Effects of different fragments of the fibronectin molecule on latex bead translocation along neural crest migratory pathways

Previous studies from this laboratory have utilized latex beads as probes of embryonic migratory pathways. After microinjection into embryos at the time of neural crest migration, uncoated latex polystyrene beads were found to translocate to ventral sites and to settle in the vicinity of endogenous neural crest derivatives. However, latex beads coated with fibronectin did not translocate ventrally, but remained associated with cells surrounding the implantation site. Fibronectin is a large glycoprotein with a variety of biological activities and multiple binding domains. Here, the binding activities which might be responsible for immobilization of the fibronectin-coated beads are examined. Latex beads were coated with three types of fragments of the fibronectin molecule representing different functional domains: (i) a 66-kDa fragment containing collagen-binding activity; (ii) a mixture of 45- and 32-kDa fragments containing heparin-binding activity; and (iii) a 120-kDa fragment containing cell-binding activity. The beads coated with fibronectin fragments were injected into the newly formed trunk somites of avian embryos. After injection, beads coated with either the heparin- or the collagen-binding domain translocated ventrally and distributed analogously to uncoated latex beads. In contrast, the majority of beads coated with the fibronectin cell-binding domain did not translocate but remained associated with dermamyotomal cells surrounding the injection site. The cell-binding fragment, however, was not as effective as the intact fibronectin molecule in preventing translocation of the beads. The results suggest that the cell-binding domain is primarily responsible for restriction of fibronectin beads from the ventral neural crest pathway. Because intact fibronectin is more effective at immobilizing beads than is the cell-binding fragment, other binding domains of fibronectin, more efficient coating with intact fibronectin, or crosslinking of intact fibronectin molecules may also play some role in immobilization of the beads at the implantation site.     

High-performance affinity beads for identifying drug receptors

We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-kappaB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-kappaB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.     

Phagocytic Activity of FRTL-5 Rat Thyroid Follicular Cells as Measured by Ingestion of Fluorescent Latex Beads

A rat thyroid cell line (FRTL-5) was used to study the phagocytic activity of thyroid follicular cells using fluorescent latex beads and flow cytometric analysis. Morphologic studies demonstrated that latex beads were engulfed and located within cytoplasmic vacuoles of thyrocytes. Flow cytometric evaluation of cell suspensions revealed high levels of fluorescence in cells engulfing latex beads. Using thyrotropin (TSH) as a stimulator of thyroid function and human interleukin-1β as an inhibitor, protocols were established for measuring the effects of these substances on either basal or TSH-induced phagocytosis. Cells exposed to latex beads over time in basal (0H) or TSH-containing medium had an increase in time-dependent phagocytic activity which was maximal after 24 or 8 h, respectively. Treatment of FRTL-5 cells with either a stimulator or an inhibitor revealed maximal change in phagocytic activity after 72 h as measured by the percentage of phagocytic cells as well as the mean fluorescence intensity. Phagocytic activity and iodide trapping by FRTL-5 cells were qualitatively similar in both sensitivity and magnitude of change in the assays used in this study. Phagocytosis of fluorescent latex beads represents a sensitive nonradioactive assay of thyrocyte function whose regulation is similar to iodide trapping.     

Latex beads as probes of a neural crest pathway: effects of laminin, collagen, and surface charge on bead translocation

In the trunk region of avian embryos, neural crest cells migrate along two pathways: dorsally just under the ectoderm, and ventrally between the neural tube and the somites. Previous work from this laboratory has shown that uncoated latex beads are able to translocate along the ventral neural crest pathway after injection into young embryos; however, beads coated with fibronectin are restricted from the ventral route ( Bronner -Fraser, M.E., 1982, Dev. Biol., 91: 50-63). Here, we extend these observations to determine the effects of other macromolecules on bead distribution. The data show that laminin-coated beads, like fibronectin-coated beads, are restricted from the ventral pathway. In contrast, beads coated with type I collagen translocate ventrally after injection. Because macromolecules have characteristic charge properties, changes in surface charge caused by coating the beads may confound interpretation of the results. Electrostatic effects on bead movement were examined by coating the latex beads with polyamino acids in order to predictably alter the initial surface charge. The surface charge before injection was measured for beads coated with amino acid polymers or with various biologically important macromolecules; the subsequent translocation ability of these beads was then monitored in the embryo. Polylysine-coated beads (positively charged) were restricted from the ventral pathway as were fibronectin and laminin-coated beads, even though fibronectin and laminin beads were both negatively charged. In contrast, polytyrosine -coated beads ( neutrally charged) translocated ventrally as did negatively charged collagen-coated or uncoated beads. The results demonstrate that no correlation exists between the charge properties on the latex bead surface and their subsequent ability to translocate along the ventral pathway. Therefore, an adhesion mechanism independent of surface charge effects must explain the restriction or translocation of latex beads on a neural crest pathway.     

