Retention of pinocytized solute by CHO cell lysosomes correlates with molecular weight

We compared the exocytosis by Chinese hamster ovary (CHO) cells of a set of fluid-phase pinocytic tracers. The tracers were horseradish peroxidase (HRP), a glycoprotein of approximately 40 kDa, lucifer yellow (LuY), a 457 dalton, membrane-impermeant fluorescent dye, and glucose polymers ranging from sucrose through higher molecular weight, fluorescein isothiocyanate (FITC) dextrans. After a long term uptake (16-20 h), each of these tracers was localized to lysosomes. Exocytosis of the majority of the small molecule tracers, LuY and [14C] sucrose, was observed over a period of a few to several h. There was no significant exocytosis of 42 kDa FITC dextran or HRP during an 18-20 h chase, while lower molecular weight dextrans were exocytosed. After co-accumulation of LuY and HRP in lysosomes, only the low molecular weight marker was exocytosed. These observations suggest retention of endocytized solutes within lysosomes is dependent on molecular size and may be limited by the rate of diffusion of molecules into shuttle vesicles.     

Coumarin-like fluorescent molecular rotors for bioactive polymers probing

Polysaccharides are interesting and often essential macromolecules but are difficult to analyse due to their lack of convenient chromophores. We propose an efficient labelling procedure for polysaccharides such as functionalized dextrans with coumarin derivatives: the fluorescent tracers present inter alia properties of emission of fluorescence dependent on the molecular environment (polarity, viscosity, temperature, pH, etc.). Hence, with in mind the understanding of cell-polysaccharide interactions, the labelled polymers were studied by in vitro tests on a line of endothelial cells sensitive to the proliferative effect of these dextran polysaccharides. Using 3D fluorescence microscopy, the fixation and internalization of fluorescent functionalized dextrans were observed in endothelial cells.     

Endocytotic uptake of fluorescent dextrans by pollen tubes grown in vitro

Summary Pollen tubes grow by tip growth, with high levels of exocytosis at the apex. The commercial availability of FITC labelled -linked dextrans provides a source of biologically inert tracers for endocytotic activity in pollen tubes. Growing tubes ofNicotiana andTradescantia were transferred to media containing 1% FD-4 for varying period of time before washing in control media and observation in a fluorescence microscope. Fluorescent material appeared to enter the pollen tubes only at the tip region, and to accumulate in vacuoles, starting with smaller vacuoles near the tip and spreading to the main vacuolated part of the tube. Mature tubes, with callose plugs, were only labelled up to the first complete plug from the tip, younger tubes without plugs were labelled into the pollen grain vacuole. The fluorescent material within the pollen tubes was shown to represent uptake of intact high molecular weight dextran by the following criteria: (i) free FITC and low molecular weight dextrans could not be detected in any of the media or pollen tubes using thin layer chromatography and (ii) pollen tube growth rates were unaffected by the fluorescent dextran, but were severely inhibited by low levels of free FITC. It was concluded that the dextrans entered the tubes by endocytosis, possibly in the tip region, and were then transferred to the vacuole system of the pollen tube.Abbreviations FITC fluorescein isothiocyanate - FD fluorescent dextran     

A method for incorporating macromolecules into adherent cells

We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes. Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold media, washed, and then replated for culture. We refer to the method as "scrape-loading." Viability of cells was 50-60% immediately after scrape-loading and was 90% for those cells remaining after 24 h of culture. About 40% of adherent, well-spread fibroblasts contained fluorescent molecules 18 h after scrape-loading of labeled dextrans, ovalbumin, or immunoglobulin-G. On average, 10(7) dextran molecules (70,000-mol wt) were incorporated into each fibroblast by scrape-loading in 10 mg/ml dextran. The extent of loading depended on the concentration and molecular weight of the dextrans used. A fluorescent analog of actin could also be loaded into fibroblasts where it labeled stress fibers. HeLa cells, a macrophage-like cell line, 1774A.1, and human neutrophils were all successfully loaded with dextran by scraping. The method of scrape-loading should be applicable to a broad range of adherent cell types, and useful for loading of diverse kinds of macromolecules.     

