Human papilloma virus transformed CaSki cells constitutively express high levels of functional SerpinB2

Many malignant tissues, including human papilloma virus (HPV)-associated cancers, express SerpinB2, also known as plasminogen activator inhibitor type-2 (PAI-2). Whether SerpinB2 is expressed by the HPV-transformed cancer cells, and if so, whether SerpinB2 is mutated or behaves aberrantly remains unclear. Here we show that HPV-transformed CaSki cells express high levels of constitutive wild-type SerpinB2, with cellular distribution, glycosylation, secretion, cleavage, induction and urokinase binding similar to that reported for primary cells. Neutralization of secreted SerpinB2 failed to affect CaSki cell migration or growth. Lentivirus-based over-expression of SerpinB2 also had no effect on growth, and we were unable to confirm a role for SerpinB2 in binding or regulating expression of the retinoblastoma protein. CaSki cells thus emerge as a useful tool for studying SerpinB2, with the physiological function of SerpinB2 expression by tumor cells remaining controversial. Using CaSki cells as a source of endogenous SerpinB2, we confirmed that SerpinB2 efficiently binds the proteasomal subunit member β1.     

Neural tissues express high levels of the cellular src gene product pp60c-src.

Chicken embryo tissues were examined for the expression of pp60c-src, the normal cellular homolog of the transforming protein of Rous sarcoma virus. Three assays, including a solid-phase radioimmunoassay, a competitive radioimmunoprecipitation assay, and an immune complex protein kinase assay, were employed. Elevated levels of pp60c-src were detected in lysates from several neural tissues, including brain, retina, and spinal ganglia. Other tissues contained 8- to 10-fold-lower levels of pp60c-src, levels comparable to those found in chicken embryo fibroblasts. Expression of pp60c-src in brain tissues was also shown to vary with the developmental stage of the embryo.     

Bone marrow-infiltrating human neuroblastoma cells express high levels of calprotectin and HLA-G proteins

Metastases in the bone marrow (BM) are grim prognostic factors in patients with neuroblastoma (NB). In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Sixty-six Italian children over 18 months of age, diagnosed with stage 4 NB, were included in the study. Metastatic NB cells were freshly isolated from patients' BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5 y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin), CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients.     

Calmodulin-binding proteins from brain and other tissues.

The calmodulin contents of rabbit brain, lung, kidney and liver, of bovine aorta and uterus, and of chicken gizzard have been determined. 2. The calmodulin in all of these tissues has been shown to be present in the form of very stable complexes with several other proteins. 3. A calmodulin-binding protein of mol.wt. 22 000 has been purified in high yield from bovine brain. It has been shown to interact with calmodulin and rabbit skeletal-muscle troponin C in a Ca2+-dependent manner. 4. The 22 000-mol.wt. protein inhibits the activation of bovine brain phosphodiesterase by calmodulin, but has very little affect on the activation of myosin light-chain kinase. 5. Calmodulin-binding proteins of mol.wts. 140000, 77000 and 61000 have also been partially purified from rabbit brain by affinity chromatography and have been shown to interact in a Ca2+-dependent manner with calmodulin. 6. The apparent molecular weights of the calmodulin-calmodulin-binding protein complexes, determined by gel filtration in the presence of 6M-urea, have been shown to be similar for most of the mammalian tissues examined. 7. By using 125I-labelled calmodulin, similar complexes have been demonstrated in rabbit skeletal muscle, although they are present at much lower concentrations.     

28 kDa adenosine-binding proteins of brain and other tissues.