Fibroblast spreading and phagocytosis: similar cell responses to different-sized substrata

Experiments were carried out to test the hypothesis that cell spreading and phagocytosis are similar cell responses to different-sized substrata. The following morphological and biochemical studies provided evidence for this supposition. Cells phagocytosed 1.09-micron and 5.7-micron latex beads, but were unable to completely ingest 15.8-micron or 25.7-micron beads. With the larger beads, the cells spread around the bead surfaces with an appearance typical of cells spread on culture dishes. Biochemical studies with cytochalasin D, azide, and iodoacetate, as well as temperature-dependence studies, demonstrated similar responses of cell spreading and phagocytosis to these treatments. Similar cell surface receptors were involved in cell spreading and phagocytosis based upon experiments using antibodies to baby hamster kidney (BHK) cell wheat germ agglutinin receptors. And finally, BHK cell variants with defective plasma fibronectin (pFN) receptors were unable to spread on pFN-coated dishes or ingest pFN-coated beads. Evidence also is presented concerning the "contact" process in cell adhesion. It was found that azide and low temperature inhibited cell attachment per se but did not block fibronectin-receptor interactions based upon cell binding of pFN-coated beads. A possible explanation for the contact process is presented based upon the resistance of cells and beads to shear forces.     

Functional response and food selection of the water flea, Bosmina longispina

The functional response and the feeding behaviour of Bosminalongispina in relation to varying food concentrations were investigatedunder laboratory conditions using radioactively labelled Scenedcsmusacutus and different sized fluorescent latex beads to measureclearance rates and size selectivity. Bosmina longispina reducedfeeding activity at low concentrations of food, having a sigmoidfunctional response. Particles of 5.18 µm were filteredmuch more efficiently than both 1.04- and 0.25-µm particles,and the selectivity of large particles increased with increasingfiltering rates. Scenedesmus acutus was filtered at approximatelythe same rate as 5.18-µm latex beads. The increased selectivityat high filtering rates is explained as an adjustment in thefiltering apparatus to allow for greater water flow. Both thesefeeding responses would tend to reduce energy expenditure insituations when energy intake is low.     

Cell-surface charge and cell-surface hydrophobicity of collagen-bindingAeromonas andVibrio strains

Partitioning in aqueous polymer two-phase systems of polyethylene glycol and dextran was used to detect and compare cell-surface charge and cell-surface hydrophobicity of Aeromonas hydrophila, A. caviae, A. sobria, Vibrio cholerae, and V. anguillarum strains. These strains have cell-surface components that bound either native or thermally denatured type I collagen (i.e., a mixture of the α1+α2 chains) and gelatin immobilized on latex beads. Our goals were: (1) to compare the possible relationship between the cell-surface charge/hydrophobicity and binding to collagen and (2) to evaluate the influence of the culture media on the expression of surface properties. There was no apparent relationship between cell-surface charge, cell-surface hydrophobicity, and binding to collagen. The expression of surface properties was dependent on the culture media. There was no relationship between binding to immobilized collagen and binding to soluble ¹²⁵I-labeled collagen. Particle-agglutination reactivity differed when using various collagen-coated microbead preparations. There were general differences in the particle-agglutination reactivity when collagen-coated latex beads were prepared using different coating procedures. The negative charge and hydrophobicity of the various collagen-coated microbead preparations were also studied by partitioning in the two-phase system of polyethylene glycol and dextran. Under these conditions, the α1+α2 collagen-chain mixture covalently immobilized on carboxy-modified latex beads was less hydrophobic and negatively charged than gelatin and native collagen immobilized on the same kind of latex beads. For latex beads passively coated with collagen preparations, the α1+α2 collagen-chain mixture was more hydrophobic than gelatin and native collagen. We suggest that for screening collagen-binding among Vibrio and Aeromonas strains, a reliable and sensitive particle-agglutination assay should consider the collagen preparation and the coating procedure for the immobilization of collagen onto the latex beads. In this regard, carboxy-modified latex beads coated with an α1+α2 collagen-chain mixture gave the best results. Received: 9 January 1995 / Accepted: 30 May 1995     

Function of fibrinogen gamma-chain dodecapeptide-conjugated latex beads under flow