Modified lysosomal compartment as carrier of slowly and non-degradable tracers in macrophages

A previous immunocytochemical study of macrophages infected with Bacillus subtilis showed that a cell wall antigen could be detected for several days in a population of small vesicles randomly distributed within the cells and apparently distinct from perinuclear lysosomes. These observations suggested the possibility that these vesicles might constitute a "storage" compartment for non-degradable compounds. In the present report we compared in pulse-chase experiments the location and fate of a series of degradable and non-degradable pinocytic tracers within the macrophages. The tracers, detected by fluorescent microscopy, were bovine serum albumin (BSA), hen egg ovalbumin (OVA), horseradish peroxidase (HRP). Lucifer Yellow, fluorescent dextran, and levan. BSA and OVA remained located in perinuclear lysosomes during the chase period until their disappearance occurring within 3 h. In contrast, the other tracers, although initially located in perinuclear lysosomes, were found after a 3 to 5-h chase in small vesicles homogeneously distributed in the macrophage cytoplasm where they remained visible for 2 to 3 days. The use of markers for different cell organelles indicated that these dispersed vesicles exhibited several of the lysosomal features. They were acidic, they contained the 100 kDa and the 120 kDa lysosomal proteins as well as some acid proteases albeit these markers were in lesser concentrations than in the perinuclear lysosomal compartment. The addition of bacteria to the macrophages previously loaded with fluorescent dextran showed that all dispersed vesicles have the same fusion property as lysosomes and that slowly degraded or non-degradable tracers turn over through the perinuclear lysosomal compartment by using the endocytic pathway. Measurement of the release of some of the tracers into the culture medium suggested that the "dispersed vesicles" were probably not implicated in exocytosis of the tracers.     

Rheology of native dextrans in relation to their primary structure

High molecular weight dextrans were synthesized at five temperatures (3, 10, 20, 25 and 30°C) using an in-vitro enzymatic method. The rheological properties of these dextrans in aqueous solution were assessed through their flow behaviour and their viscoelastic characteristics. The results were interpreted in relation to their primary structure and particularly to their branching.

It was shown that the relatively expanded conformation of the dextrans synthesized at 3, 10 and 20°C gives to these dextrans comparable properties which are not too different from those described in literature for random-coil linear polysaccharides. Dextran synthesized at 30°C exhibited flow properties which are typical of particle suspensions in dilute and semi-dilute solution. In the concentrated domain, this dextran yielded structured systems with properties typical of weak gels. This unexpected behaviour could be related to the highly-ramified structure of this dextran in comparison with the dextrans synthesized between 3 and 20°C. On the other hand, the dextran synthesized at 25°C displayed rheological behaviour which could also be related to an intermediate primary structure between those of dextran synthesized at 20°C and dextran synthesized at 30°C.     

