A single centrifugation method for isolating fat droplets from cells and tissues

Fat droplets (FDs) have important roles in cellular energy regulation. Isolating FDs from either cells or tissue continues to be important for studying these organelles. Here, we describe a procedure wherein whole homogenates of cultured cells or tissue are fractionated with a single centrifugation step in a standard microcentrifuge. This procedure reproducibly yields three fractions highly enriched in either FDs, soluble cellular components, or sedimentable organelles/membranes.     

Distribution of the inducer of differentiation bis-acetyl-diaminopentane in murine erythroleukemia cells

Exposure of murine leukemia cells in culture to bis-acetyl-diaminopentane (BADP) caused erythroid maturation as measured by the accumulation of hemoglobin in treated cells. The appearance of differentiated cells in cultures exposed to BADP occurred 18 to 20 hours earlier than in those treated with dimethylsulfoxide (DMSO), a standard inducer of differentiation in this system. Studies with [³H]BADP indicated the occurrence of relatively rapid association of the inducer with cells, and subsequent linear accumulation. Fractionation of cellular components and measurement of radioactivity from BADP therein demonstrated that this agent preferentially associates with a fraction enriched for plasma membrane. In addition, [³H]BADP was capable of binding to the plasma membrane-enriched fraction isolated from murine erythroleukemia cells as measured by gel filtration. These findings support the concept that interaction of inducers of murine erythroleukemia differentiation such as BADP with components of the surface membrane may be important in the cascade of events that lead to the erythroid maturation of these leukemic cells.     

Molecular and cellular studies on the absorption,function, and safety of food components in intestinal epithelial cells

The intestinal tract comes into direct contact with the external environment despite being inside the body. Intestinal epithelial cells, which line the inner face of the intestinal tract, have various important functions, including absorption of food substances, immune functions such as cytokine secretion, and barrier function against xenobiotics by means of detoxification enzymes. It is likely that the functions of intestinal epithelial cells are regulated or modulated by these components because they are frequently exposed to food components at high concentrations. This review summarizes our research on the interaction between intestinal epithelial cells and food components at cellular and molecular levels. The influence of xenobiotic contamination in foods on the cellular function of intestinal epithelial cells is also described in this review.     

Evidence for alteration of the membrane-bound ribosomes in Micrococcus luteus cells exposed to lead

Micrococcus luteus cells exposed to Pb(NO₃)₂ contained cytosol ribosomal particles and disaggregated membranal ribosomal particles as determined by ultracentrifugation and spectral studies. Approx. 60% of the membrane ribosome fraction from lead exposed cells had a sedimentation value of 8.4S. Cytosol ribosomes from lead exposed cells as well as membranal and cytosol ribosomes from control cells were comparable by their contents of predominantly the 70S type with the 50S and 100S present in relatively small amounts. The lead content of the 8.4S component was more than 200 times higher than the components with higher sedimentation coefficients from lead exposed cells and approx. 650 times more than that of control cell ribosomes. The cells exposed to lead, however, showed no adverse effects from the lead in respect to their growth rates and cellular yields. These results indicate that lead is interacting only at specific sites of the membrane and is inducing events initiated only in strategic cellular regions. These data further substantiate that subtle changes do occur in lead exposed cells that show no obvious effects. It is assumed that these ‘minor’ alterations are, in toto, biologically significant.     

Analysis of the Mechanism of Gram Differentiation by Use of a Filter-Paper Chromatographic Technique.

Bartholomew, J. W. (University of Southern California, Los Angeles), Thomas Cromwell, and Richard Gan. Analysis of the mechanism of Gram differentiation by use of a filter-paper chromatographic technique. J. Bacteriol. 90:766-777. 1965.-Data are presented which demonstrate that the mechanism of gram-positivity could not be due solely to factors such as a single, specific gram-positive substrate, specific affinities of crystal violet for certain cellular components, a specific crystal violet-iodine-substrate complex, or to any specific characteristic of the dye, iodine, or solvent molecules. Ruptured cells of gram-positive organisms stain gram-negatively when subjected to a standard Gram-stain procedure. However, when stained fragments of broken cells were deposited in thick layers on the surface of filter-paper strips and exposed to decolorizers, the rate of dye release correlated with the Gram characteristic of the intact cell. Therefore, the intact cell in itself is not an absolute requirement for Gram differentiation. The data are interpreted as indicating that the mechanism of Gram differentiation primarily involves the rate of permeation of molecules (dye, iodine, solvent) through the interstitial spaces of cell-wall material.     

