Mild Sonication Research Articles

Regulation of WRN Protein Cellular Localization and Enzymatic Activities by SIRT1-mediated Deacetylation

Werner syndrome is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutations of the WRN gene. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Sir2, an NAD-dependent histone deacetylase, has been proven to extend life span in yeast and Caenorhabditis elegans. Mammalian Sir2 (SIRT1) has also been found to regulate premature cellular senescence induced by the tumor suppressors PML and p53. SIRT1 plays an important role in cell survival promoted by calorie restriction. Here we show that SIRT1 interacts with WRN both in vitro and in vivo; this interaction is enhanced after DNA damage. WRN can be acetylated by acetyltransferase CBP/p300, and SIRT1 can deacetylate WRN both in vitro and in vivo. WRN acetylation decreases its helicase and exonuclease activities, and SIRT1 can reverse this effect. WRN acetylation alters its nuclear distribution. Down-regulation of SIRT1 reduces WRN translocation from nucleoplasm to nucleoli after DNA damage. These results suggest that SIRT1 regulates WRN-mediated cellular responses to DNA damage through deacetylation of WRN.

Thermal oxidative cutting of multi-walled carbon nanotubes

Commercially available, multi-walled carbon nanotubes grown by CVD are usually inherently entangled, but can be separated by cutting. However, most cutting methods both cause damage to the nanotubes and involve a lengthy work-up procedure. The use of abrupt, repeated exposure to oxidising conditions in air proved to be an efficient (68% yield) means of producing material with open ends, moderate functionalisation, and enhanced solvent dispersibility; the average lengths were reduced from over 5 μm to approximately 650 nm. Additionally, the character of the surface oxides can be tuned to have either an acidic or basic character by using a simple thermal treatment. These approaches could be deliberately integrated into conventional CVD processes, but also have implications for the products of standard nanotube syntheses. Raman spectroscopy and electron microscopy were used to study the impact of cutting on the intrinsic graphitic structure and the length distribution. X-ray photoelectron spectroscopy was used to determine the extent of functionalisation. The cut carbon nanotubes were dispersed in dimethylformamide (DMF), a Lewis basic solvent, and chloroform, a Lewis acidic solvent, using mild sonication. Through the use of an experimentally determined extinction coefficient ( ε = 35.10 ml mg −1 cm −1), the relative dispersibility of the cut and functionalised carbon nanotubes in DMF and chloroform was determined.

Mechanistic Studies of MDM2-mediated Ubiquitination in p53 Regulation

As a central regulator for cell cycle arrest, apoptosis, and cellular senescence, p53 requires multiple layers of regulatory control to ensure correct temporal and spatial functions. It is well accepted that Mdm2-mediated ubiquitination plays a crucial role in p53 regulation. In addition to proteasome-mediated degradation, ubiquitination of p53 by Mdm2 acts a key signal for its nuclear export. Nuclear export has previously been thought to require the disassociation of the p53 tetramer and exposure of the intrinsic nuclear export signal. To elucidate the molecular mechanism of degradation-independent repression on p53 by Mdm2, we have developed a two-step approach to purify ubiquitinated forms of p53 induced by Mdm2 from human cells. Surprisingly, however, we found that ubiquitination has no effect on the tetramerization/oligomerization of p53, arguing against this seemingly well accepted model. Moreover, nuclear export of p53 alone is not sufficient to completely abolish p53 activity. Ubiquitination-mediated repression of p53 by Mdm2 acts at least, in part, through inhibiting the sequence-specific DNA binding activity. Thus, our results have important implications regarding the mechanisms by which Mdm2 acts on p53.

Biomimetic Growth of Biomorphic CaCO3 with Hierarchically Ordered Cellulosic Structures

Biomorphic CaCO3 with different hierarchically ordered micro- and nanostructures was fabricated with natural cellulose substances as the host templates. The fabrication involves two mild sonication processes in which calcium and carbonate ions are sequentially added to the template, together with subsequent calcination in air. The specific pseudo-1D and pseudo-2D structures of the resultant crystalline calcite can be tailored by mimicking both natural and artificially woven cellulosic substances, with the former following the ribbon/tube form of cellulose fiber and the latter following the netlike architectures of the woven cotton cloth. The building block of these structures is a layer of CaCO3 grains grown on the surfaces of each cellulose fiber. By choosing Ca2+, CO32-, or HCO3- ions as the first adsorption species on the cellulose template, we show that the resultant CaCO3 grain size can be fine-tuned in a nanoscopic scale, most probably due to the differences in the nature of ion adsorption on the cellulose molecules and the resultant CaCO3 nucleation and growth. The impact of this new route is that we can precisely predict the morphologies of the final CaCO3 products that were not realized in other chemical approaches.

