Interaction between pathogenic amebas and fibronectin: substrate degradation and changes in cytoskeleton organization

Invasion of human tissues by the parasitic protozoan Entamoeba histolytica is a multistep process involving, as a first step, the recognition of surface molecules on target tissues by the amebas or trophozoites. This initial contact is followed by the release of proteolytic and other activities that lyse target cells and degrade the extracellular matrix. In other parasitic diseases, as well as in certain cancers, the interaction of invasive organisms or cells with fibronectin (FN) through specific receptors has been shown to be the initial step in target cell recognition. Interaction with FN triggers the release of proteolytic activities necessary for the effector cell migration and invasion. Here, we describe the specific interaction of Entamoeba histolytica trophozoites with FN, and identify a 37-kD membrane peptide as the putative receptor for FN. The interaction between the parasite and FN leads to a response reaction that includes the secretion of proteases that degrade the bound FN and the rearrangement of amebic actin into "adhesion plates" at sites of contact with FN-coated surfaces. The kinetics of the interaction was determined by measuring the binding of soluble 125I-FN to the trophozoites and visualization of the bound protein using specific antibodies. Degradation of FN was measured by gel electrophoresis and the release of radioactivity into the incubation medium. Focal degradation of FN was visualized as black spots under the trophozoites at contact sites with fluorescent FN. We conclude that the interaction of E. histolytica with FN occurs through a specific surface receptor. The interaction promotes amebic cytoskeleton changes and release of proteases from the parasite. The binding and degradation of extracellular matrix components may facilitate the migration and penetration of amebas into tissues, causing the lesions seen in human hosts.     

Human-Xenopus chimeras of Gsα reveal a new region important for its activation of adenylyl cyclase

G proteins are heterotrimeric GTPases that play a key role in signal transduction. The α subunit of G_s bound to GTP is capable of activating adenylyl cyclase. The amino acid sequences derived from two X. laevis cDNA clones that apparently code for G_sα subunits are 92% identical to those found in the short form of human G_sα. Despite this high homology, the X. laevis G_sα clones expressed in vitro, yielded a protein that are not able to activate the adenylyl cyclase present in S49 cyc⁻ membranes in contrast with human G_sα similarly expressed. This finding suggested that the few amino acid substitutions found in the amphibian subunit are important in defining the functionality of the human G_sα. The construction of chimeras composed of different fractions of the cDNAs of the two species was adopted as an approach in determining the regions of the molecule important in its functionality in this assay. Four pairs of chimeras were constructed using reciprocal combinations of the cDNAs coding for human and Xenopus G_sα. These eight constructs were expressed in vitro and equivalent amounts of the resulting proteins were assayed in the activation of adenylyl cyclase with GTPγs and isoproterenol. The results obtained here clearly indicate that the Gα sequence that extends from amino acid 70 to 140, is important for the functionality of human G_sα in activating adenylyl cyclase.     

Participation of Rho, ROCK-2, and GAP activities during actin microfilament rearrangements in Entamoeba histolytica induced by fibronectin signaling

In Entamoeba histolytica little is known about the microfilament rearrangements formed by actin and ABPs. Fibronectin regulates many aspects of cell behavior involving the actin cytoskeleton and members of the Rho family of small GTPases. Using trophozoites interacted with fibronectin and glass, we present evidence related with the formation and regulation of different microfilament rearrangements and their cellular distribution, the effect of actin affecting drugs on these arrangements, and on trophozoites adhesion; we also demonstrate that actin isoforms are induced after adhesion, and also the selective participation of specific actin binding proteins such as ABP-120 and phospho-paxillin, regarding their location in the different actin structures. In addition, we show results that confirm the participation of EhRho, ROCK-2, and GAP activities. We propose that fibronectin induced signaling in E. histolytica trophozoites have important consequences in the actin cytoskeleton that may affect its behavior during the invasive process in the host.     