Remote effects at cell and subcell level in the hemopoietic system after chronic radiation exposure in man

The parameters characterizing the state of hemopoietic cells obtained from chronically exposed residents of the Techa riverside villages studied at late time after the exposure included: the level of somatic mutations in the TCR gene, the level of chromosome aberrations, the intensity of peripheral blood lymphocyte apoptosis. Exposed versus unexposed subjects (controls) showed an increased frequency of CD3-CD4+ T-lymphocytes, chromosome aberrations of stable type (translocations) and unstable type (dicentrics, rings), and also increased intensity of lymphocyte apoptosis. The findings of tests using a standard additional gamma-irradiation (1 Gy) accompanied by 24-hour incubation indicated that the rate of apoptosis of lymphocytes was significantly higher in exposed individuals in comparison with unexposed ones. It was suggested basing on the obtained data that at late time the chronic (for over 50 years) exposure at RBM doses from 0.01 to 3.22 Sv was a factor inducing the damage to the genetic apparatus of hemopoietic cells. Evidently, the initial chronic low-intensity irradiation in the above-indicated dose range activates adaptive processes at the cellular level in hemopoietic cells. Late time after the onset of exposure the adaptation reserves are depleted in chronically exposed persons which brings about its failure in the case of a challenge by additional external exposures.     

Appraisal of the methodology and applications for measurement of the zinc content of blood components as indicators of zinc status

Methods for the measurement of the zinc content of blood components (plasma, erythrocytes, platelets, mononuclear leukocytes, and polymorphonuclear neutrophils) as indicators of the zinc status of animals have been reviewed and evaluated. The values of plasma or serum zinc concentrations as indicators of zinc status in experimental animals or humans is questionable. Consequently, the zinc content of blood cellular components has been suggested as better indicators of zinc status. Methodological problems, such as incomplete cellular separation or zinc contamination, occur in some of the procedures applied to the quantitation of blood cellular component zinc. Specialized microprobe procedures involving, for example, proton-induced X-ray fluorescence, scanning ion technology, mass spectrometery, or laser microprobe mass analysis might be used to measure the absolute zinc level in individual cells subjected to minimal handling. Then, more conventional methods can be tested against the true standard. Nevertheless, there appears to be species differences in regard to the response of blood cellular zinc concentrations to dietary zinc deficiency. Blood cellular component zinc is conserved during severe zinc deficiency in the rat. In contrast, the zinc content of blood cellular components in humans may reflect the whole body zinc status.     

Effects of cyclic mechanical stimulation of the cellular components of the heart: In vitro

Summary The response of the cellular components of the heart to cyclic mechanical stimulation is of particular importance because these cells are continually subjected to mechanical forces as a result of changes in blood volume and pressure. To directly investigate how mechanical tension affects these cellular components of the heart, an in vitro system that exposes the particular cell type (cardiac myocytes, endothelial cells, or fibroblasts) to a calibrated increase in cyclical linear stretch was developed. Cells were grown on silastic membranes coated with laminin and subjected to a 10% cyclical distention 10 times a minute for 72 h. Within 24 h of being exposed to the mechanical stretch, the cells became elongated and oriented perpendicular to the direction of the stretch. These results indicate that cyclical mechanical stimulation directly influences the cellular organization of the heart cells in vitro. This work was supported in part by grants HL 33656 and HL 24935 from the National Institutes of Health, Bethesda, MD.     

A microfluidic biochip for the nanoporation of living cells

This paper deals with the development of a microfluidic biochip for the exposure of living cells to nanosecond pulsed electric fields (nsPEF). When exposed to ultra short electric pulses (typical duration of 3-10ns), disturbances on the plasma membrane and on the intra cellular components occur, modifying the behavioral response of cells exposed to drugs or transgene vectors. This phenomenon permits to envision promising therapies. The presented biochip is composed of thick gold electrodes that are designed to deliver a maximum of energy to the biological medium containing cells. The temporal and spectral distributions of the nsPEF are considered for the design of the chip. In order to validate the fabricated biochip ability to orient the pulse towards the cells flowing within the exposition channels, a frequency analysis is provided. High voltage measurements in the time domain are performed to characterize the amplitude and the shape of the nsPEF within the exposition channels and compared to numerical simulations achieved with a 3D Finite-Difference Time-Domain code. We demonstrate that the biochip is adapted for 3 ns and 10 ns pulses and that the nsPEF are homogenously applied to the biological cells regardless their position along the microfluidic channel. Furthermore, biological tests performed on the developed microfluidic biochip permit to prove its capability to permeabilize living cells with nanopulses. To the best of our knowledge, we report here the first successful use of a microfluidic device optimized for the achievement and real time observation of the nanoporation of living cells.     