Effect of 100% O2 on passage of uncharged dextrans from blood to lung lymph

Since charge as well as size may influence the passage of plasma proteins from blood to lung lymph, we used uncharged dextrans as tracers to study the effects of hyperoxic lung injury on the molecular sieving properties of the pulmonary microcirculation in unanesthetized sheep. Polydisperse [3H]dextran was infused intravenously into five sheep before and after the animals breathed 100% O2 until lymph flow increased threefold (66-84 h). Lymph-to-plasma concentration ratios (L/P) were determined for [3H]dextran fractions of graded molecular sizes (1.6-8.4 nm effective radius) from samples obtained during the infusions. Before hyperoxia the blood-lymph barrier was highly restrictive to transport of [3H]dextrans above 5.0 nm in radius; steady-state L/P for these molecules averaged 0.03 or less. After the sheep breathed 100% O2, [3H]dextrans as large as 8.4 nm radius appeared in the lymph. Posthyperoxia, the L/P were significantly increased relative to prehyperoxia base-line values for every [3H]dextran fraction larger than 2.0 nm radius (P less than 0.05). In contrast, neither the L/P for albumin or total protein changed significantly. At autopsy, electron microscopy showed widespread damage to the endothelium of the alveolar capillaries with infrequent gaps between endothelial cells. In two control sheep, inhalation of compressed air for 96 h had no effect on lymph flow or L/P for the [3H]dextrans. We conclude that O2 poisoning reduced the selective sieving of uncharged dextrans across the blood-lymph barrier of the lungs and allowed larger dextrans to enter the lymph. These larger molecules may have leaked from the pulmonary microcirculation via disruptions in the continuity of the endothelial lining.     

Size determination of red blood cell aggregates induced by dextran using ultrasound backscattering phenomenon.

Different methods are commonly used to study the red blood cell aggregation phenomenon. The major interest of the ultrasonic method presently discussed is to assess the mean size of red blood cell (RBC) aggregates by measuring ultrasonic intensity backscattered by blood. Applying Rayleigh theory of sound to blood medium, one can show that the scattered ultrasonic intensity is proportional to the 6th power of the size of the RBC aggregates. The ultrasonic method is used to evaluate the mean size of RBC aggregates induced by dextrans. RBCs are suspended at various hematocrits H, in solution of dextrans of different molecular weights M and at different weight concentrations Cw. Results are presented by using the ultrasonic backscattering coefficient chi which is a relevant quantity in a scattering experiment. For suspensions of RBCs aggregated with dextran of molecular weight 70,000 dalton (dextran 70) at concentration Cw = 40 g/l, variations of chi as a function of H are similar to those obtained for normal blood. At a fixed hematocrit, variation of chi versus Cw for dextran 70 exhibits a maximum at 40 g/l. In the case of RBCs suspended at hematocrit 20% and aggregated with dextrans of molecular weight M, 70,000 less than or equal to M less than or equal to 2,000,000, the variations of chi versus molar concentration Cm are similar to those of the microscopic aggregation index defined by Chien (1). Finally, a statistical model of the blood structure previously described (2) is applied to evaluate the mean size of the aggregates. According to this model, the mean size of aggregates is independent of hematocrit for H less than or equal to 40% and independent of the molecular weight of dextran for M greater than or equal to 150,000 dalton.     

The mechanism of dextransucrase action: Activation of dextransucrase from Streptococcus mutans OMZ 176 by dextran and modified dextran and the nonexistence of the primer requirement for the synthesis of dextran

Streptococcus mutans OMZ 176 was grown in a sucrose-free medium containing fructose as a carbohydrate source. Dextransucrase was precipitated from the culture supernatant with 40% saturated ammonium sulfate. The activity of dextransucrase was shown to be stimulated by exogenous dextran. Maximum activity was reached when the concentration of exogenous dextran was 2 mg/ml. Dextrans modified at the nonreducing ends by reaction with tripsyl chloride and/or by hydrolysis with an exodextranase also activated dextransucrase four to six times over that of a control. The exodextranasemodified dextrans have nonreducing chains that are very short in comparison with unmodified dextran and the tripsyl-modified dextrans have chains that are blocked at the nonreducing ends with a triisopropylbenzenesulfonyl group on the C₆ hydroxyl group. Because the nonreducing ends of the modified dextrans are not available for reaction, the activation of dextransucrase by these modified dextrans cannot be due to primer reactions with the nonreducing ends. The activation of dextransucrase, thus, must be by an alternate mechanism. Two alternative mechanisms discussed are an allosteric effect and nucleophilic displacement reactions by the added dextran. It was also found that the addition of increasing amounts of dextran shifted the synthesis from an insoluble dextran to a soluble dextran.     