Membranes prepared from calf brain were solubilized and chromatographed on a column containing 5'-amino-5'-deoxyadenosine covalently linked to agarose through the 5'-amino group. When the column was eluted with adenosine, a pure protein emerged with subunit molecular mass of 28 kDa. The protein was extracted from the membranes with sodium cholate, but not with 100 microM-adenosine or 0.5 M-NaCl. A similar 28 kDa protein was isolated from the soluble fraction of calf brain. The yield of membrane-bound and soluble 28 kDa protein per gram of tissue was about the same. The 28 kDa protein was also found in membrane and soluble fractions of rabbit heart, rat liver and vascular smooth muscle from calf aorta. The yield per gram of tissue fell into the order brain greater than heart approximately vascular smooth muscle greater than liver for the 28 kDa protein from the membrane fraction, and brain approximately heart greater than vascular smooth muscle greater than liver for the 28 kDa protein from the soluble fraction. Polyclonal antibodies to pure 28 kDa protein from calf brain membranes cross-reacted with the 28 kDa protein from calf brain soluble fraction and with 28 kDa proteins isolated from other tissues. The 28 kDa protein from calf brain membranes was also eluted from the affinity column by AMP and 2',5'-dideoxyadenosine, but at a concentration higher than that at which adenosine eluted the protein, but N6-(R-phenylisopropyl)adenosine, 5'-N-ethylcarboxamidoadenosine, ADP, ATP, GTP, NAD+, cyclic AMP and inosine failed to elute the protein at concentrations up to 1 mM. The 28 kDa protein from the soluble fraction was not eluted by 3 mM-AMP or 1 mM-N6-(R-phenylisopropyl)adenosine,-5'-N-ethylcarboxamidoadenosine or -cyclic AMP. Unexpectedly, the soluble 28 kDa protein was eluted by AMP in the presence of sodium cholate. Soluble 28 kDa protein from calf brain had a KD for adenosine of 12 microM. Membrane 28 kDa protein from calf brain had a KD of 14 microM in the presence of 0.1% sodium cholate. Amino acid compositions of the 28 kDa proteins were similar, but not identical.     

Lines of BPV-transformed murine cells that constitutively express influenza virus hemagglutinin.

We have developed and characterized several murine cell lines that constitutively express either the full-length, membrane-bound form of influenza virus hemagglutinin (HA) or a truncated version of the protein (HAsec) that lacks the carboxyterminal anchoring sequences and is secreted from cells. cDNAs encoding HA or HAsec were linked to the murine metallothionein-I promoter or the SV40 early promoter, and inserted into plasmids containing the transforming DNA fragment of bovine papilloma virus (BPV). The resulting vectors were introduced into three cultured lines of murine cells--C127, NIH3T3 and MME--either alone or in the presence of a plasmid that carries the aminoglycoside transferase gene of Tn5. The resulting lines of MME cells contained 1-5 copies of the vector in an integrated state and expressed low levels of HA (approximately 10(4) molecules/cell). In contrast, lines of C127 and NIH3T3 cells were obtained that express up to 5 X 10(6) molecules of HA per cell or secrete approximately 10(7) molecules of HAsec per cell per 24 h. Some of these cell lines carry multiple (30-200) copies of the vector in an integrated state; in others, the vector is propagated as unit-length episomes or as oligomers. Both the membrane-bound and secreted forms of HA expressed in these cell lines display a more extensive pattern of glycosylation than HA or HAsec synthesized in simian cells and they are transported to the cell surface more slowly. Pulse-chase experiments suggest that the step which limits the rate at which HA and HAsec travel down the secretory pathway occurs in the rough endoplasmic reticulum before the molecules are transferred to the Golgi apparatus. Using indirect immunofluorescence in combination with a cell sorter, we have shown that the level of expression of HA within cloned populations of producing cells can be variable. However, greater than 90% of the cells in certain cell lines display considerable quantities of HA on their surface, as judged by their ability to bind red blood cells in large numbers. We have taken advantage of the membrane fusion activity of HA to effect the fusion of erythrocytes to these cells and to deliver the contents of red cell ghosts into the cells' cytoplasm.     