Arachidonic acid and Docosahexanoic acid enhance platelet formation from human apheresis-derived CD34+ cells

An Aberration in megakaryopoiesis and thrombopoiesis, 2 important processes that maintain hemostasis, leads to thrombocytopenia. Though platelet transfusions are used to treat this condition, blood banks frequently face a shortage of platelets. Therefore, methods to generate platelets on a large scale are strongly desirable. However, to generate megakaryocytes (MKs) and platelets (PLTs) in numbers sufficient for clinical application, it is essential to understand the mechanism of platelet production and explore efficient strategies accordingly. We have earlier reported that the N-6 and N-3 poly-unsaturated fatty acids (PUFAs), Arachidonic acid (AA)/Docosahexanoic acid (DHA) have beneficial effect on the generation of MKs and PLTs from umbilical cord blood derived CD34⁺ cells. Here we tested if a similar effect is observed with peripheral blood derived CD34⁺ cells, which are more commonly used in transplantation settings. We found a significant enhancement in cell numbers, surface marker expression, cellular ploidy and expression of cytoskeletal components during PLT biogenesis in cultures exposed to media containing AA/DHA than control cultures that were not exposed to these PUFAs. The test cells engrafted more efficiently in NOD/SCID mice than control cells. AA/DHA appears to have enhanced MK/PLT generation through upregulation of the NOTCH and AKT pathways. Our data show that PUFAs could be valuable additives in the culture system for large scale production of platelets for clinical applications.     

P0 and PMP22 mark a multipotent neural crest-derived cell type that displays community effects in response to TGF-beta family factors.

Protein zero (P0) and peripheral myelin protein 22 (PMP22) are most prominently expressed by myelinating Schwann cells as components of compact myelin of the peripheral nervous system (PNS), and mutants affecting P0 and PMP22 show severe defects in myelination. Recent expression studies suggest a role of P0 and PMP22 not only in myelination but also during embryonic development. Here we show that, in dorsal root ganglia (DRG) and differentiated neural crest cultures, P0 is expressed in the glial lineage whereas PMP22 is also detectable in neurons. In addition, however, P0 and PMP22 are both expressed in a multipotent cell type isolated from early DRG. Like neural crest stem cells (NCSCs), this P0/PMP22-positive cell gives rise to glia, neurons and smooth-muscle-like cells in response to instructive extracellular cues. In cultures of differentiating neural crest, a similar multipotent cell type can be identified in which expression of P0 and PMP22 precedes the appearance of neural differentiation markers. Intriguingly, this P0/PMP22-positive progenitor exhibits fate restrictions dependent on the cellular context in which it is exposed to environmental signals. While single P0/PMP22-positive progenitor cells can generate smooth muscle in response to factors of the TGF-(beta) family, communities of P0/PMP22-positive cells interpret TGF-(beta) factors differently and produce neurons or undergo increased cell death instead of generating smooth-muscle-like cells. Our data are consistent with a model in which cellular association of postmigratory multipotent progenitors might be involved in the suppression of a non-neural fate in forming peripheral ganglia.     

Metallothionein as a clonable high-density marker for cryo-electron microscopy

Cryo-electron microscopy is expanding its scope from macromolecules towards much larger and more complex cellular specimens such as organelles, cells and entire tissues. While isolated macromolecular specimens are typically composed of only very few different components that may be recognized by their shape, size or state of polymerization, cellular specimens combine large numbers of proteinaceous structures as well as nucleic acids and lipid arrays. Consequently, an unambiguous identification of these structures within the context of a whole cell may create a very difficult challenge. On plastic-embedded specimens, or Tokuyasu sections (Tokuyasu, 1980), epitopes that are exposed at the surface can be tagged by antibodies. However, vitrified sections have to be kept at strict cryo-conditions (below -140 °C) and therefore do not allow any post-sectioning treatment of the specimens other than data acquisition in the microscope. Hence, the labels have to be placed into the specimen before freezing. Here we report on the application of a small metal-clustering protein, metallothionein (MTH), as a clonable label capable of clustering metal atoms into a high-density particle with high spatial resolution. We tested MTH as a label for kinesin-decorated microtubules (MTs) as well as the building blocks of desmin intermediate filaments (IFs).     

71-Kilodalton Heat Shock Cognate Protein Acts as a Cellular Receptor for Syncytium Formation Induced by Human T-Cell Lymphotropic Virus Type 1