Priming effects in soil size fractions of a podzol Bs horizon after addition of fructose and alanine

AbstractIt is well known that the addition of easily available substrates to soils can affect microbial activity and thus the mineralization of soil organic carbon (SOC). Up to now, little is known about the processes leading to these priming effects and which fractions of organic matter (OM) are affected. The objectives of this study were to determine if SOC associated with isolated soil size fractions showed different susceptibility to priming effects, whether these pools are easily depleted, or whether the amount of substrate addition affects the extent of priming effects. In an incubation experiment, the effect of the uniformly 14C‐labeled substrates fructose and alanine on the mineralization of the SOC of a Bs horizon of a Haplic Podzol was investigated. The soil sample was fractionated into the three soil size fractions sand, silt, and clay by a mild sonication followed by sieving and sedimentation. Additionally, nonfractionated soil of the horizon was included in the experiment. Every soil sample received four substrate additions repeated at weekly intervals with 3.325 μg substrate‐C (mg SOC)–1 and a final addition of 13.3 μg substrate‐C (mg SOC)–1 after 4 weeks. The respiration was determined hourly and 14CO2 was analyzed every 2, 4, and 7 d after the respective substrate addition. After 56 d, between 42% and 58% of the added substrates had been mineralized. Both substrates strongly increased the mineralization of the OM in all fractions (positive priming effects). The priming effects were always higher after the addition of the high substrate dose than during the first 4 weeks when four small doses were added. In general, the priming effects increased with decreasing particle size. Alanine generally caused higher priming effects than fructose in the soil size fractions (up to 280% vs. 231%, respectively). This indicates that alanine serves not only as an energy substrate but also as a N source and, thus, also promotes microbial growth. The strong priming effects in the silt and clay fraction (133% and 125% with fructose, 172% and 168% with alanine) showed, that not only the labile pool of OM is affected, but also a more stable pool characterized by higher 14C ages. We assume that the stability of the OM in these fractions is not only due to recalcitrance or to interactions with the minerals, but that it may also be caused by a substrate limitation of the degrading microorganisms.

Functional Heterogeneity of Photosystem II in Domain Specific Regions of the Thylakoid Membrane of Spinach (Spinacia oleracea L.)

A mild sonication and phase fractionation method has been used to isolate five regions of the thylakoid membrane in order to characterize the functional lateral heterogeneity of photosynthetic reaction centers and light harvesting complexes. Low-temperature fluorescence and absorbance spectra, absorbance cross-section measurements, and picosecond time-resolved fluorescence decay kinetics were used to determine the relative amounts of photosystem II (PSII) and photosystem I (PSI), to determine the relative PSII antenna size, and to characterize the excited-state dynamics of PSI and PSII in each fraction. Marked progressive increases in the proportion of PSI complexes were observed in the following sequence: grana core (BS), whole grana (B3), margins (MA), stroma lamellae (T3), and purified stromal fraction (Y100). PSII antenna size was drastically reduced in the margins of the grana stack and stroma lamellae fractions as compared to the grana. Picosecond time-resolved fluorescence decay kinetics of PSII were characterized by three exponential decay components in the grana fractions, and were found to have only two decay components with slower lifetimes in the stroma. Results are discussed in the framework of existing models of chloroplast thylakoid membrane lateral heterogeneity and the PSII repair cycle. Kinetic modeling of the PSII fluorescence decay kinetics revealed that PSII populations in the stroma and grana margin fractions possess much slower primary charge separation rates and decreased photosynthetic efficiency when compared to PSII populations in the grana stack.

Ecto‐glycanases and metabolic stability of the capsule in Cryptococcus neoformans