Aromatase-immunoreactive cells are present in mouse brain areas that are known to express high levels of aromatase activity

The transformation of testosterone into estradiol in the brain plays a key role in several behavioral and physiological processes, but it has been so far impossible to localize precisely the cells of the mammalian brain containing the relevant enzyme, viz., aromatase. We have recently established an immunohistochemical technique that allows the visualization of aromatase-immunoreactive cells in the quail brain. In this species, a marked increase in the optical density of aromatase-immunoreactive cells is observed in subjects that have been treated with the aromatase inhibitor, R76713 or racemic Vorozole. This increased immunoreactivity, associated with a total blockade of aromatase activity, has been used as a tool in the present study in which the distribution of aromatase-immunoreactive material has been reassessed in the brain of mice pretreated with R76713. As expected, the aromatase inhibitor increases the density of the immunoreactive signal in mice. Strongly immunoreactive cells are found in the lateral septal region, the bed nucleus of the stria terminalis, the central amygdala, and the dorso-lateral hypothalamus. A less dense signal is also present in the medial preoptic area, the nucleus accumbens, several hypothalamic nuclei (e.g., paraventricular and ventromedial nuclei), all divisions of the amygdala, and several regions of the cortex, especially the cortex piriformis. These data demonstrate that, contrary to previous claims, aromatase-immunoreactive cells are present in all brain regions that have been shown previously to contain high aromatase activity.     

Detection of high molecular weight vinculin binding proteins in muscle and nonmuscle tissues with an electroblot-overlay technique

In this study, we examined binding of radiolabelled vinculin to proteins separated on sodium dodecyl sulfate-polyacrylamide gels, and then electrophoretically transferred onto nitrocellulose sheets. We detected saturable binding of vinculin to polypeptides with apparent Mr's of 215,000, 205,000 and 185,000 in a low ionic strength extract from chicken gizzard membranes. Binding of vinculin to proteins with apparent Mr's of 205,000, 185,000, and 165,000 in human platelets was also detected. In addition, we found that [125I]vinculin binds to unlabelled vinculin and to alpha-actinin, although these interactions appear to be of lower affinity than those with the higher molecular weight proteins.     

Troponin C-like proteins (calmodulins) from mammalian smooth muscle and other tissues.

1. An acidic protein with properties similar to those of troponin C from rabbit skeletal muscle has been shown to be present in bovine and rabbit smooth muscles, chicken gizzard and rabbit liver, kidney and lung. 2. A simple new method involving the use of organic solvents is described for the purification of the troponin C-like proteins from various tissues. 3. The troponin C-like proteins can be distinguished from rabbit skeletal-muscle toponin C by their electrophoretic behaviour on polyacrylamide gels at pH 8.3 in the presence and absence of Ca2+. The troponin C-like proteins have been shown to form complexes with rabbit skeletal-muscle troponin I that migrate on electrophoresis in polyacrylamide gels. 4. Behaviour on electrophoresis, amino acid analysis and the patterns of CNBr digests on polyacrylamide gels indicate that the troponin C-like proteins from bovine uterus and aorta, rabbit uterus, and liver and chicken gizzard are very similar to, if not identical with, bovine brain modulator protein. 5. With bovine cardiac muscle the organic-solvent method yields a preparation consisting of roughly similar amounts of troponin C and troponin C-like protein. 6. By the isotope-dilution technique, troponin C-like protein has been shown to represent 0.42% of the total protein in rabbit uterus. 7. In homogenates of smooth muscle, rabbit lung, kidney and brain, the troponin C-like proteins form a complex with other protein (or proteins) that requires Ca2+ for its formation and that is not dissociated in 9M-urea.     