In order to perform a fundamental study of platelet substitutes, novel particles that bound to activated platelets were prepared using two oligopeptides conjugated to latex beads. The oligopeptides were CHHLGGAKQAGDV (H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411), and CGGRGDF (RGD), which contains a fibrinogen alpha-chain sequence (alpha 95-98 RGDF). Both peptides contained an additional amino-terminal cysteine to enable conjugation. Human serum albumin was adsorbed onto the surface of latex beads (average diameter 1microm) and pyridyldisulfide groups were chemically introduced into the adsorbed protein. H12 or RGD peptides were then chemically linked to the modified surface protein via disulfide linkages. H12- or RGD-conjugated latex beads prepared in this way enhanced the in vitro thrombus formation of activated platelets on collagen-immobilized plates under flowing thrombocytopenic-imitation blood. Based on the result of flow cytometric analyses of agglutination, PAC-1 binding, antiP-selectin antibody binding, and annexin V binding, the H12-conjugated latex beads showed minimal interaction with non-activated platelets. These results indicate the excellent potential of H12-conjugated particles as a candidate for a platelet substitute.     

Measurement of the effects of cadmium stress on protozoan grazing of bacteria (bacterivory) in activated sludge by fluorescence microscopy

The effect of cadmium stress on protozoan bacterivory in sewage sludge was measured by experimentally exposing sludge communities to 0 to 150 mg of Cd per liter for up to 6 h and then determining the rates of protozoan grazing on bacteria, using a double-staining technique and epifluorescence microscopy. Bacterivory was measured by incubating the sludge with fluorescently labeled bacterium-sized latex beads and directly observing ingestion of the beads and bacterial cells in the sludge by epifluorescence microscopy of preserved samples. Staining with 4',6-diamidino-2-phenylindole and acridine orange permitted the simultaneous determination of protozoan numbers and bacterivory activity as estimated by the number of bacterial cells and bacterium-sized latex beads ingested by the representative ciliate Aspidisca costata. Enumeration with latex beads proved to be an effective way of estimating bacterivory in sludges subjected to heavy-metal stress. This technique should prove useful for determining the effects of other chemical stresses on protozoan numbers and bacterivory in organic-rich environments. Although the number of protozoa declined significantly only after exposure to 100 mg of Cd per liter for 4 h, grazing, as indicated by bead ingestion, was significantly inhibited by Cd concentrations of greater than 25 mg/liter in less than 1 h, and exposure to 100 mg of Cd per liter effectively stopped protozoan grazing within 1 h of exposure. Protozoan ingestion of latex beads and bacteria was inversely correlated to Cd concentration and exposure time. The reduction of protozoan bacterivory by Cd provides a possible explanation for the increase in suspended bacteria in the effluents of metal-stressed treatment facilities.     

Measurement of the effects of cadmium stress on protozoan grazing of bacteria (bacterivory) in activated sludge by fluorescence microscopy.

The effect of cadmium stress on protozoan bacterivory in sewage sludge was measured by experimentally exposing sludge communities to 0 to 150 mg of Cd per liter for up to 6 h and then determining the rates of protozoan grazing on bacteria, using a double-staining technique and epifluorescence microscopy. Bacterivory was measured by incubating the sludge with fluorescently labeled bacterium-sized latex beads and directly observing ingestion of the beads and bacterial cells in the sludge by epifluorescence microscopy of preserved samples. Staining with 4',6-diamidino-2-phenylindole and acridine orange permitted the simultaneous determination of protozoan numbers and bacterivory activity as estimated by the number of bacterial cells and bacterium-sized latex beads ingested by the representative ciliate Aspidisca costata. Enumeration with latex beads proved to be an effective way of estimating bacterivory in sludges subjected to heavy-metal stress. This technique should prove useful for determining the effects of other chemical stresses on protozoan numbers and bacterivory in organic-rich environments. Although the number of protozoa declined significantly only after exposure to 100 mg of Cd per liter for 4 h, grazing, as indicated by bead ingestion, was significantly inhibited by Cd concentrations of greater than 25 mg/liter in less than 1 h, and exposure to 100 mg of Cd per liter effectively stopped protozoan grazing within 1 h of exposure. Protozoan ingestion of latex beads and bacteria was inversely correlated to Cd concentration and exposure time. The reduction of protozoan bacterivory by Cd provides a possible explanation for the increase in suspended bacteria in the effluents of metal-stressed treatment facilities.     

