Cell membrane patches are supported by proteoglycans

A cell membrane patch isolated on a patch clamp pipette incorporates in addition to the phospholipid bilayer, an extracellular matrix and cytoskeletal components. The significance of the extracellular matrix for the patch formation was studied in aortic smooth muscle and cerebellar granule cells grow in the presence of an inhibitor of proteoglycan synthesis, -d -xyloside. The xyloside improved the seal success rate, and after patch excision membrane vesicles were formed instead of inside-out patches. When amphotericin B was included in the pipette solution, perforated outside-out vesicles were formed in 96% of cells. The findings suggest, that membrane patches are supported by the extracellular matrix or by structures that relate to this matrix.     

Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody

A novel monoclonal antibody, SM/C-2.6, specific for mouse muscle satellite cells was established. SM/C-2.6 detects mononucleated cells beneath the basal lamina of skeletal muscle, and the cells co-express M-cadherin. Single fiber analyses revealed that M-cadherin+ mononucleated cells attaching to muscle fibers are stained with SM/C-2.6. SM/C-2.6+ cells, which were freshly purified by FACS from mouse skeletal muscle, became MyoD+ in vitro in proliferating medium, and the cells differentiated into desmin+ and nuclear-MyoD+ myofibers in vitro when placed under differentiation conditions. When the sorted cells were injected into mdx mouse muscles, donor cells differentiated into muscle fibers. Flow cytometric analyses of SM/C-2.6+ cells showed that the quiescent satellite cells were c-kit-, Sca-1-, CD34+, and CD45-. More, SM/C-2.6+ cells were barely included in the side population but in the main population of cells in Hoechst dye efflux assay. These results suggest that SM/C-2.6 identifies and enriches quiescent satellite cells from adult mouse muscle, and that the antibody will be useful as a powerful tool for the characterization of cellular and molecular mechanisms of satellite cell activation and proliferation.     

Mass spectrometry images acylcarnitines,phosphatidylcholines, and sphingomyelin in MDA-MB-231 breast tumor models

The lipid compositions of different breast tumor microenvironments are largely unknown due to limitations in lipid imaging techniques. Imaging lipid distributions would enhance our understanding of processes occurring inside growing tumors, such as cancer cell proliferation, invasion, and metastasis. Recent developments in MALDI mass spectrometry imaging (MSI) enable rapid and specific detection of lipids directly from thin tissue sections. In this study, we performed multimodal imaging of acylcarnitines, phosphatidylcholines (PC), a lysophosphatidylcholine (LPC), and a sphingomyelin (SM) from different microenvironments of breast tumor xenograft models, which carried tdTomato red fluorescent protein as a hypoxia-response element-driven reporter gene. The MSI molecular lipid images revealed spatially heterogeneous lipid distributions within tumor tissue. Four of the most-abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1), and PC(18:0/18:1), were localized in viable tumor regions, whereas LPC(16:0/0:0) was detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoylcarnitine, stearoylcarnitine, PC(16:0/22:1), and SM(d18:1/16:0) sodium adduct, which colocalized primarily with hypoxic tumor regions. For the first time, we have applied a multimodal imaging approach that has combined optical imaging and MALDI-MSI with ion mobility separation to spatially localize and structurally identify acylcarnitines and a variety of lipid species present in breast tumor xenograft models.     

Optical imaging of pancreatic beta cells in living mice expressing a mouse insulin I promoter-firefly luciferase transgene

We generated transgenic mice expressing firefly (Photinus pyralis) luciferase (luc) under the control of the mouse insulin I promoter (MIP). The mice have normal glucose tolerance and pancreatic islet architecture. The luciferase-expressing beta cells can be readily visualized in living mice using whole-body bioluminescent imaging. The MIP-luc transgenic mice may be useful for monitoring changes in beta cell function or mass in living animals with normal or altered metabolic states.     

Cell-mediated fas-based lysis of dendritic cells which are apparently resistant to anti-Fas antibody

In this report, the controversy concerning the sensitivity of dendritic cells (DCs) to Fas-dependent induction of apoptosis was examined using murine DCs. Although DCs could not be lysed when exposed to an anti-Fas antibody, Jo2, the observed resistance turned out to reflect their lack of the expression of Fc(gamma)R necessary for crosslinking the antibody, rather than their intrinsic resistance. Thus, at least a fraction of DCs was sensitive to Jo2 in the presence of Fc(gamma)R-expressing by-standers. Consistently, a significant fraction of DCs was sensitive to Fas-dependent lysis mediated by T cells including the antigen-specific killing by CD4+ T cells. Both immature (class II MHClow) and mature (class II MHChigh) DCs were sensitive to the Fas-based induction of apoptosis.     

