Surface immunoglobulin on rabbit lymphoid cells. III. Double expression and separate endocytosis of surface immunoglobulin allotypes on heterozygous lymphocytes demonstrated by immunoelectron microscopic labeling.

The expression of immunoglobulin b locus (k chain) allotypes on the surface of rabbit peripheral blood lymphocytes (PBL's) is examined using an indirect double immunoelectron microscopic labeling technique. Ferritin and whelk hemocyanin individually conjugated to allotypically specific IgG are used as ultrastructurally identifiable molecular markers. These indicators are coupled to lymphocyte surface immunoglobulin (Ig) allotypic determinants by an antiallotype antibody linkage. Human red blood cells, conjugated with IgG of a specific allotype and used as test cells, demonstrate the absolute specificity and high efficiency of the ultrastructural labeling technique. Specific labeling on rabbit PBL's shows that 65–75% of the cells are positive for surface Ig. Lymphocytes from homozygous donors (b4b4 or b6b6) are labeled specifically with only the appropriate allotypic labeling system. Thirty-three percent of the PBL's from heterozygous donors (b4b6) express both allotypes (allelic inclusion) on the cell surface; the remaining proportion of Ig-bearing cells have only one detectable allotype present (allelic exclusion). We conclude that approximately 50% of the Ig-bearing PBL's demonstrate allelic inclusion for the b locus allotypes. On allelically included heterozygous lymphocytes, both allotypic determinants can undergo specific endocytosis. Endocytosis of one allotype on heterozygous cells can be induced by stimulation with antiallotypic serum without affecting the surface appearance of the other allelic marker (separate endocytosis).     

Magnesium binding in the digesta of the chicken.

Two experiments involving laying hens and mature roosters were carried out to determine the effect of pH on the ultrafilterable fraction of Mg((Mg)u) present in the digesta along the gastrointestinal tract. Concurrently, the approximate molecular weight of the (Mg)u was determined. The average pH's for laying hens were: crop 4.88, proventriculus 5.27, ventriculus 4.77, duodenum 5.57, jejunum 6.15, ileum 7.82, and colon 6.65. Similar pH values were obtained for mature roosters. The (Mg)u fraction decreased significantly in the ileum where the pH was greater than 7.00. However, treating the ileal digesta with an acid pH buffer released about 70% of the nonfilterable Mg. The molecular weight of the (Mg)u was found to be less than 700, suggesting that the Mg was present in digesta as an inorganic salt or as an organic complex of low molecular weight.     

Studies on rabbit lymphocytes in vitro: XVII. Kinetics of reversible concanavalin a stimulation and restimulation of blast transformation after blocking with α-methyl-d-mannopyranoside

The stimulation of transformation of rabbit peripheral blood lymphocytes by Concanavalin A (ConA) has a narrow dose optimum, is reversible by α-d-methyl-mannopyranoside (MAM) and cultures that have been stimulated and reversed may be restimulated by removal of the blocking saccharide and re-addition of ConA. The kinetics of the stimulatory dose of ConA, the blocking dose of MAM, and the time of stimulation and blocking indicate a competitive binding of lymphocyte receptors and blocking saccharide for ConA. Most of the lymphocytes that respond to ConA become enlarged during the first 16–24 h after stimulation, although fully developed ‘blast’ cell transformation and mitosis do not occur until after approx. 40 h. Lymphocytes that are held in vitro prior to ConA stimulation gradually lose the ability to respond to ConA stimulation (delayed stimulation). Morphologic and metabolic analysis of ConA-stimulated and MAM blocked cultures demonstrate (1) that RNA synthesis gradually decreases in blocked cultures at a time that it is increasing in stimulated cultures; (2) that cells enlarged after ConA stimulation become smaller following MAM inhibition; (3) that the ability of blocked cells to be restimulated by ConA gradually decreases following MAM block (delayed restimulation). Lymphocyte activation requires the continued presence of the stimulant for consumation of the transformation process, and activated cells that have been blocked have a temporary ability to respond to restimulation at a time when cells that have not been preactivated are unable to respond. The requirement of increasing amounts of blocking MAM to reverse stimulation by ConA as the time of contact of ConA with the cells in culture increases is consistent with the concept that internalization, or stripping of mitogen or cellular receptors is the important cellular event initiating transformation. Blocking is achieved by permitting re-externalization of lectin or cell surface receptors. Stimulation requires the continued internalization or stripping of newly formed receptors by reaction with the stimulating mitogen during the entire culture period prior to initiation of DNA synthesis.     

Studies on rabbit lymphocytes in vitro : XX. Demonstration of cells reactive to more than one mitogen by mixed sequential stimulation

Rabbit peripheral blood lymphocyte (PBL) cultures stimulated by ConA and then blocked by the addition of competing sugar or antiserum after 6–15 h of ConA prestimulation respond to restimulation by PHA or PWM to a much greater extent than to continuous stimulation or delayed stimulation with PHA or PWM. This effect of mixed lectin sequential stimulation indicates that many of the same PBLs will respond to more than one mitogen, but that some cells require preactivation by one mitogen in order to respond fully to another mitogen. Thus, the number of PBLs which respond to PHA or PWM alone is much less than the number which respond following pretreatment with ConA when the pretreatment effect of ConA alone is blocked. Rabbit PBLs do not respond to LPS and preactivation by ConA does not prepare rabbit PBLs to respond to LPS.     