Homer proteins and InsP(3) receptors co-localise in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres

Striated muscle represents one of the best models for studies on Ca(2+) signalling. However, although much is known on the localisation and molecular interactions of the ryanodine receptors (RyRs), far less is known on the localisation and on the molecular interactions of the inositol trisphosphate receptors (InsP(3)Rs) in striated muscle cells. Recently, members of the Homer protein family have been shown to cluster type 1 metabotropic glutamate receptors (mGluR1) in the plasma membrane and to interact with InsP(3)R in the endoplasmic reticulum of neurons. Thus, these scaffolding proteins are good candidates for organising plasma membrane receptors and intracellular effector proteins in signalosomes involved in intracellular Ca(2+) signalling. Homer proteins are also expressed in skeletal muscle, and the type 1 ryanodine receptor (RyR1) contains a specific Homer-binding motif. We report here on the relative sub-cellular localisation of InsP(3)Rs and Homer proteins in skeletal muscle cells with respect to the localisation of RyRs. Immunofluorescence analysis showed that both Homer and InsP(3)R proteins present a staining pattern indicative of a localisation at the Z-line, clearly distinct from that of RyR1. Consistent herewith, in sub-cellular fractionation experiments, Homer proteins and InsP(3)R were both found in the fractions enriched in longitudinal sarcoplasmic reticulum (LSR) but not in fractions of terminal cisternae that are enriched in RyRs. Thus, in skeletal muscle, Homer proteins may play a role in the organisation of a second Ca(2+) signalling compartment containing the InsP(3)R, but are apparently not involved in the organisation of RyRs at triads.     

Modulation of synaptic signalling complexes by Homer proteins

The number of neurotransmitter receptors in the postsynaptic membrane and their functional coupling to intracellular signalling cascades are important determinants of synaptic strength--and hence potential targets for plasticity related modulation. In this context, Homer/Vesl proteins have gained particular interest for three main reasons: (i) they constitute part of the molecular scaffold at postsynaptic densities of excitatory synapses in the mammalian brain; (ii) they physically link type-I metabotropic glutamate receptors to the postsynaptic density and to inositol 1,4,5-triphosphate receptors in the subsynaptic endoplasmic reticulum; and (iii) Homer-1a, which has been categorized as an immediate early gene isoform, exerts dominant-negative activity, suggesting that it is involved in activity dependent rearrangements at synaptic junctions. Although these fundamental aspects have been reviewed previously by Xiao et al., this review will address primarily more recent studies on the regulation of Homer 1a expression and on the role of Homer/Vesl proteins in spine morphogenesis and receptor targeting and signalling.     

Transition of Homer isoforms during skeletal muscle regeneration

Homer represents a new and diversified family of proteins that includes several isoforms, Homer 1, 2, and 3; some of these isoforms have been reported to be present in striated muscles. In this study, the presence of Homer isoforms 1a, 1b/c/d, 2b, and 3 was thoroughly investigated in rat skeletal muscles under resting conditions. Transition in Homer isoforms compositon was studied under experimental conditions of short-term and long-term adaptation, e.g., fatigue and regeneration, respectively. First, we show that Homer 1a was constitutively expressed and was transiently upregulated during regeneration. In C₂C₁₂ cell cultures, Homer 1a was also upregulated during formation of myotubes. No change of Homer 1a was observed in fatigue. Second, Homer 1b/c/d and Homer 2b were positively and linearly related to muscle mass change during regeneration, and third, Homer 3 was not detectable under resting conditions but was transiently expressed during regeneration although with a temporal pattern distinct from that of Homer 1a. Thus a switch in Homer isoforms is associated to muscle differentiation and regeneration. Homers may play a role not only in signal transduction of skeletal muscle, in particular regulation of Ca²⁺ release from sarcoplasmic reticulum (Ward CW, Feng W, Tu J, Pessah IN, Worley PF, and Schneider MF. Homer protein increases activation of Ca²⁺ sparks in permeabilized skeletal muscle. J Biol Chem 279: 5781–5787, 2004), but also in adaptation. fatigue; immediate early gene; muscle adaptation; myogenesis     