We previously reported that the region corresponding to amino acids 197 to 216 of the gp46 surface glycoprotein (gp46-197) served as a binding domain for the interaction between gp46 and trypsin-sensitive membrane components of the target cell, leading to syncytium formation induced by human T-cell lymphotropic virus type 1 (HTLV-1)-bearing cells. Our new evidence shows that the 71-kDa heat shock cognate protein (HSC70) acts as a cellular receptor for syncytium formation. Using affinity chromatography with the peptide gp46-197, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we isolated three components (bands A, B, and C) from MOLT-4 cell lysate which exhibited specific interactions with gp46 and inhibitory activities for syncytium formation induced by HTLV-1-bearing cells. Band A and B components were identified as HSC70 and β-actin, respectively, through amino acid sequencing by tandem mass spectrometry and immunostaining with specific monoclonal antibodies. Band C is likely to be a nonprotein component, because full activity for syncytium formation was seen after extensive trypsin digestion. Anti-HSC70 monoclonal antibody clearly blocked syncytium formation in a coculture of HTLV-1-bearing cells and indicator cells, whereas no inhibition was seen with anti-β-actin monoclonal antibody. Furthermore, flow cytometric analysis indicated that anti-HSC70 antibody reacted with MOLT-4 cells. Thus, we propose that HSC70 expressed on the target cell surface acts as a cellular acceptor to gp46 exposed on the HTLV-1-infected cell for syncytium formation, thereby leading to cell-to-cell transmission of HTLV-1.     

The expression of hex A and hex B isozymes of hexosaminidase in parental and experimental human fibroblast cells and their components

The expression of the two major isozyme forms of hexosaminidase (EC 3.2.1.30), hesoxaminidase A and hexosaminidase B, has been examined. The parental cells and/or cellular components of parental cells are individually fused using inactivated Sendai virus with the aid of a micromanipulator. The progeny cells produced from such hybrids are subjected to a microenzymatic assay which allows measurements at the single cell level. The lysosomal-deficient cells used in this study are Tay-Sachs and Sandhoff fibroblasts, and the normal cells used are WI-38 (fetal lung fibroblasts), amniotic fluid cells (GM 473), and JASD₃ (normal human foreskin). The results show that the ratio of cell components which are fused to form the experimental cell affects the percentage of hexosaminidase A expressed in the progeny cells. Furthermore, our results imply the presence of a “factor” in the Sandhoff cell's cytoplasm which, together with the Tay-Sachs nucleus, is necessary for hexosaminidase A expression in the experimental cell's progeny.     

Effects of extremely low frequency (50 Hz) magnetic field on morphological and biochemical properties of human keratinocytes

We investigated the effects on human keratinocytes (HaCaT) of exposure to a sinusoidal magnetic field of 2 mT (50 Hz). These cells are a good model for studying interaction of nonionising radiation, because they are not shielded from fields in vivo and also because they are resistant to both mechanical and thermal stimuli. We performed scanning microscopy which showed modification in shape and morphology in exposed cells. This modification is related to differential actin distribution as revealed by phalloidin fluorescence analysis. Moreover, the exposed cells show increased clonogenic capacity, as well as increased cellular growth as showed by clonogenicity assays and growth curves. Indirect immunofluorescence analysis using a fluorescent antibody against involucrin and beta4 integrin, which are respectively differentiation and adhesion markers, revealed an increase of involucrin expression and segregation of beta4 integrin in the cell membrane in cells exposed to 50 Hz; a higher percentage of the exposed cells shows a modified pattern of adhesion and differentiation markers. We also present evidence that exposure of HaCaT cells can interfere with protein kinase activity. Our observations confirm the hypothesis that electromagnetic fields at 50 Hz may modify cell membrane morphology and interfere with initiation of the signal cascade pathway and cellular adhesion.     

Biotin and choline replace the growth requirement of Madin-Darby canine kidney cells for high-density lipoproteins

MDCK cells maintained on extracellular matrix (ECM)-coated dishes and exposed to Dulbecco's modified Eagle's medium (DME) supplemented with transferrin and either high-density lipoproteins (HDLs) or phosphatidyl choline (PC) liposomes have a growth rate and final cell density similar to those of cultures exposed to serum-supplemented DME. When MDCK cells are exposed to a medium consisting of a mixture (1:1) of DME and F12 medium (D/F), the addition of transferrin (10 μg/ml) alone supports cell growth and the presence of HDLs or PC liposomes is no longer required. MDCK cells exposed to D/F medium supplemented with transferrin can be passaged for more than 50 generations in total absence of serum. The F12 components that support growth in the absence of HDLs or PC liposomes are biotin (which is absent in DME) and choline (which is present in insufficient concentration in DME). Supplementation of DME with transferrin, biotin (3.6 ng/ml), and choline (10 μg/ml) allows optimal growth of MDCK cells and permits serial propagation through more than 50 generations. The growth requirement of MDCK cells for HDLs or PC liposomes can therefore be replaced by adequate concentrations of biotin and choline. The widely observed fact that a combination of DME/F12 medium is more effective than DME alone in supporting cell growth may be due in part to the lack of biotin and suboptimal choline concentration in DME.     

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling^1,2, while LRRK2 is implicated in the pathogenesis of Parkinson''s disease^3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with ³²P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing ³²P-orthophosphate. The ³²P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (³²P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.