O-methylated glycans from Toxocara are specific targets for antibody binding in human and animal infections

The parasitic helminth Toxocara canis is a widely distributed nematode of mammals. Larval parasites, which infect a wide range of hosts including mice and humans, export glycosylated macromolecules bearing novel methylated oligosaccharide structures, similar to the mammalian blood group antigen H but bearing one or two O-methylated substitutions on the terminal fucose and subterminal galactose residues. We have studied the reactivity of synthetic forms of these glycans to parasite-specific antibodies and mammalian immune system lectins. Murine antibodies, generated to T. canis infection, predominantly recognise the mono-O-methylated form with the beta-configuration of the GalNAc residue (MoMbeta), and antibodies are entirely IgM isotype. The mAb Tcn-2 reproduces this pattern, and shows little reactivity to either the alpha isomer (MoMalpha) or the di-O-methylated form (DiM). Antibodies generated to helminth infections other than T. canis were unreactive with the glycans, except antibodies to other members of the Toxocara genus. Hence, the carbohydrate structures represent immunogenic, genus-specific antigens. Antibodies from human toxocariasis patients are reactive with the same sugars, although preferentially towards DiM. Sera from unrelated helminth infections do not react, confirming the status of these structures as Toxocara-specific glycans. The human dendritic cell lectin, DC-SIGN, was found to bind both Toxocara excretory/secretory products and mammalian blood group antigen H3. However, DC-SIGN did not bind the synthetic glycans, indicating additional non-methylated carbohydrates may also play a role in the interaction between T. canis and its host.     

Ceramide and sphingomyelin species of fibroblasts and neurons in culture

The ceramide (Cer) and sphingomyelin (SM) species of cultured differentiated rat cerebellar granule cells and human fibroblasts were characterized by electrospray ionization-mass spectrometry. We identified 35 different species of Cer and 18 species of SM in human fibroblasts, and 35 different species of Cer and 9 species of SM were characterized in rat neurons. The main Cer species of rat cerebellar granule cells contained d18:1 sphingosine linked with palmitic, stearic, or nervonic fatty acid, and the two main SM species were d18:1,16:0 and d18:1,18:0. Both sphingolipids were enriched in detergent-resistant membranes (DRMs; or lipid rafts), and significant differences were found in the sphingolipid patterns of DRMs and of detergent-soluble fractions (DSF) from these cells. In human fibroblasts, the main Cer species were d18:1,16:0, d18:2,16:0, d18:1,24:0, d18:2,24:0, d18:1,24:1, and d18:2,24:1; the most represented species of SM were d18:1,16:0, d18:1,24:0, and d18:1,24:1. In these cells, SM was highly enriched in DRMs and Cer was mainly associated with DSF, and the species found in DRMs were markedly different from those found in DSF.     

Yeast actin patches are networks of branched actin filaments

Cortical actin patches are the most prominent actin structure in budding and fission yeast. Patches assemble, move, and disassemble rapidly. We investigated the mechanisms underlying patch actin assembly and motility by studying actin filament ultrastructure within a patch. Actin patches were partially purified from Saccharomyces cerevisiae and examined by negative-stain electron microscopy (EM). To identify patches in the EM, we correlated fluorescence and EM images of GFP-labeled patches. Patches contained a network of actin filaments with branches characteristic of Arp2/3 complex. An average patch contained 85 filaments. The average filament was only 50-nm (20 actin subunits) long, and the filament to branch ratio was 3:1. Patches lacking Sac6/fimbrin were unstable, and patches lacking capping protein were relatively normal. Our results are consistent with Arp2/3 complex-mediated actin polymerization driving yeast actin patch assembly and motility, as described by a variation of the dendritic nucleation model.     

A Lipidomic Study of the Effects of N-methyl-N＇-nitro-N-nitrosoguanidine on Sphingomyelin Metabolism

Systems biology is a new and rapidly developing research area in which, by quantitatively describing the interaction among all the individual components of a cell, a systems-level understanding of a biological response can be achieved. Therefore, it requires high-throughput measurement technologies for biological molecules, such as genomic and proteomic approaches for DNA/RNA and protein, respectively.Recently, a new concept, lipidomics, which utilizes the mass spectrometry （MS） method for lipid analysis,has been proposed. Using this lipidomic approach, the effects of N-methyl-N＇-nitro-N-nitrosoguanidine （MNNG） on sphingomyelin metabolism, a major class of sphingolipids, were evaluated. Sphingomyelin molecules were extracted from cells and analyzed by matrix-assisted laser desorption ionization-time of flight MS. It was found that MNNG induced profound changes in sphingomyelin metabolism, including the appearance of some new sphingomyelin species and the disappearance of some others, and the concentrations of several sphingomyelin species also changed. This was accompanied by the redistribution of acid sphingomyelinase （ASM）, a key player in sphingomyelin metabolism. On the other hand, imipramine, an inhibitor of ASM,caused the accumulation of sphingomyelin. It also prevented some of the effects of MNNG, as well as the redistribution of ASM. Taken together, these data suggested that the lipidomic approach is highly effective for the systematic analysis of cellular lipids metabolism.     