In vitro fermentation of linear and alpha-1,2-branched dextrans by the human fecal microbiota

The role of structure and molecular weight in fermentation selectivity in linear α-1,6 dextrans and dextrans with α-1,2 branching was investigated. Fermentation by gut bacteria was determined in anaerobic, pH-controlled fecal batch cultures after 36 h. Inulin (1%, wt/vol), which is a known prebiotic, was used as a control. Samples were obtained at 0, 10, 24, and 36 h of fermentation for bacterial enumeration by fluorescent in situ hybridization and short-chain fatty acid analyses. The gas production of the substrate fermentation was investigated in non-pH-controlled, fecal batch culture tubes after 36 h. Linear and branched 1-kDa dextrans produced significant increases in Bifidobacterium populations. The degree of α-1,2 branching did not influence the Bifidobacterium populations; however, α-1,2 branching increased the dietary fiber content, implying a decrease in digestibility. Other measured bacteria were unaffected by the test substrates except for the Bacteroides-Prevotella group, the growth levels of which were increased on inulin and 6- and 70-kDa dextrans, and the Faecalibacterium prausnitzii group, the growth levels of which were decreased on inulin and 1-kDa dextrans. A considerable increase in short-chain fatty acid concentration was measured following the fermentation of all dextrans and inulin. Gas production rates were similar among all dextrans tested but were significantly slower than that for inulin. The linear 1-kDa dextran produced lower total gas and shorter time to attain maximal gas production compared to those of the 70-kDa dextran (branched) and inulin. These findings indicate that dextrans induce a selective effect on the gut flora, short-chain fatty acids, and gas production depending on their length.     

Opening of size-selective pores in endosomes during human rhinovirus serotype 2 in vivo uncoating monitored by single-organelle flow analysis

The effect of virus uncoating on endosome integrity during the early steps in viral infection was investigated. Using fluid-phase uptake of 10- and 70-kDa dextrans labeled with a pH-dependent fluorophore (fluorescein isothiocyanate [FITC]) and a pH-independent fluorophore (cyanine 5 [Cy5]), we determined the pHs of labeled compartments in intact HeLa cells by fluorescence-activated cell sorting analysis. Subsequently, the number and pH of fluorescent endosomes in cell homogenates were determined by single-organelle flow analysis. Cointernalization of adenovirus and 70-kDa FITC- and Cy5-labeled dextran (FITC/Cy5-dextran) led to virus-induced endosomal rupture, resulting in the release of the marker from the low-pH environment into the neutral cytosol. Consequently, in the presence of adenovirus, the number of fluorescent endosomes was reduced by 40% compared to that in the control. When human rhinovirus serotype 2 (HRV2) was cointernalized with 10-and 70-kDa FITC/Cy5-dextrans, the 10-kDa dextran was released, whereas the 70-kDa dextran remained within the endosomes, which also maintained their low pH. These data demonstrate that pores are generated in the membrane during HRV2 uncoating and RNA penetration into the cytosol without gross damage of the endosomes; 10-kDa dextran can access the cytosol through these pores. Whereas rhinovirus-mediated pore formation was prevented by the vacuolar ATPase inhibitor bafilomycin A1, adenovirus-mediated endosomal rupture also occurred in the presence of the inhibitor. This finding is in keeping with the low-pH requirement of HRV2 infection; for adenovirus, no pH dependence for endosomal escape was found with this drug.     