Gαo/i-stimulated proteosomal degradation of RGS20: A mechanism for temporal integration of Gs and Gi pathways

The G_s and G_i pathways interact to control the levels of intracellular cAMP. Although coincident signaling through G_s and G_i-coupled receptors can attenuate G_s-stimulated cAMP levels, it is not known if prior activation of the G_i pathway can affect signaling by G_s-coupled receptors. We have found that activated Gα_o/i interact with RGS20, a GTPase activating protein for members of the Gα_ο/i family. Interaction between Gα_o/i and RGS20 results in decreased cellular levels of RGS20. This decrease was induced by activated Gα_o and Gα_i2 but not by Gα_q, Gα_i1 or Gα_i3. The Gα_o/i-induced decrease in RGS20 can be blocked by proteasomal inhibitors lactacystin or MG132. Activated Gα_o stimulates the ubiquitination of RGS20. The serotonin-1A receptor that couples to G_o/i reduces the levels of RGS20 and this effect is blocked by lactacystin, suggesting that G_o/i promotes the degradation of RGS20. Expression of RGS20 attenuates the inhibition of β-adrenergic receptor-induced cAMP levels mediated by the serotonin-1A receptor. Prior activation of the serotonin-1A receptor results in loss of the RGS20-mediated attenuation, and the loss of attenuation is blocked when lactacystin is included during the prior treatment. These observations suggest that G_o/i-coupled receptors, by stimulating the degradation of RGS20, can regulate how subsequent activation of the G_s and G_i pathways controls cellular cAMP levels, thus allowing for signal integration.     

Differentiation of Entamoeba histolytica: a possible role for enolase

The study of the encystation process of Entamoeba histolytica has been hampered by the lack of experimental means of inducing mature cysts in vitro. Previously we have found that cytoplasmic vesicles similar to the encystation vesicles of Entamoeba invadens are present in E. histolytica trophozoites only in amebas recovered from experimental amebic liver abscesses. Here we report that a monoclonal antibody (B4F2) that recognizes the cyst wall of E. invadens also identifies a 48 kDa protein in vesicles of E. histolytica trophozoites recovered from hepatic lesions. This protein is less expressed in trophozoites continuously cultured in axenical conditions. As previously reported for E. invadens, the B4F2 specific antigen was identified as enolase in liver-recovered E. histolytica, by two-dimensional electrophoresis, Western blot and mass spectrometry. In addition, the E. histolytica enolase mRNA was detected by RT PCR. The antigen was localized by immunoelectron microscopy in cytoplasmic vesicles of liver-recovered amebas. The B4F2 antibody also recognized the wall of mature E. histolytica cysts obtained from human samples. These results suggest that the enolase-containing vesicles are produced by E. histolytica amebas, when placed in the unfavorable liver environment that could be interpreted as an attempt to initiate the encystation process.     

Characterization of a Cytochalasin D-Resistant Mutant of Entamoeba histolytica

Characterization of a cytochalasin D-resistant mutant of the human parasite Entamoeba histolytica capable of growing at 10 μM cytochalasin is described. The mutant cells also show resistance to 5 mM colchicine and 100 μM cytochalasin B, drugs proved deleterious for wild type trophozoites. The mutants show increased osmotic fragility and electric mobility but reduced phagocytic activity, and agglutination by Concanavalin A. On the other hand pinocytic activity remains unaltered when compared with the wild type cells. Polymerized actin, seen by staining with phalloidin, often appears polarized to one end of the trophozoites and forms few of the endocytic invaginations found in wild type amebas. An altered distribution of part of the actin could explain the differences in surface properties and motility observed in the mutant amebas.     

Characterization of a cytochalasin D-resistant mutant of Entamoeba histolytica

Characterization of a cytochalasin D-resistant mutant of the human parasite Entamoeba histolytica capable of growing at 10 microM cytochalasin is described. The mutant cells also show resistance to 5 mM colchicine and 100 microM cytochalasin B, drugs proved deleterious for wild type trophozoites. The mutants show increased osmotic fragility and electric mobility but reduced phagocytic activity, and agglutination by Concanavalin A. On the other hand pinocytic activity remains unaltered when compared with the wild type cells. Polymerized actin, seen by staining with phalloidin, often appears polarized to one end of the trophozoites and forms few of the endocytic invaginations found in wild type amebas. An altered distribution of part of the actin could explain the differences in surface properties and motility observed in the mutant amebas.     