Mitochondria are targets of photodynamic therapy

Photodynamic Therapy (PDT) is an evolving cancer treatment that depends on three known and variable components: photosensitizer, light and oxygen. Optimization of these variables yields reactive oxygen species, mainly singlet oxygen, that damage cellular components leading to cytotoxicity. Our research has demonstrated that porphyrin sensitizers, in particular, significantly inhibit the inner mitochondrial membrane enzymes cytochrome c oxidase and F ₀ F ₁ ATP synthase. These results were obtained from an in vivo-in vitro experimental protocol that exposes sensitizers to metabolic and pharmacokinetic events. The resulting inhibition of oxidative phosphorylation was expected to reduce ATP levels, which were quantitated in cells and were confirmed by ³¹P-NMR spectroscopy of tumors in situ in animals treated with PDT. Based on these findings, and more recent investigations of apoptosis, there is little doubt that mitochondria are critical targets in the actions of PDT.     

Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry

An advanced two-dimensional liquid chromatography/mass spectrometry platform was used to quantify individual host cell proteins (HCPs) present at various purification steps for several therapeutic monoclonal antibodies (mAbs) produced in Chinese hamster ovary cells. The methodology produced reproducible identifications and quantifications among replicate analyses consistent with a previously documented individual limit of quantification of ~13 ppm. We were able to track individual HCPs from cell culture fluid to protein A eluate pool to subsequent viral inactivation pool and, in some cases, further downstream. Approximately 500 HCPs were confidently identified in cell culture fluid and this number declined progressively through the purification scheme until no HCPs could be confidently identified in polishing step cation-exchange eluate pools. The protein A eluate pool of nine different mAbs contained widely differing numbers, and total levels, of HCPs, yet the bulk of the total HCP content in each case consisted of a small subset of normally intracellular HCPs highly abundant in cell culture fluid. These observations hint that minimizing cell lysis during cell culture/harvest may be useful in minimizing downstream HCP content. Clusterin and actin are abundant in the protein A eluate pools of most mAbs studied. HCP profiling by this methodology can provide useful information to process developers and lead to the refinement of existing purification platforms.     

Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry

An advanced two-dimensional liquid chromatography/mass spectrometry platform was used to quantify individual host cell proteins (HCPs) present at various purification steps for several therapeutic monoclonal antibodies (mAbs) produced in Chinese hamster ovary cells. The methodology produced reproducible identifications and quantifications among replicate analyses consistent with a previously documented individual limit of quantification of ~13 ppm. We were able to track individual HCPs from cell culture fluid to protein A eluate pool to subsequent viral inactivation pool and, in some cases, further downstream. Approximately 500 HCPs were confidently identified in cell culture fluid and this number declined progressively through the purification scheme until no HCPs could be confidently identified in polishing step cation-exchange eluate pools. The protein A eluate pool of nine different mAbs contained widely differing numbers, and total levels, of HCPs, yet the bulk of the total HCP content in each case consisted of a small subset of normally intracellular HCPs highly abundant in cell culture fluid. These observations hint that minimizing cell lysis during cell culture/harvest may be useful in minimizing downstream HCP content. Clusterin and actin are abundant in the protein A eluate pools of most mAbs studied. HCP profiling by this methodology can provide useful information to process developers and lead to the refinement of existing purification platforms.     

Serum sphingomyelin levels are related to the clearance of postprandial remnant-like particles

It is known that sphingomyelin (SM) content is higher in apolipoprotein B-containing particles (BLps) than in high density lipoproteins and that BLp levels, including chylomicrons and their remnant particles, are positively related to atherosclerosis. To evaluate the relationship between serum SM and postprandial remnant particle levels, we determined SM, triglyceride (TG), and cholesterol levels in serum and in remnant-like particles (RLPs) before and 3, 5, 7, and 10 h after a high-fat meal in 31 healthy subjects. We found that serum SM, like serum TG, was increased to its maximum 3 h after fat loading and then gradually decreased to basal levels after 10 h. More important, we determined that SM and TG levels in RLPs were parallel. Serum SM was positively correlated with serum TG (P <0.001), RLP SM (P <0.001), RLP TG (P <0.001), and RLP cholesterol (P <0.001) levels. It is our conclusion that serum SM is a marker for the clearance of RLPs.     