Synthesis of ultrasmall superparamagnetic iron oxides using reduced polysaccharides

Investigation into the effect of the reducing sugar of dextran on formation and stability of dextran-coated ultrasmall superparamagnetic iron oxides (USPIO) has demonstrated that reduction of the terminal reducing sugar can have a significant effect on particle size, coating stability, and magnetic properties. Four aspects of polysaccharide-coated USPIO particle synthesis were investigated: (i) the effect reduction of the terminal polysaccharide sugar has upon polysaccharide usage, particle size, stability, and magnetic susceptibility; (ii) the effect an exogenous reducing sugar can have upon particle synthesis; (iii) the effect the molecular weight of the reduced polysaccharide has on particle synthesis; and (iv) the effectiveness of reduced and native dextrans in stabilizing a preformed magnetic sol. For low molecular weight dextrans (MW 20,000 x 10(-6) cgs). Similar results were obtained with a 12 kDa pullulan. The effect of polysaccharide molecular weight on particle size was studied, wherein higher molecular weight reduced dextrans produced larger particles. The effectiveness of the reduced and native dextrans in stabilizing a preformed magnetic sol was compared. Reduced dextrans were found to be superior for stabilizing the magnetic sol. The observed effects of reduction of the terminal sugar in dextran compared with the native dextran were modeled using the Langmuir adsorption isotherm. A good fit of experimental data with this model was found.     

Neutral and DEAE dextrans as tracers for assessing lung microvascular barrier permeability and integrity.

Steady-state lymph-to-plasma concentration ratios (L/Ps) of neutral dextrans, cationic DEAE dextrans, and endogenous proteins were determined under normal and increased permeability conditions in six unanesthetized yearling sheep prepared with chronic lung lymph fistulas. Fluorescent dextrans with radii ranging from 1 to 30 nm were intravenously infused, and after 24 h, perilla ketone (PK) was given to alter permeability while the dextran infusion was maintained. Plasma and lymph samples were collected before and after PK administration and analyzed for dextran and protein concentrations after high-performance liquid chromatography size separation. Under both baseline and increased permeability conditions, DEAE dextrans had higher L/Ps than neutral dextrans of similar size but lower L/Ps than proteins of similar size. Comparison of L/Ps before and after PK revealed that the percentage change in permeability for neutral and DEAE dextrans was significantly larger than that for proteins. These results suggest that 1) the pulmonary microvascular barrier behaves as a net negative barrier, 2) some transport mechanisms for proteins and dextrans are different, and 3) neutral and cationic dextrans are more sensitive markers than proteins of the same size for assessing changes in pulmonary capillary permeability.     

Erythrocytes sedimentation profiles under gravitational field as determined by He-Ne laser. VII. Influence of dextrans, albumin and saline on cellular aggregation and sedimentation rate

The erythrocytes sedimentation profiles (ESP) of normal blood and of blood mixed with saline, albumin (7%), and various molecular weight dextrans of different concentrations, at various height and widths of the sample holder are determined. These observations show that the sedimentation characteristics of the erythrocytes depend on the influence of these substitutes on the plasma and cellular constituents. The normalised aggregation and the sedimentation rate, as determined from these profiles, show that the dextran 40 and dextran 70 retard the erythrocytes sedimentation, for high molecular weight it is similar to that of normal blood and is the maximum for saline. This change for high molecular weight dextrans could be attributed to the enhanced aggregation tendency of erythrocytes and for saline to the enhanced sedimentation due to decrease in the viscosity and density of suspending medium. The influence of the various concentrations of dextrans on these parameters has been determined.     