Correlation of relaxation time with water content in muscle and brain tissues

Proton T₁ of water in muscle and brain tissues was studied as a function of water content at 25°C and 5, 30, and 100 MHz. Muscle samples were bathed in modified Krebs solutions that have the same ionic strength but different osmolarity in order to change the water content. At all three frequencies their 1/T₁ extrapolated to the same value of 0.6 s^?1 for zero dry weight, indicating that T₁ for the majority of water molecules in normal muscle and brain is frequency-independent. Dehydrated muscle, homogenized brain tissue suspended in modified Krebs solution and dehydrated brain tissue provide samples with wide ranges of water content. Their T₁ values at all three frequencies can be well accounted for by a simple equation derived from the two-state model.     

Mouse meningiocytes express Sox2 and yield high efficiency of chimeras after nuclear reprogramming with exogenous factors

Induced pluripotent stem cell technology, also termed iPS, is an emerging approach to reprogram cells into an embryonic stem cell-like state by viral transduction with defined combinations of factors. iPS cells share most characteristics of embryonic stem cells, counting pluripotency and self-renewal, and have so far been obtained from mouse and humans, including patients with genetic diseases. Remarkably, autologous transplantation of cell lineages derived from iPS cells will eliminate the possibility of immunological rejection, as well as current ethical issues surrounding human embryonic stem cell research. However, before iPS can be used for clinical purposes, technical problems must be overcome. Among other considerations, full and homogeneous iPS reprogramming is an important prerequisite. However, despite the fact that cells from several mouse tissues can be successfully induced to iPS, the overall efficiency of chimera formation of these clones remains low even if selection for Oct4 or Nanog expression is applied. In this report, we demonstrate that cells from the mouse meningeal membranes express elevated levels of the embryonic master regulator Sox2 and are highly amenable to iPS. Meningeal iPS clones, generated without selection, are fully and homogeneously reprogrammed based on DNA methylation analysis and 100% chimera competent. Our results define a population of somatic cells that are ready to undergo iPS, thus highlighting a very attractive cell type for iPS research and application.     

Pericytes of the brain microvasculature express gamma-glutamyl transpeptidase.

The expression of gamma-glutamyl transpeptidase (GGT) is a specific property of the brain capillary endothelium that constitutes the blood-brain barrier. We report here the detection of GGT, not only in endothelial cells, but also in pericytes, demonstrating that a brain capillary-specific pericyte population exists. We raised antibodies to GGT using a porcine brain microvessel GGT-protein-A (staphylococcal protein A) fusion protein as antigen which was expressed in Escherichia coli. The immunohistochemical analysis of the subcapillary distribution of GGT in porcine brain cortex and cerebellum sections by both light and electron microscopy revealed the expression of GGT in the capillary-adjacent pericytes in addition to the GGT-positive endothelial layer. We confirmed these data for cultured porcine brain microvascular endothelial cells and pericytes. GGT immunofluorescence could be detected in both cell types in culture. Endothelial cells exhibited a weak staining, whereas pericytes were strongly positive for GGT. Due to the high phagocytotic activity of pericytes and their location on the abluminal surface of the microvessels, we propose a possible protective or detoxifying function of GGT in cerebrovascular pericytes.     

Homer/vesl proteins and their roles in CNS neurons

Since their initial discovery in 1997, Homer/Vesl proteins have become increasingly investigated as putative regulators of receptor and ion-channel function in the central nervous system. Within a relatively brief period, numerous research reports have described manifold effects of Homer proteins, including the modulation of the trafficking of type I metabotropic glutamate receptors (mGluRs), axonal pathfinding, mGluR coupling to calcium and potassium channels, agonist-independent mGluR activity, ryanodine receptor regulation, locomotor activity, and behavioral plasticity. This review summarizes our current knowledge on the induction, expression, and structure of the various forms of Homer proteins, as well as their roles in neuronal function. In addition, we provide an outlook on novel developments with regard to the involvement of Homer-1a in hippocampal synaptic function.