We have identified a number of ecto-glycanases (glycosylhydrolases) associated with the capsule and/or the cell wall of Cryptococcus neoformans. The enzyme activities detected included alpha-mannosidase, alpha-, and beta -glucosidase, alpha-, and beta-galactosidase, beta-xylosidase, beta-glucuronidase, and endo-beta-1,3-glucanase. Small portions of the enzymes were also secreted into the growth medium. Cell-wall associated endo-beta-1,3-glucanases exhibited highest activity in the acidic range between pH 2.5 and 5.0. The products of laminarin hydrolysis by the enzymes located on the cell surface were glucose and beta-1,3-linked glucooligosaccharides. The same products were released from isolated cell walls incubated in the buffer. Endo-beta-1,3-glucanase activity extracted from the cell surfaces by mild sonication consisted of six isoforms separable by isoelectric focusing. In spite of the presence of the whole array of glycanase activities on the cell surfaces, capsular polysaccharides released from C. neoformans cells into the growth medium were practically metabolically stable. From the defined polysaccharides tested, only laminarin (beta-1,3-glucan) and to some extent also mixed-linkage beta-1,3/beta-1,4-glucan and/or 4-O-methyl-D-glucurono-D-xylan were able to support the yeast growth. The activities of majority of identified ecto-glycanases were low when the yeast was grown on glucose but were considerably elevated when the cells were grown on glycerol indicating that their synthesis is regulated by catabolite repression.

Robust hybrid thin films that incorporate lamellar phospholipid bilayer assemblies and transmembrane proteins

This study describes facile methods based on sol-gel processing for the formation of robust thin films that incorporate phospholipid bilayer membranes and transmembrane proteins as multilamellar assemblies in cross-linked silica matrices. Transmission electron microscopy and x-ray diffraction were used to examine the lamellar structure of the hybrid thin films containing1, 2-dioleyl-sn-glycero-3-phospoethanolamine (DOPE), an unsaturated lipid, and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), a saturated lipid. While the d spacing measured for DOPE containing films varied (from 35 to 48 A) depending on the amount of DOPE added to the coating solution (10-1 wt %), similar changes were not observed for the films containing saturated lipid, DMPC (d spacing approximately 43 A). Addition of purple membrane containing bacteriorhodopsin to the DOPE/silica coating solution led to the formation of multilamellar vesicle-like structures within the thin films. Mild sonication of these solutions containing the purple membrane prior to coating led to the formation thin films with planar multilamellar structures that exhibit uniform d spacing. The study further investigates the effects of incorporation of gramicidin and sonication on the structure of hybrid films and speculates on the eventual application of thin films prepared in this manner.

Reducing Liposome Size with Ultrasound: Bimodal Size Distributions

Sonication is a simple method for reducing the size of liposomes. We report the size distributions of liposomes as a function of sonication time using three different techniques. Liposomes, mildly sonicated for just 30 sec, had bimodal distributions when surface-weighted with modes at about 140 and 750 nm. With extended sonication, the size distribution remains bimodal but the average diameter of each population decreases and the smaller population becomes more numerous. Independent measurements of liposome size using Dynamic Light Scattering (DLS), transmission electron microscopy (TEM), and the nystatin/ergosterol fusion assay all gave consistent results. The bimodal distribution (even when number-weighted) differs from the Weibull distribution commonly observed for liposomes sonicated at high powers over long periods of time and suggests that a different mechanism may be involved in mild sonication. The observations are consistent with the following mechanism for decreasing liposome size. During ultrasonic irradiation, cavitation, caused by oscillating microbubbles, produces shear fields. Large liposomes that enter these fields form long tube-like appendages that can pinch-off into smaller liposomes. This proposed mechanism is consistent with colloidal theory and the observed behavior of liposomes in shear fields.

The Protozoan Parasite Cryptosporidium parvum Possesses Two Functionally and Evolutionarily Divergent Replication Protein A Large Subunits

Very little is known about protozoan replication protein A (RPA), a heterotrimeric complex critical for DNA replication and repair. We have discovered that in medically and economically important apicomplexan parasites, two unique RPA complexes may exist based on two different types of large subunit RPA1. In this study, we characterized the single-stranded DNA binding features of two distinct types (i.e. short and long) of RPA1 subunits from Cryptosporidium parvum (CpRPA1A and CpRPA1B). These two proteins differ from human RPA1 in their intrinsic single-stranded DNA binding affinity (K) and have significantly lower cooperativity (omega). We also identified the RPA2 and RPA3 subunits from C. parvum, the latter of which had yet to be reported to exist in any protozoan. Using fluorescence resonance energy transfer technology and pull-down assays, we confirmed that these two subunits interact with each other and with CpRPA1A and CpRPA1B. This suggests that the heterotrimeric structure of RPA complexes may be universally conserved from lower to higher eukaryotes. Bioinformatic analyses indicate that multiple types of RPA1 are present in the other apicomplexans Plasmodium and Toxoplasma. Apicomplexan RPA1 proteins are phylogenetically more related to plant homologues and probably arose from a single gene duplication event prior to the expansion of the apicomplexan lineage. Differential expression during the life cycle stages in three apicomplexan parasites suggests that the two RPA1 types exercise specialized biological functions.

Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor 1 Signaling Requires G Protein-coupled Receptor Kinase 2 Binding to the Second Intracellular Loop

Metabotropic glutamate receptors (mGluRs) are members of a unique class of G protein-coupled receptors (class III) that include the calcium-sensing and gamma-aminobutyric acid type B receptors. The activity of mGluRs is regulated by second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). The attenuation of both mGluR1a and mGluR1b signaling by GRK2 is phosphorylation- and beta-arrestin-independent and requires the concomitant association of GRK2 with both the receptor and Galpha(q/11). G protein interactions are mediated, in part, by the mGluR1 intracellular second loop, but the domains required for GRK2 binding are unknown. In the present study, we showed that GRK2 binds to the second intracellular loop of mGluR1a and mGluR1b and also to the mGluR1a carboxyl-terminal tail. Alanine scanning mutagenesis revealed a discrete domain within loop 2 that contributes to GRK2 binding, and the mutation of either lysine 691 or 692 to an alanine within this domain resulted in a loss of GRK2 binding to both mGluR1a and mGluR1b. Mutation of either Lys(691) or Lys(692) prevented GRK2-mediated attenuation of mGluR1b signaling, whereas the mutation of only Lys(692) prevented GRK2-mediated inhibition of mGluR1a signaling. Thus, the mGluR1a carboxyl-terminal tail may also be involved in regulating the signaling of the mGluR1a splice variant. Taken together, our findings indicated that kinase binding to an mGluR1 domain involved in G protein-coupling is essential for the phosphorylation-independent attenuation of signaling by GRK2.

A diversity of bacteriophage forms and genomes can be isolated from the surface sands of the Sahara Desert

The surface sands of the Sahara Desert are exposed to extremes of ultraviolet light irradiation, desiccation and temperature variation. Nonetheless, the presence of bacteria has recently been demonstrated in this environment by cultivation methods and by 16S rDNA analyses from total DNA isolated from surface sands. To discern the presence of bacteriophages in this harsh environment, we searched for extracellular phages and intracellularly located phages present as prophages or within pseudolysogens. Mild sonication of the sand, in different liquid culture media, incubated with and without Mitomycin-C, was followed by differential centrifugation to enrich for dsDNA phages. The resulting preparations, examined by electron microscopy, revealed the presence of virus-like particles with a diversity of morphotypes representative of all three major double-stranded DNA bacteriophage families (Myoviridae, Siphoviridae and Podoviridae). Moreover, pulsed-field gel electrophoresis of DNA, extracted from the enriched bacteriophage preparations, revealed the presence of distinct bands suggesting the presence of putative dsDNA phage genomes ranging in size from 45 kb to 270 kb. Characterization of the bacteriophages present in the surface sands of the Sahara Desert extends the range of environments from which bacteriophages can be isolated, and provides an important point of departure for the study of phages in extreme terrestrial environments.

Functionalization of Multiwalled Carbon Nanotubes by Mild Aqueous Sonication

Sonication has been widely used in the dispersal of carbon nanotubes (CNTs) in various liquids as well as in their functionalization in aqueous acids. Here, for the first time, we study the sonication of multiwalled CNTs (MWCNTs) in deionized water. Our results indicate an improvement in the aqueous dispersal of MWCNTs as well as an increase in their adhesive interaction with Au substrates. Field emission scanning electron and high-resolution transmission electron microscopies as well as X-ray photoelectron, photoacoustic Fourier transform IR, and Raman spectroscopies have shown this to be due to the production of low concentrations of O-containing functionalizations (alcohol, carbonyl, acid, with the total O concentration being approximately 2%), without damaging the basic CNT structure; this production of functional groups is mirrored by the disappearance of -CH(n) groups existing on the pristine CNTs. These new functional groups are capable of hydrogen bonding, which plays an important role in their aqueous dispersal and enhanced substrate interactions.

Activation of Stat3 Sequence-specific DNA Binding and Transcription by p300/CREB-binding Protein-mediated Acetylation

Signal transducers and activators of transcription (Stat) belong to a family of latent cytoplasmic factors that can be activated by tyrosine phosphorylation by members of the Jak tyrosine kinase family in response to a variety of cytokines and growth factors. Activated Stats form dimers and translocate into nucleus to induce expression of critical genes essential for normal cellular events. Here we report for the first time that Stat3 can be modified by acetylation both in vivo and in vitro. A major site of Stat3 that is acetylated by its coactivator, p300/CREB-binding protein (CBP), resides in the C-terminal transcriptional activation domain at lysine 685. Furthermore, the acetylation of Stat3 can stimulate its sequence-specific DNA binding ability and transactivation activity. Inhibition of histone deacetylase activity in cells results in increased Stat3 nuclear localization. These observations clearly indicate a novel mechanism for Stat3 activation in mammalian cells.