Production and characterization of monoclonal antibodies to rabbit lymphocyte subpopulations. I. Tissue immunofluorescence and flow cytometric analysis

Nine monoclonal antibodies to rabbit T cells and B subpopulations have been generated from three separate fusions of spleen cells from mice immunized with fractionated populations of rabbit lymphocytes. These monoclonal antibodies, as well as a previously described rabbit T cell monoclonal antibody, 9AE10, have been analyzed by immunofluorescence staining on frozen tissue sections of rabbit thymus, spleen, and appendix. This screening method permits rapid identification of the lymphocyte subdomains in each tissue which is not possible by other screening methods. Each monoclonal antibody selected has a unique tissue staining pattern. Flow cytometric analysis of these monoclonal antibodies, using indirect immunofluorescence techniques on thymocytes, splenocytes, and PBL, revealed varying percentages of positive cells and individual mean fluorescence intensities indicating different epitope densities for each antigen. These monoclonal antibodies are now being used to characterize normal lymphocyte function and the role of specific lymphocyte subpopulations in experimental disease models in the rabbit.     

Integration of ion exchange resin materials for a downstream-processing approach of an imine reductase-catalyzed reaction

In this study, an ion exchange resin-based downstream-processing concept for imine reductase (IRED)-catalyzed reactions was investigated. As a model reaction, 2-methylpyrroline was converted to its corresponding product (S)-2-methylpyrrolidine with >99% of conversion by the (S)-selective IRED from Paenibacillus elgii B69. Under optimized reaction conditions full conversion was achieved using a substrate concentration of 150 and 500 mmol/L of d -glucose. Seven commercially available cation- and anion-exchange resins were studied with respect to their ability to recover the product from the reaction solution. Without any pretreatment, cation-exchange resins Amberlite IR-120(H), IRN-150, Dowex Monosphere 650C, and Dowex Marathon MSC showed high recovery capacities (up to >90%). A 150-ml preparative scale reaction was performed yielding ~1 g hydrochloride salt product with >99% purity. Any further purification steps, for example, by column chromatography or recrystallization, were not required.     

Membrane Properties of Striatal Direct and Indirect Pathway Neurons in Mouse and Rat Slices and Their Modulation by Dopamine

D1 and D2 receptor expressing striatal medium spiny neurons (MSNs) are ascribed to striatonigral (“direct”) and striatopallidal (“indirect”) pathways, respectively, that are believed to function antagonistically in motor control. Glutamatergic synaptic transmission onto the two types is differentially affected by Dopamine (DA), however, less is known about the effects on MSN intrinsic electrical properties. Using patch clamp recordings, we comprehensively characterized the two pathways in rats and mice, and investigated their DA modulation. We identified the direct pathway by retrograde labeling in rats, and in mice we used transgenic animals in which EGFP is expressed in D1 MSNs. MSNs were subjected to a series of current injections to pinpoint differences between the populations, and in mice also following bath application of DA. In both animal models, most electrical properties were similar, however, membrane excitability as measured by step and ramp current injections consistently differed, with direct pathway MSNs being less excitable than their counterparts. DA had opposite effects on excitability of D1 and D2 MSNs, counteracting the initial differences. Pronounced changes in AP shape were seen in D2 MSNs. In direct pathway MSNs, excitability increased across experimental conditions and parameters, and also when applying DA or the D1 agonist SKF-81297 in presence of blockers of cholinergic, GABAergic, and glutamatergic receptors. Thus, DA induced changes in excitability were D1 R mediated and intrinsic to direct pathway MSNs, and not a secondary network effect of altered synaptic transmission. DAergic modulation of intrinsic properties therefore acts in a synergistic manner with previously reported effects of DA on afferent synaptic transmission and dendritic processing, supporting the antagonistic model for direct vs. indirect striatal pathway function.     

Energetics of liposomes encapsulating silica nanoparticles

Nanoparticles may be taken up into cells via endocytotic processes whereby the foreign particles are encapsulated in vesicles formed by lipid bilayers. After uptake into these endocytic vesicles, intracellular targeting processes and vesicle fusion might cause transfer of the vesicle cargo into other vesicle types, e.g., early or late endosomes, lysosomes, or others. In addition, nanoparticles might be taken up as single particles or larger agglomerates and the agglomeration state of the particles might change during vesicle processing. In this study, liposomes are regarded as simple models for intracellular vesicles. We compared the energetic balance between two liposomes encapsulating each a single silica nanoparticle and a large liposome containing two silica nanoparticles. Analytical expressions were derived that show how the energy of the system depends on the particle size and the distance between the particles. We found that the electrostatic contributions to the total energy of the system are negligibly small. In contrast, the van der Waals term strongly favors arrangements where the liposome snugly fits around the nanoparticle(s). Thus the two separated small liposomes have a more favorable energy than a larger liposome encapsulating two nanoparticles.