WIF-B cells: an in vitro model for studies of hepatocyte polarity

We have evaluated the utility of the hepatoma-derived hybrid cell line, WIF-B, for in vitro studies of polarized hepatocyte functions. The majority (> 70%) of cells in confluent culture formed closed spaces with adjacent cells. These bile canalicular-like spaces (BC) accumulated fluorescein, a property of bile canaliculi in vivo. By indirect immunofluorescence, six plasma membrane (PM) proteins showed polarized distributions similar to rat hepatocytes in situ. Four apical PM proteins were concentrated in the BC membrane of WIF-B cells. Microtubules radiated from the BC (apical) membrane, and actin and foci of gamma-tubulin were concentrated in this region. The tight junction- associated protein ZO-1 was present in belts marking the boundary between apical and basolateral PM domains. We explored the functional properties of this boundary in living cells using fluorescent membrane lipid analogs and soluble tracers. When cells were incubated at 4 degrees C with a fluorescent analog of sphingomyelin, only the basolateral PM was labeled. In contrast, when both PM domains were labeled by de novo synthesis of fluorescent sphingomyelin from ceramide, fluorescent lipid could only be removed from the basolateral domain. These data demonstrate the presence of a barrier to the lateral diffusion of lipids between the PM domains. However, small soluble FITC- dextrans (4,400 mol wt) were able to diffuse into BC, while larger FITC- dextrans were restricted to various degrees depending on their size and incubation temperature. At 4 degrees C, the surface labeling reagent sNHS-LC-biotin (557 mol wt) had access to the entire PM, but streptavidin (60,000 mol wt), which binds to biotinylated molecules, was restricted to only the basolateral domain. Such differential accessibility of well-characterized probes can be used to mark each membrane domain separately. These results show that WIF-B cells are a suitable model to study membrane trafficking and targeting in hepatocytes in vitro.     

Reagents specific for cell surface components.

Mercury, diazonium ions and dyes which bind nucleic acids were covalently linked to dextrans using methods that resulted in non-hydrolyzable reagent-dextran bonds without impairing the binding abilities of the reagents, i.e. these dextran derivatives reacted with thiols, phenols/imidazoles and nucleic acids respectively. Since these dextran derivatives cannot penetrate into cells and since dextran itself does not bind to cells, these compounds represent reagents specific for the cell surface. They may be used both to evaluate cell surface constituents of intact cells and to affect viable cells via an interaction with those constituents. Mercury-dextran was found to bind to cells; the amount of mercury thus attached to the cells was about ten times smaller than when an equivalent concentration of free mercury ions was used. Mercury-dextran, bound to cells after a 30-min exposure at room temperature, was localized on the surface of these cells, as sodium borohydride reduced this complex giving rise to the intact cells, elementary mercury and free dextran which was released into medium. When cells were constantly exposed to the mercury-dextran, its toxic effects were comparable to that of free mercury ions. Diazonium-dextran, which also binds tightly to the cell surface, was also considerably toxic. Dextrans substituted with dyes which bind to nucleic acids were less toxic than the parent dyes themselves; it was shown that the attachment of such a dye to dextran decreased the binding of dye to cells under detection limits.     

Novel fluo-4 analogs for fluorescent calcium measurements

We report new fluorescent calcium indicators based on fluo-4. Attachment of a carboxamide or methylenecarboxamide moiety to the BAPTA chelator portion of fluo-4 allowed for the attachment of dextrans, protein-reactive moieties, and biotin. In particular, a high affinity fluo-4 dextran conjugate was prepared and shown to be functional in brain slices. All new probes were characterized spectroscopically and exhibited large fluorescence increases upon calcium-binding. The biotinylated version of fluo-4 formed a persistent streptavidin complex which still responded to increasing calcium concentrations with a large fluorescence increase.     

Histology of plastic embedded amphibian embryos and larvae

Amphibians including the South African clawed frog Xenopus laevis, its close relative Xenopus tropicalis, and the Mexican axolotl (Ambystoma mexicanum) are important vertebrate models for cell biology, development, and regeneration. For the analysis of embryos and larva with altered gene expression in gain-of-function or loss-of-function studies histology is increasingly important. Here, we discuss plastic or resin embedding of embryos as valuable alternatives to conventional paraffin embedding. For example, microwave-assisted tissue processing, combined with embedding in the glycol methacrylate Technovit 7100, is a fast, simple, and reliable method to obtain state-of-the-art histology with high resolution of cellular details in less than a day. Microwave-processed samples embedded in Epon 812 are also useful for transmission electron microscopy. Finally, Technovit-embedded samples are well suited for serial section analysis of embryos labeled either by whole-mount immunofluorescence, or with tracers such as GFP or fluorescent dextrans. Therefore, plastic embedding offers a versatile alternative to paraffin embedding for routine histology and immunocytochemistry of amphibian embryos.     