Spectroscopic Evidence for π−π Interaction between Poly(diallyl dimethylammonium) Chloride and Multiwalled Carbon Nanotubes

The interaction between multiwalled carbon nanotubes (MWCNTs) and aqueous poly(diallyl dimethylammonium) chloride (PDDA) was studied by X-ray photoelectron (XPS) and photoacoustic Fourier transform infrared (PA-FTIR) spectroscopies. We have found that the mild sonication of MWCNTs in aqueous PDDA results in a significant improvement of CNT dispersibility and greatly enhances their adhesion to Au and Si substrates. The MWCNT-PDDA interaction is due to the presence of an unsaturated contaminant in the PDDA chain, as confirmed by both XPS and PA-FTIR, which enters into a pi-pi interaction with the CNTs. Electrostatic group repulsions of the coated CNTs then provide the dispersibility and adhesion.

Synthesis and Self-assembly Properties of Acylated Cyclodextrins and Nitrilotriacetic Acid (NTA)-modified Inclusion Ligands for Interfacial Protein Crystallization

Three different β-cyclodextrins, peracylated on the primary hydroxymethyl rim of the cyclodextrin (6-C n )7β-CD, and nine different nitrilotriacetic acid-modified inclusion ligands (R-NTA) have been prepared and their self-assembly properties characterized. These modular amphiphiles have been developed to promote two-dimensional crystallization of proteins at the lipid-water interface. Pressure-area isotherm data suggest that the occupancy of the host sites within the (6-C n )7β-CD monolayers vary as a function of the known host-guest binding constant for the unmodified β-CD host and R guest substituents. Negative-stain TEM experiments show that vesicles are formed upon mild sonication of (6-C10)7β-CD and (6-C16)7β-CD in 10 mM Tris, pH 7.4 buffer. These results indicate that (6-C n )7β-CD forms stable lyotropic phases that may be useful for templating the interfacial crystallization of histidine-tagged proteins or other molecules capable of interacting with the R-NTA guest ligands.

The plasma membrane is the site of selective phosphatidylserine oxidation during apoptosis: role of cytochrome C.

Phosphatidylserine (PS) externalization, a functional end point of apoptosis that triggers phagocytic recognition of dying cells, may be modulated by oxidative stress in biological membranes. We previously observed selective oxidation of PS during apoptosis, but the intracellular location and molecular mechanisms responsible for PS oxidation remain to be described. Peroxidation in individual classes of cellular phospholipids was monitored in whole cells and various subcellular fractions obtained from HL-60 cells undergoing apoptosis in response to tert-butyl hydroperoxide (t-BuOOH) after metabolic acylation of phospholipids with the oxidation-sensitive fluorescent fatty acid, cis-parinaric acid. Nonrandom selective oxidation of PS was observed in whole cells, as well as in plasma membrane. PS in mitochondria appeared selectively resistant to oxidation during apoptosis. All phospholipids in nuclear membranes appeared resistant to oxidation after t-BuOOH treatment. Selective PS oxidation was accompanied by cytochrome c release and PS externalization. PS oxidation and externalization were followed by caspase activation and other end points of apoptosis. HL-60 cells "loaded" with exogenous cytochrome c by mild sonication showed selective oxidation of PS in both the absence and presence of t-BuOOH. Cytochrome c/hydrogen peroxide could effectively oxidize purified PS but not phosphatidylcholine in a cell-free model system. Selective plasma membrane-based PS oxidation and subsequent externalization during oxidant-induced apoptosis may be mediated through the redox activity of cytochrome c.

Fate of postacrosomal perinuclear theca recognized by monoclonal antibody MN13 after sperm head microinjection and its role in oocyte activation in mice.