牙源性干细胞复合微渠多孔羟基磷灰石支架成骨性能研究*

目的: 探讨牙源性干细胞复合微渠多孔羟基磷灰石支架(grooved porous hydroxyapatite scaffolds, HAG支架)的成骨性能,为骨缺损修复治疗提供新手段。方法: 从健康成人第三磨牙中提取牙周膜干细胞(periodontal ligament stem cells, PDLSCs)及牙髓干细胞(dental pulp stem cells, DPSCs)分别接种于HAG支架上,进行多向分化鉴定及碱性磷酸酶(alkaline phosphatase,ALP)活性测定;并通过CCK-8检测细胞增殖能力;逆转录聚合酶链反应(qRT-PCR)检测骨形态发生蛋白2(bone morphogenetic protein 2, BMP-2)、骨钙素(osteocalcin, OCN)和骨桥蛋白(osteopontin, OPN)等成骨相关基因的表达。体内研究中将搭载PDLSCs和DPSCs的HAG支架移植到裸鼠的背部皮下,8周后取材,组织切片后采用苏木精-伊红(HE)染色观察新骨形成,提取组织蛋白采用Western blot检测ALP、OCN等成骨相关蛋白的表达。结果: 体外研究中DPSCs复合HAG支架组的细胞增殖能力、ALP活性,以及成骨相关基因ALP、BMP2、OCN等的表达均高于PDLSCs复合HAG支架组。体内研究中HE染色显示,PDLSCs复合HAG支架组及DPSCs复合HAG支架组均较空白HAG支架组有更多细胞生长区、纤维细胞增生及骨基质形成,且DPSCs复合HAG支架组的骨基质面积更大,成纤维细胞数量更多;PDLSCs复合HAG支架组及DPSCs复合HAG支架组成骨相关蛋白的表达量均高于空白HAG组,且DPSCs复合HAG支架组中ALP蛋白表达量显著高于PDLSCs复合HAG支架组。结论: PDLSCs、DPSCs复合HAG支架在体内外均表现出良好的成骨性能,其中DPSCs复合HAG支架的成骨性能更为优异。     

Rapid determination of bovine lactoferrin in dairy products by an automated quantitative agglutination assay based on latex beads coated with F(ab′)2 fragments

This study evaluated an automated immunoassay for bovine lactoferrin (LF) in dairy products based on latex beads coated with F(ab')2 fragments. Methods: F(ab')2 fragments were obtained by pepsin digestion of rabbit anti-bovine LF (IgG fraction) and polystyrene latex beads were coated with the F(ab')2 fragments. We used the beads to develop a rapid and homogeneous light scatter immunoassay employing an autoanalyzer (the Automated Latex assay). The Automated Latex assay was easy to perform and could rapidly determine bovine lactoferrin in lactoferrin-supplemented products. It was sensitive enough for testing products and showed good precision.     

Lack of immune stimulation by immobilized CpG-oligodeoxynucleotide.

Selected phosphorothioate oligodeoxynucleotides containing CpG (CpG-ODN) activate immune responses, including B-cell proliferation and cytokine production. The mechanism by which cells detect CpG-motifs is not known. There are conflicting reports in the literature concerning the ability of CpG-ODN linked to solid supports to stimulate immunity. We prepared a fluorescent, biotinylated CpG-ODN, a reagent that will support the growth of 7TD1 cells, a murine B-cell hybridoma line that requires CpG-ODN or interleukin-6 (IL-6) for survival. Stimulation of 7TD1 cell growth was not reduced by complexing biotinylated CpG-ODN to streptavidin, but cell growth was not supported by CpG-ODN coupled to streptavidin-coated latex, magnetic, gold, or agarose beads. A fluorescent CpG-ODN was also covalently attached to cyanogen bromide-activated Sepharose beads via a 5'-amine group. These derivatized Sepharose beads did support 7TD1 cell growth, but incubation of the beads with 7TD1 cells resulted in the appearance of fluorescence within the cells, suggesting that growth stimulation may be due to CpG-ODN leached from the beads. Our results are consistent with the need for CpG-ODN to be internalized into cells to be immunostimulatory.     