巨桉和天竺桂幼树对不同浓度SO2的光合生理响应

二氧化硫是大气主要污染物之一,可对植物的关键生理过程光合作用产生重要影响。利用密闭环境控制室熏气处理,研究不同浓度(自然状态下浓度、0.5mg·L-1、1.5mg·L-1、3.0mg·L-1)SO2对盆栽巨桉和天竺桂幼树叶绿素含量、光响应曲线、光合特征参数、光合日变化及硫含量的影响。结果表明:(1)SO2胁迫显著减少了巨桉叶绿素a、b含量,且叶绿素a/b值显著降低,而天竺桂在SO2胁迫下叶绿素a、b含量显著增加,叶绿素a/b值无显著影响。(2)SO2胁迫显著抑制了两树种的净光合速率(Pn);在SO2胁迫下巨桉气孔导度(Gs)、胞间CO2浓度(Ci)和蒸腾速率(Tr)显著上升,而天竺桂的Gs和Tr显著被SO2抑制,Ci随SO2浓度的增加先升高后降低。(3)巨桉表观量子效率(AQY)、暗呼吸速率(Rd)、光补偿点(LCP)和光饱和点(LSP)及天竺桂Rd和LCP均随着SO2浓度的增加而先升高后降低,而天竺桂的AQY和LSP逐渐降低。(4)一天中,SO2胁迫显著提高了巨桉Pn、Gs和Tr,而对天竺桂Pn无显著影响,较低浓度SO2胁迫显著促进了天竺桂Gs和Tr,高浓度SO2胁迫则显著抑制其Gs和Tr;SO2胁迫显著抑制了两种植物的Ci。(5)SO2胁迫下,巨桉和天竺桂幼树叶片硫含量均显著增加。研究认为,巨桉对较低浓度的SO2胁迫有一定的适应能力,但对高浓度SO2胁迫的抗性不如天竺桂强,这可能与二者不同的叶片形态及生理特性有关。     

甘肃马先蒿寄生对禾草内生真菌共生体光合特性的影响

[目的]甘肃马先蒿与感染内生真菌的禾草(紫花针茅和麦宾草)建立根寄生关系,有关内生真菌对根寄生危害禾草光合作用调控方面的研究较少。[方法]本研究以紫花针茅和麦宾草带菌(E+)、不带菌(E-)植株为研究对象,研究甘肃马先蒿寄生和未寄生处理对紫花针茅和麦宾草E+、E-植株不同生长阶段光合特性影响的动态变化。[结果]甘肃马先蒿寄生显著降低紫花针茅和麦宾草的净光合速率、蒸腾速率和气孔导度,而胞间二氧化碳浓度和水分利用率却显著增加,这与禾草是否感染内生真菌无关。甘肃马先蒿寄生后E+紫花针茅的净光合速率、气孔导度和蒸腾速率高于E-植株,而麦宾草E+植株的净光合速率、气孔导度和蒸腾速率却低于E-植株;同时,根寄生条件下E+紫花针茅的胞间二氧化碳浓度和水分利用率低于E-植株;而E-麦宾草植株的胞间二氧化碳浓度和水分利用率却低于E+植株。[结论]内生真菌侵染影响甘肃马先蒿根寄生危害禾草的光合作用;甘肃马先蒿和内生真菌同时成为禾草营养消耗库时,内生真菌与禾草的共生关系处于一种互惠共生和相互拮抗的动态变化。     

All-atom molecular dynamics simulations reveal significant differences in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc1 complexes

Antimycin A is the most frequently used specific and powerful inhibitor of the mitochondrial respiratory chain. We used all-atom molecular dynamics (MD) simulations to study the dynamic aspects of the interaction of antimycin A with the Q_i site of the bacterial and bovine bc₁ complexes embedded in a membrane. The MD simulations revealed considerable conformational flexibility of antimycin and significant mobility of antimycin, as a whole, inside the Q_i pocket. We conclude that many of the differences in antimycin binding observed in high-resolution x-ray structures may have a dynamic origin and result from fluctuations of protein and antimycin between multiple conformational states of similar energy separated by low activation barriers, as well as from the mobility of antimycin within the Q_i pocket. The MD simulations also revealed a significant difference in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc₁ complexes. The strong hydrogen bond between antimycin and conserved Asp-228 (bovine numeration) was observed to be frequently broken in the bacterial bc₁ complex and only rarely in the bovine bc₁ complex. In addition, the distances between antimycin and conserved His-201 and Lys-227 were consistently larger in the bacterial bc₁ complex. The observed differences could be responsible for a weaker interaction of antimycin with the bacterial bc₁ complex.     

