MHC class II+ cells in the mouse cornea

Membrane nanotubes are a recently discovered form of cellular protrusion between two or more cells whose functions include cell communication, environmental sampling, and protein transfer. Although clearly demonstrated in vitro, evidence of the existence of membrane nanotubes in mammalian tissues in vivo has until now been lacking. Confocal microscopy of whole-mount corneas from wild-type, enhanced GFP chimeric mice, and Cx3cr1(gfp) transgenic mice revealed long (>300 microm) and fine (<0.8 microm diameter) membrane nanotube-like structures on bone marrow-derived MHC class II(+) cells in the corneal stroma, some of which formed distinct intercellular bridges between these putative dendritic cells. The frequency of these nanotubes was significantly increased in corneas subjected to trauma and LPS, which suggests that nanotubes have an important role in vivo in cell-cell communication between widely spaced dendritic cells during inflammation. Identification of these novel cellular processes in the mammalian cornea provides the first evidence of membrane nanotubes in vivo.     

Fumarate reductase is a soluble enzyme in anaerobically grown Shewanella putrefaciens MR-1

Abstract The expression and distribution of fumarate reductase activity was examined in Shewanella putrefaciens MR-1. Fumarate reductase was expressed at very low levels in aerobically grown cell and was markedly induced by growth under anaerobic conditions. Cells were fractionated into soluble and purified membrane components by four different methods. For all four methods used, and in marked contrast to the membrane-bound fumarate reductases of other bacteria, ≧ 98% of the fumarate reductase activity was localized in the soluble fraction. In cells subjected to osmotic shock or treated with lysozyme and EDTA to form spheroplasts, the specific activity of fumarate reductase was highest in the periplasmic fraction, while the majority of total fumarate reductase activity was in the cytoplasmic fraction.