A differential cell capture assay for evaluating antibody interactions with cell surface targets

Many biological and biomedical laboratory assays require the use of antibodies and antibody fragments that strongly bind to their cell surface targets. Conventional binding assays, such as the enzyme-linked immunosorbent assay (ELISA) and flow cytometry, have many challenges, including capital equipment requirements, labor intensiveness, and large reagent and sample consumption. Although these techniques are successful in mainstream biology, there is an unmet need for a tool to quickly ascertain the relative binding capabilities of antibodies/antibody fragments to cell surface targets on the benchtop at low cost. We describe a novel cell capture assay that enables several candidate antibodies to be evaluated quickly as to their relative binding efficacies to their cell surface targets. We used chimeric rituximab and murine anti-CD20 monoclonal antibodies as cell capture agents on a functionalized microscope slide surface to assess their relative binding affinities based on how well they capture CD20-expressing mammalian cells. We found that these antibodies’ concentration-dependent cell capture profiles correlate with their relative binding affinities. A key observation of this assay involved understanding how differences in capture surfaces affect the assay results. This approach can find utility when an antibody or antibody fragment against a known cell line needs to be selected for targeting studies.     

Primary and secondary vegetation patches as contributors to floristic diversity in a tropical deciduous forest landscape

The floristic diversity of Mexican tropical deciduous forests (TDF) is of critical importance given the high species richness (alpha diversity), species turnover (beta diversity), and the intense deforestation rates. Currently, most TDF landscapes are mosaics of agricultural land, secondary vegetation, and patches of relatively undisturbed primary vegetation. Here we illustrate how both primary forest remnants and secondary vegetation patches contribute to the floristic diversity of TDF in a landscape of volcanic origin in central Veracruz, Mexico. Our objectives were to assess sampling efficiency and inventory completeness, to compare mean and cumulative species richness between primary forest and secondary vegetation sites, and to analyze beta diversity between vegetation types. In an area of 12,300 m2 we recorded 105 families, 390 genera, and 682 species. Species inventories for both vegetation types were about 80% complete. Secondary vegetation is more alpha diverse than primary forest, both in terms of cumulative and mean species richness. We found a remarkably high beta diversity between vegetation types (75% of complementarity, 91.60% of mean dissimilarity). We also identified the species that contribute the most to similarity within vegetation types and to dissimilarity between vegetation types. Our results support the idea that assessing biodiversity on the landscape scale is an appropriate way to ascertain the impact of human activities. For this land mosaic, conservation of the flora would not be possible by focusing solely on primary forest remnants. We propose the implementation of a network of small conservation areas with a flexible structure, following the “archipelago reserve” model.     

Monoclonal antibody K312-based depletion of pluripotent cells from differentiated stem cell progeny prevents teratoma formation

Human pluripotent stem cells (PSCs) have been utilized as a promising source in regenerative medicine. However, the risk of teratoma formation that comes with residual undifferentiated PSCs in differentiated cell populations is most concerning in the clinical use of PSC derivatives. Here, we report that a monoclonal antibody (mAb) targeting PSCs could distinguish undifferentiated PSCs, with potential teratoma-forming activity, from differentiated PSC progeny. A panel of hybridomas generated from mouse immunization with H9 human embryonic stem cells (hESCs) was screened for ESC-specific binding using flow cytometry. A novel mAb, K312, was selected considering its high stem cell-binding activity, and this mAb could bind to several human induced pluripotent stem cells and PSC lines. Cell-binding activity of K312 was markedly decreased as hESCs were differentiated into embryoid bodies or by retinoic acid treatment. In addition, a cell population negatively isolated from undifferentiated or differentiated H9 hESCs via K312 targeting showed a significantly reduced expression of pluripotency markers, including Oct4 and Nanog. Furthermore, K312-based depletion of pluripotent cells from differentiated PSC progeny completely prevented teratoma formation. Therefore, our findings suggest that K312 is utilizable in improving stem cell transplantation safety by specifically distinguishing residual undifferentiated PSCs.     

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.     

Identification of mouse sphingomyelin synthase 1 as a suppressor of Bax-mediated cell death in yeast

We have identified mouse sphingomyelin synthase 1 as a novel suppressor of the growth inhibitory effect of heterologously expressed Bax. Yeast cells expressing sphingomyelin synthase 1 were also found to show an increased resistance to a variety of cytotoxic stimuli including hydrogen peroxide, osmotic stress and elevated temperature. Sphingomyelin synthase 1 functions by catalyzing the conversion of ceramide and phosphatidylcholine to sphingomyelin and diacylglycerol. Ceramide is an antiproliferative and proapoptotic sphingolipid whose level increases in response to a variety of stresses. Consistent with its biochemical function, yeast cells expressing sphingomyelin synthase 1 have an enhanced ability to grow in media containing the cell-permeable C2-ceramide analog as well as the ceramide precursor phytosphingosine. We also show that overexpression of AUR1, a potential yeast functional homolog of sphingomyelin synthase, also protects cells from osmotic stress. Taken together, these results suggest that sphingomyelin synthase 1 likely prevents cell death by counteracting stress-mediated accumulation of endogenous sphingolipids.