Extracellular matrix-induced signaling in Entamoeba histolytica: its role in invasiveness

Entamoeba histolytica trophozoites can invade the mucosa of the host large intestine to cause disease. Here, Isaura Meza describes recent work indicating that the trophozoites respond to the binding of extracellular matrix proteins by activating receptor-mediated signal transduction pathways. Activation of phosphokinase C or adenylyl cyclase induces protein phosphorylation, actin gene expression and reorganization of the actin cytoskeleton to form adhesive structures that promote interaction with the substrate and the release of proteases. The release of proteases at the sites of contact, with the subsequent degradation of the substrate and generation of chemotactic peptides, facilitates locomotion and dissemination of the invading trophozoites.     

Disruption of the Plasma Membrane Integrity by Cholesterol Depletion Impairs Effectiveness of TRH Receptor-Mediated Signal Transduction via Gq/G11α Proteins

We monitored the radioligand-binding characteristics of thyrotropin-releasing hormone (TRH) receptors, functional activity of G_q/11α proteins, and functional status of the whole signaling cascade in HEK293 expressing high levels of TRH receptors and G₁₁α. Our analyses indicated that disruption of plasma membrane microdomains by cholesterol depletion did not markedly influence the binding parameters of TRH receptors, but it altered efficacy of signal transduction. The functional coupling between TRH receptor and G_q/11α was assessed by agonist-stimulated [³⁵S]GTPγS binding, and results of these measurements pointed out to significantly lower potency of TRH to mediate G protein activation in the plasma membrane fraction isolated from cholesterol-depleted cells; there was a shift in sensitivity by one order of magnitude to the higher concentrations. A markedly lower sensitivity to stimulation with TRH was also observed in our experiments dealing with determination of hormone-induced Ca²⁺ response. These data suggest that the intact structure of plasma membranes is an important optimum signal transduction initiated by TRH receptors and mediated by G_q/11α proteins.     

Chlorophyll fluorescence imaging of photosynthetic activity in sun and shade leaves of trees

The differences in pigment levels, photosynthetic activity and the chlorophyll fluorescence decrease ratio R _Fd (as indicator of photosynthetic rates) of green sun and shade leaves of three broadleaf trees (Platanus acerifolia Willd., Populus alba L., Tilia cordata Mill.) were compared. Sun leaves were characterized by higher levels of total chlorophylls a + b and total carotenoids x + c as well as higher values for the weight ratio chlorophyll (Chl) a/b (sun leaves 3.23–3.45; shade leaves: 2.74–2.81), and lower values for the ratio chlorophylls to carotenoids (a + b)/(x + c) (with 4.44–4.70 in sun leaves and 5.04–5.72 in shade leaves). Sun leaves exhibited higher photosynthetic rates P _N on a leaf area basis (mean of 9.1–10.1 μmol CO₂ m⁻² s⁻¹) and Chl basis, which correlated well with the higher values of stomatal conductance G _s (range 105–180 mmol m⁻² s⁻¹), as compared to shade leaves (G _s range 25–77 mmol m⁻² s⁻¹; P _N: 3.2–3.7 μmol CO₂ m⁻² s⁻¹). The higher photosynthetic rates could also be detected via imaging the Chl fluorescence decrease ratio R _Fd, which possessed higher values in sun leaves (2.8–3.0) as compared to shade leaves (1.4–1.8). In addition, via R _Fd images it was shown that the photosynthetic activity of the leaves of all trees exhibits a large heterogeneity across the leaf area, and in general to a higher extent in sun leaves than in shade leaves.     