Distribution of latex beads and retinal pigment epithelial cells along the ventral neural crest pathway

Neural crest cells migrate along defined pathways in the trunk of avian embryos. Previous studies have demonstrated that crest-derived pigment cells migrated ventrally after injection onto the ventral neural crest pathway (M. E. Bronner-Fraser and A. M. Cohen, 1980, Develop. Biol.77, 130–141). In the present study, latex polystyrene beads and retinal pigment epithelial (RPE) cells were injected onto the ventral pathway in order to probe the environment along this migratory route. Although the RPE cells are nonmotile and not derived from the neural crest, they also translocated ventrally. Thus, factors independent of active migration may affect the localization of RPE cells (and endogenous crest cells). To test this possibility, latex polystyrene beads were injected onto the ventral pathway. Three types of beads were used: (a) uncoated latex beads; (b) latex beads coated with bovine serum albumin (BSA-beads); and (c) latex beads coated with fibronectin (FN-beads). Uncoated and BSA-beads distributed along the ventral pathway similarly to RPE cells and endogenous crest cells. However, FN-beads remained near the site of implantation and did not move ventrally. The results suggest (1) that molecules like fibronectin on the cell surface might serve as a recognition mechanism that prevents entrance onto the ventral pathway; and (2) that crest cell localization may, in part, be influenced by a driving force imparted by the embryonic environment.     

Latex bread technique for detection of the plaque-forming cell response to teratocarcinoma tumor antigens

Summary A latex bead technique modified for measuring the plaque-forming cell (PFC) response to teratocarcinoma tumor antigens in syngeneic animals is described.With this method one can detect both the primary (IgM) and the secondary (IgG) immune response to tumor antigens. Optimal detection of the PFC response depends on the proper ratio of sheep red blood cells to latex beads and the dose of tumor cell antigen used for immunization. The presence of fetal calf serum interfered with immunization of animals and the coating of the latex beads with the tumor cell antigens. The plaques obtained in response to immunization with teratocarcinoma cell antigens varied in size, probably reflecting the complex immune response to more than one class of antigens on tumor cells.     

Latex bread technique for detection of the plaque-forming cell response to teratocarcinoma tumor antigens

A latex bead technique modified for measuring the plaque-forming cell (PFC) response to teratocarcinoma tumor antigens in syngeneic animals is described. With this method one can detect both the primary (IgM) and the secondary (IgG) immune response to tumor antigens. Optimal detection of the PFC response depends on the proper ratio of sheep red blood cells to latex beads and the dose of tumor cell antigen used for immunization. The presence of fetal calf serum interfered with immunization of animals and the coating of the latex beads with the tumor cell antigens. The plaques obtained in response to immunization with teratocarcinoma cell antigens varied in size, probably reflecting the complex immune response to more than one class of antigens on tumor cells.     

Fine structural aspects of phagocytotic activity in inverted follicle cells of cultured porcine thyroids

Inversion of thyroid follicles took place when they were isolated by collagenase and trypsin and cultured in suspension in Eagle's medium supplemented with 10% fetal calf serum without TSH. The apical surface facing the culture medium contained numerous microvilli and a central cilium, while the luminal surface became flattened. Phagocytotic activity by pseudopods was promoted after addition of TSH to the culture medium. When the inverted follicles were incubated in culture medium containing TSH (50 mU/ml) and human red blood cells, or TSH and polystyrene latex beads (2.02 micron in diameter) for 1-3 h, numerous red blood cells or latex beads respectively were observed to be taken up by the epithelial follicle cells by scanning electron microscopy, as well as conventional thin-section electron microscopy. These results show that the apical surface (culture medium side) of the epithelial cell of the cultured thyroid follicle whose polarity is reversed phagocytoses red blood cells and latex beads.     

SMV对大豆花粉表面的污染及对花粉活力的影响

应用PALLAS将病毒粒体转化为乳胶颗粒通过扫描电镜直接观察,成功地检测到了大豆花粉表面污染的大豆花叶病毒(SMV)。田间春大豆宁镇1号的重花叶、轻花叶及无症状植株花粉所吸附的乳胶颗粒依次递减,而健株花粉上极少观测到乳胶颗粒,花粉数量测定及萌发试验表明,SMV的侵染对大豆花粉形成及活力都有明显的影响。     

Variants deficient in phagocytosis of latex beads isolated from the murine macrophagelike cell line J774

Variants that lack the ability to ingest latex beads were isolated from the mouse macrophagelike cell line J774. Carboxylated latex beads were derivatized with polylysine and then daunomycin by a carbodiimide method. Cells that ingested such beads were killed; variants that survived were isolated. Variants were detected at very low frequency and only after nitrosoguanidine mutagenesis. Of 11 independent isolates, 10 showed a lowered rate of uptake of polystyrene beads (without daunomycin). All of these proved normal in rate and extent of Fc-mediated phagocytosis. There was essentially no change in sensitivity to free daunomycin in the variants compared to the parent. These results support the previous hypothesis that there are differences in the metabolic routes of receptor-mediated and nonspecific phagocytosis.