Changes in macromolecular movement accompany organogenesis in thin cell layers of <Emphasis Type="Italic">Torenia fournieri</Emphasis>

A range of fluorescently labelled probes of increasing molecular weight was used to monitor diffusion via the symplast in regenerating thin cell layer (TCL) explants of Torenia fournieri. An increase in intercellular movement of these molecules was associated with the earliest stages of vegetative shoot regeneration, with the movement of a 10 kDa dextran (FD 10000) observed between epidermal cells prior to the appearance of the first cell divisions. A low frequency of dextran movement in thin cell layers maintained under non-regenerating conditions was also observed, indicating a possible wound induced increase in intercellular movement. Dextran movement between epidermal cells reached a peak by day 4 of culture and then declined as cell division centres (CDCs) formed, became meristematic regions and finally emerged as adventitious shoots. Within CDCs, testing with small fluorescent probes (CF: carboxyfluorescein, mw 376 Da and F(Glu)₃: fluorescein-triglutamic acid, mw 799 Da) revealed a mosaic of cell isolation and regions of maintained symplastic linkage. Within shoots, surface cells of the presumptive apical meristem permitted the intercellular movement of 10 kDa dextrans but epidermal cells of the surrounding leaf primordia did not permit dextran movement. In some cases, intercellular movement of CF was maintained within leaf primordia. Symplastic movement of labelled dextrans during regeneration in Torenia thin cell layers represents a significant increase in the basal size exclusion limit (SEL) of this tissue and reveals the potential for intercellular trafficking of developmentally related endogenous macromolecules.     

An immunochemical study of the combining site specificities of C57BL/6J monoclonal antibodies to alpha (1----6)-linked dextran B512

This is the first report of an immunochemical study of the combining site specificities of a set of monoclonal antibodies to dextran B512 from C57BL/6J mice. The results confirm previous observations on antidextran combining sites and reveal specificities not seen earlier extending the observed repertoire of antibody combining sites to the single alpha (1----6)-linked glucosyl antigenic determinant. Eight C57BL/6J anti-dextran B512 hybridomas, four IgM,kappa and four IgA,kappa, were produced by PEG fusion of immune spleen cells with the nonproducer myeloma cell line P3X63Ag8 6.5.3. Antibody combining site specificities were determined by quantitative precipitin assays with 14 dextrans. Native dextrans with high percentages of linear alpha (1----6)-linked glucoses, similar to the immunogen B512, were the best precipitinogens; dextrans with alternating alpha (1----3), alpha (1----6) linkages, and highly branched dextrans were less effective. All antibodies precipitated with a synthetic, unbranched alpha (1----6)-linked dextran, suggesting their combining sites were "groove-like" and directed toward internal sequences of alpha (1----6)-linked residues, rather than "cavity-like" and directed toward a nonreducing terminal glucose. Two of the IgA hybridomas gave biphasic precipitin curves with dextran B512; this was shown to be due to differences in the precipitability of IgA monomers and polymers. Differences were observed in the reactivities of several dextrans considered previously to be structurally similar, and a newly proposed structural model of dextran B1299S was assessed. Quantitative precipitin inhibition studies with alpha (1----6)-linked isomaltosyl (IM) oligosaccharides, IM2 to IM9, showed that maximum inhibition was reached with IM6 or IM7, consistent with earlier estimates of the upper limit for the sizes of anti-B512 combining sites. Two IgM hybridomas showed a unique pattern, with inhibition being obtained only with IM5 or larger IM oligosaccharides. Association constants of the antidextrans for dextran B512 and for IM7, determined by affinity gel electrophoresis, ranged from 10(2) to 10(4) ml/g, comparable to earlier findings with antidextrans and other anticarbohydrate antibodies.