Substrate specificities of a bacterial sialidase and rat liver ganglioside GM3 sialyltransferase

Sialidases cleave off sialic acid residues from the oligosaccharide chain of gangliosides in their catabolic pathway while sialyltransferases transfer sialic acid to the growing oligosaccharide moiety in ganglioside biosynthesis. Ganglioside G_M3 is a common substrate for both types of enzymes, for sialidase acting on ganglioside G_M3 as well as for ganglioside G_D3 synthase. Therefore, it is possible that both enzymes recognize similar structural features of the sialic acid moiety of their common substrate, ganglioside G_M3. Based on this idea we used a variety of G_M3 derivatives as glycolipid substrates for a bacterial sialidase (Clostridium perfringens) and for G_D3 synthase (of rat liver Golgi vesicles). This study revealed that those G_M3 derivatives that were poorly degraded by sialidase also were hardly recognized by sialyltransferase (G_D3 synthase). This may indicate similarities in the substrate binding sites of these enzymes.     

Scanning electron microscopy of attached trophozoites of pathogenic Entamoeba histolytica

The surface morphology of Entamoeba histolytica trophozoites of HM 1:IMSS (axenic and monoxenic) and HK9 (axenic) strains cultured on plastic and MDCK cell substrates was examined using scanning electron microscopy (SEM). The conditions for processing trophozoites were determined by comparing the SEM observations with the morphology of living amebas examined by light microscopy. The most frequent surface differentiations in all the amebas observed with SEM were lobopodia. Round cytoplasmic projections were found in approximately half of the axenic amebas. Endocytic stomas and filopodia were more common in monoxenic cultures while the uroid was found in only 2-8% of all examined amebas. The basal surfaces of the trophozoites, involved in both attachment and cytolysis, showed no unusual features, except for the presence of a small number of short filopodia at the outer edge. No differences were found in the morphology of amebas grown on artificial and natural substrates. These observations demonstrate that there are significant quantitative differences in the surface morphology of cultured trophozoites of different strains of E. histolytica and that association with bacteria produces an increase in the relative number of surface specializations of the parasite.     

ADP-ribosylation of actin from the green algaChara corallina byClostridium botulinum C2 toxin andClostridium perfringens iota toxin

Summary The ability of two bacterial toxins to modify a plant actin by covalent ADP-ribosylation was tested in the green algaChara corallina. Using [³²P]NAD, bothClostridium botulinum C2 toxin andClostridium perfringens iota toxin labelled a protein of M_r 42 kDa which comigrated with actin and was immunoprecipitated by a monoclonal anti-actin antibody. ADP-ribosylation ofChara actin was more efficient with iota toxin than with C2 toxin. The actin bundles in perfusedChara cells were not affected by toxin-containing media competent for ADP-ribosylation. The data indicate that monomeric plant actin is substrate for ADP-ribosylation by the bacterial toxins.Abbreviations ADP adenosine-diphosphate - EGTA ethyleneglycol-bis-(-aminoethyl)N,N,N,N-tetraacetic acid - NAD nicotinamide dinucleotide - pCA -log [Ca²⁺] - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Molecular Mechanism of Resistance of Equine (Equus caballus) Platelets to α2-Adrenergic Regulation

Adrenaline is a weak aggregating agonist for human platelets acting through G-protein-coupled α₂-adrenoceptors to inhibit adenylate cyclase and thus reduce cyclic AMP levels. Studies of equine platelets have shown that adrenaline is unable to promote their aggregation. We now confirm that adrenaline is without effect on equine platelet aggregation and demonstrate that it is also without effect on equine platelet membrane adenylate cyclase activity. We have previously shown that equine platelet membranes contain conventionally regulated adenylate cyclase activity, with both stimulatory ligands (forskolin and PGE₁) and inhibitory ligands (collagen and PAF) each showing substantial and dose-dependent effects. We now show, in Western blots, that equine platelet membranes contain G proteins, including G_i2 (which mediates inhibition of adenylate cyclase by adrenaline in human platelets), G_i3, G_s, and G_q. Hence, all the necessary components and responses are in place in equine platelets to provide for a conventional role for cyclic AMP and adenylate cyclase in modulating platelet aggregation. The basis for the failure of adrenaline, unlike other ligands, to deliver such a signal, appears to be a marked lack of α₂-adrenoceptors. This is supported by the low receptor density we found in idazoxan binding studies.     

Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line

Prolyl oligopeptidase (POP) is a post-proline cleaving enzyme, which is widely distributed in various organs, with high levels in the brain. In this study, we investigated the effects of a selective POP inhibitor, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746), on the growth of NB-1 human neuroblastoma cells. SUAM-14746 treatment for 24–72 h suppresses the growth of NB-1 cells without cell death in a dose-dependent manner (10–60 μM). Similar suppressive effects were observed with another POP inhibitor benzyloxycarbonyl-thioprolyl-thioprolinal. The SUAM-14746-induced growth inhibition in NB-1 cells was associated with pronounced G₀/G₁ arrest and reduced levels of phosphorylated retinoblastoma protein (pRb), cyclin E, and cyclin dependent kinase (CDK) 2, and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. SUAM-14746 also induced transient inhibition of S and G₂/M phase progression, which was correlated with retardation of the decrease in the levels of cyclins A and B. Moreover, RNAi-mediated knockdown of POP also led to inhibition of NB-1 cell growth and the effect was accompanied by G₀/G₁ arrest. These results indicate that POP is a part of the machinery that controls the cell cycle.     

Entamoeba invadens: Identification of ADF/cofilin and their expression analysis in relation to encystation and excystation

The differentiation processes of excystation and encystation of Entamoeba are essential for infection and completion of their life-cycle, and the processes need cell motility and its control by actin cytoskeletal reorganization. This study investigated actin depolymerizing factor (ADF)/cofilin (Cfl) family proteins, which are important molecules in actin cytoskeletal reorganization, in Entamoeba invadens in relation to the encystation and excystation. Axenic culture systems were used to induce encystation and excystation. A homology search of the E. invadens genome database and molecular cloning identified three ADF/Cfl family proteins of the parasite (named for short as EiCfl-1, EiCfl-2, and EiCfl-3). This is different from other Entamoeba species, i.e. Entamoeba histolytica and Entamoeba dispar, each of which has only one ADF/Cfl family protein. These ADF/Cfl of E. invadens do not have Ser3 (serine locates third from first methionine), similar to E. histolytica, E. dispar, Saccharomyces cerevisiae and Schizosaccharomyces pombe, although the activity of ADF/Cfl is negatively regulated by phosphorylation of the Ser3 in metazoans. Phylogenetic analysis revealed that Entamoeba Cfl formed a distinctive clade that is separate from other organisms, and the branches of the tree were separated in two consistent with the presence and absence of Ser3. Rabbit anti-EiCfl-2 serum reacted with all recombinant EiCfls and EiCfl in lysates of cysts, trophozoites and metacystic amoebae. Immunofluorescence staining with this antiserum showed co-localization of EiCfl with actin beneath the cell membrane through the life stages. Both proteins proved to be rich in pseudopodia of trophozoites and metacystic amoebae. Real-time RT-PCR showed that mRNAs of EiCfl-2 and actins were highly expressed, but there were few mRNA of EiCfl-1 and EiCfl-3. Remarkably decreased mRNA levels were observed in EiCfl-2 and actins during encystation. All three EiCfls and actins became transcribed after the induction of excystation. The mRNAs of only EiCfl-1 and EiCfl-3 increased remarkably when the excystation was induced in the presence of cytochalasin D. These findings demonstrate that EiCfl-2 and actins co-localize beneath the cell membrane in trophozoites and cysts as well as metacystic amoebae being rich in pseudopodia, that EiCfl-1 and EiCfl-3 are expressed only after the induction of excystation, and that enhanced excystation by cytochalasin D is associated with high expression of EiCfl-1 and EiCfl-3.     

Proteomic analysis of Gal/GalNAc lectin-associated proteins in Entamoeba histolytica

Contact-dependent killing and phagocytosis of target cells by Entamoeba histolytica trophozoites is mediated by the galactose (Gal) and N-acetyl-d-galactosamine (GalNAc)-inhibitable lectin. Previous work has suggested that this lectin functions as part of a signal transduction complex. To identify proteins that might be part of this complex, amebic trophozoites were bound to GalNAc-BSA-labeled magnetic beads and lysed. Bound proteins were eluted from the beads and analyzed by tandem mass spectrometry. Along with the Gal/GalNAc lectin subunits, several cytoskeletal proteins, potential signaling proteins, and a novel transmembrane protein, consistently purified with the GalNAc-BSA beads.