Monoclonal antibody (mAb) MN13 labels mouse sperm head postacrosomal perinuclear theca (PT), which is possibly involved in oocyte activation during fertilization. The antigenic site is expressed after mild sonication followed by treatment with dithiothreitol (DTT) or heat (45 degrees C), and is visible as a thick band in the postacrosomal region. The presence of protease inhibitors in the sonication medium suppresses the exposure of MN13 epitope (MN13p), suggesting the involvement of a proteolytic reaction in this process. Spermatozoa do not express MN13p after the induction of acrosome exocytosis by Ca(2+) ionophore, zona binding, or during zona penetration, a strategy that ensures safe delivery of postacrosomal PT proteins to oocytes after fusion. MN13 labeling was not detectable during fertilization by zona-free in vitro fertilization, suggesting that the antigenic site does not react with proteolytic enzymes during sperm-oocyte fusion and the antibody does not recognize the nascent epitope. Microinjection of sperm heads prepared by sonication and DTT treatment led to the activation of metaphase II oocytes. The oocyte activating function of such sperm heads was significantly diminished after labeling with MN13 prior to intracytoplasmic sperm injection (ICSI), but labeling with antiequatorin antibody MN9 activated oocytes with a frequency similar to that of unlabeled sperm heads. The sperm heads in inactive oocytes formed premature chromosome condensations (PCCs), which were invested by independent metaphase-like spindles. These observations indicate that the postacrosomal PT recognized by mAb MN13 is involved in oocyte activation. MN13p is dissociated from sperm heads during the early stages of decondensation after ICSI. In activated oocytes, MN13-labeled fine granules were redistributed in the midzone spindle region, whereas in inactive oocytes they formed a ring around the polar regions of the metaphase II and PCC spindles.

Mechanical damage of roots provides enhanced colonization of the wheat endorhizosphere by the dinitrogen-fixing cyanobacterium Nostoc sp. strain 2S9B

When wheat (Triticum aestivum L.) seedlings are co-cultivated with Nostoc sp. strain 2S9B in hydroponics, the cyanobacterium colonizes the endorhizosphere at low frequency. Mild sonication of the roots dramatically increased the number of cyanobacteria within the root tissue. The cyanobacteria penetrated the roots in the form of motile filaments (hormogonia), and once inside, they divided and transformed into aseriate packages, which showed nitrogenase activity. Cyanobacteria were found in the intercellular spaces as well as inside the epidermal and cortex cells. By prolonging the sonication time the number of cyanobacterial packages inside the roots increased. However, after reaching a certain abundance the total cyanobacterial biomass within the roots did not change regardless of the sonication time. Besides sonication, other methods of root surface damage also brought about enhanced internal root colonization. Sonication of roots proved to be an efficient way of amplifying the internal root colonization by cyanobacteria and can be suggested as a method for creating a model system in non-legume plant-microbe studies.

Tris(3-mercaptopropyl)-N-glycylaminomethane as a new linker to bridge antibody with metal particles for biological cell separations.

Conjugates of nickel beads with CD8 and anti-red blood cell KC16 antibody were prepared by using the aminotrithiolate "spider" ligand, tris(3-mercaptopropyl)-N-glycylaminomethane, in its new function as a linker between the surface of nickel beads and antibody via activation of spider ligand attached to nickel beads with the common, heterobifunctional cross-linker, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). Raw nickel beads were cleaned by either mild sonication in a bath or by stronger probe sonication to remove surface nickel oxide layers, before attachment of the spider ligand. Scanning electron micrographs of the nickel beads before and after probe sonication showed a marked change from a corrugated to a smooth bead surface. Analyses of the supernatants of conjugation mixtures for antibody gave surface densities of 2.5-5.2 mg/m(2) for CD8 and 0.6-12 mg/m(2) for KC16 antibody runs. The antibody-spider-nickel bead conjugates were used in magnetic bead depletions of targeted CD8+ lymphocytes or red blood cells (rbcs) in whole blood of normal donors. For CD8 cell depletions, the undepleted controls and supernatants of depleted samples were analyzed for CD8/CD4 cell populations by flow cytometry with appropriate fluorescent antibody markers. Enumeration of red blood cells, white blood cells (wbcs), and platelets (plts) in undepleted controls and supernatants of depleted samples were carried out on appropriate hematology counters. Whole blood titer results with various lots of either CD8-spider-nickel or KC16-spider-nickel bead conjugates showed varying degrees of depletion ability as indicated by bead-to-cell ratios of 2-32 for CD8 beads and by rbc-to-bead ratios of 1.2-10 for KC16 beads. Moreover, varying degrees of specificity of CD8 beads for CD8+ cells over CD4+ cells and of KC16 beads for rbcs over white blood cells and platelets were observed from the normalized nontargeted cell population figures in undepleted controls versus supernatants of depleted samples.