Enhanced antigen-antibody binding affinity mediated by an anti-idiotypic antibody

We previously described the production of four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol. One of these antibodies, 5B7 (IgG2a, kappa), was used to raise anti-idiotypic antisera in rabbits. In contrast to the expected results, one of the anti-idiotypic antisera (R9) promotes [125I]iodocyanopindolol (ICYP) binding to antibody 5B7. In the presence of R9, the dissociation constant decreases 100-fold from 20 to 0.3 nM. This increase in binding affinity of antibody 5B7 for ICYP is not observed in the presence of preimmune, rabbit anti-mouse or anti-idiotypic antisera generated to a monoclonal antibody of a different specificity. Furthermore, R9 in the absence of 5B7 does not bind ICYP. The F(ab) fragments of 5B7 and R9 behaved in a similar manner, and the soluble complex responsible for the high-affinity interaction with ICYP can be identified by gel filtration chromatography. The elution position of the complex is consistent with a 5B7 F(ab)-R9 F(ab) dimer, indicating that polyvalency is not responsible for the enhanced ligand binding. Kinetic analysis of ICYP-5B7 binding revealed that the rate of ICYP dissociation from 5B7 in the presence of R9 is approximately 100 times slower than in the absence of R9 [k-1(+R9) = 0.025 min-1 vs. k-1(-R9) = 2.04 min-1], consistent with the 100-fold change in binding affinity of 5B7 for ICYP. The available data best fit a model in which an anti-idiotypic antibody binds at or near the binding site of the idiotype participating in the formation of a hybrid ligand binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cytoskeleton of isolated murine primitive erythrocytes

Summary Cytoskeletons of primitive erythrocytes have been isolated from the embryos of day 12 pregnant C57/Bl mice and examined by transmission electron microscopy, immunofluorescence microscopy, and SDS-polyacrylamide gel electrophoresis. Microtubules are the most prominent cytoskeletal component. They are found either singly or organized into loose bundles just under the plasma membrane, but do not form classical marginal bands in most cells. Immunofluorescence with a polyclonal tubulin antiserum confirms this distribution and further reveals numerous mitotic figures among the cells. Rhodamine-conjugated phalloidin and heavy meromyosin labeling reveal that actin is localized in the cortex of the primitive erythrocyte in the form of 6 nm filaments. Antibody directed against avian erythrocyte alpha spectrin demonstrates that spectrin is also found in the cortex. Occasional 10-nm intermediate filaments, observed in the primitve erythrocytes by electron microscopy, are believed to be of the vimentin class based on positive reaction of the cells with vimentin-specific antiserum. In addition, a band in erythrocyte cytoskeletons comigrates in SDS-polyacrylamide gels with vimentin isolated from mouse kidney. Spectrin and actin were also found to be associated with the membrane of primitive erythrocytes when membrane ghost preparations were analyzed by SDS-polyacrylamide gel electrophoresis.     

Heterogeneity in lymphocyte spectrin distribution: ultrastructural identification of a new spectrin-rich cytoplasmic structure

Spectrin-like proteins are found in a wide variety of non-erythroid cells where they generally occur in the cell cortex near the plasma membrane. To determine the intracellular distribution of alpha-spectrin (alpha-fodrin) in lymphocytes, we have developed an immunoperoxidase method to localize this protein at the ultrastructural level. Of considerable interest, particularly with regard to our efforts to determine the function of spectrin in this cell type, was the finding that its subcellular localization and its relationship with the plasma membrane can vary dramatically. Based on its position in the cell, alpha-spectrin can occur in two forms in lymphocytes: one that associates closely with the plasma membrane and another that occurs at some distance from the cell periphery, either as a single large aggregate or as several smaller ones. The single large aggregate of spectrin is a stable feature in a number of lymphocyte cell lines and hybrids which were used to examine its ultrastructural characteristics. A previously undescribed cellular structure, consisting of a meshwork of spectrin filaments and membranous vesicles, was identified in these cells. This structure could be induced to dissipate in response to membrane perturbants (e.g., hyperthermia and phorbol esters, known effectors of lymphocyte function and differentiation) and the patterns resulting from the redistribution of spectrin were a reflection of those observed routinely in lymphocytes in situ. The correlation between naturally occurring spectrin localization patterns and those seen after membrane perturbation suggested the possibility that spectrin distribution is indicative of particular maturation stages or functional states in lymphocytes. The implications of these findings with regard to the role of spectrin in lymphocyte function are discussed.     

The role of erythropoietin in the production of principal erythrocyte proteins other than hemoglobin during terminal erythroid differentiation

Erythropoietin (EP) controls the terminal phase of differentiation in which proerythroblasts and their precursors, the colony forming units-erythroid (CFU-e), develop into erythrocytes. Biochemical studies of this hormone-directed terminal differentiation have been hindered by the lack of a homogeneous population of erythroid cells at the developmental stages of CFU-e and proerythroblasts that will synchronously differentiate in response to EP. Such a population of cells can be prepared from the spleens of mice with the acute erythroblastosis resulting from infection with anemia-inducing Friend virus (FVA). Using these FVA-infected erythroid cells, which were induced to differentiate with EP, four proteins other than hemoglobin that have key functions in mature erythrocytes were monitored during the 48-hour period of terminal differentiation. Synthesis of spectrin and membrane band 3 proteins were determined by immunoprecipitation and SDS-polyacrylamide gel electrophoresis; accumulation of the cytoskeletal protein band 4.1 was monitored by immunoblotting; carbonic anhydrase activity was measured electrometrically. Band 3 synthesis and band 4.1 accumulation could be detected only after exposure of the cells to EP. Spectrin synthesis was ongoing prior to culture with EP, but it did increase after exposure to the hormone. Carbonic anhydrase-specific activity changed very little throughout the terminal differentiation process. These results reveal at least three patterns of production of principal erythrocyte proteins during EP-mediated terminal differentiation of FVA-infected erythroid cells. Depending on the specific protein examined, de novo synthesis can be induced by EP, an ongoing production can be enhanced by EP, or the production of a protein can be completed at a developmental stage prior to EP-mediated differentiation in these cells.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation     

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.     

Region-Specific Targets of p42/p44MAPK Signaling in Rat Brain

Abstract: In vitro studies indicate that p42/p44^MAPK phosphorylate both nuclear and cytoplasmic proteins. However, the functional targets of p42/p44^MAPK activation in vivo remain unclear. To address this question, we localized activated p42/p44^MAPK in hippocampus and cortex and determined their signaling effects after electroconvulsive shock treatment (ECT) in rats. Phosphorylated p42/p44^MAPK content increased in the cytoplasm of hippocampal neurons in response to ECT. Consistent with this cytoplasmic localization, inhibition of ECT-induced p42/p44^MAPK activation by the extracellular signal-regulated kinase kinase inhibitor PD098059 blocked phosphorylation of the cytoplasmic protein microtubule-associated protein 2c (MAP2c), but failed to inhibit the induction of the nuclear protein c-Fos in response to ECT. In contrast to hippocampal neurons, cortical neurons exhibited an increase in amount of phosphorylated p42/p44^MAPK in both the nucleus and cytoplasm after ECT. Accordingly, PD098059 blocked the induction of Fos-like immunoreactivity in the nuclei of cortical neurons as well as MAP2c phosphorylation in the cytoplasm. Our data indicate that both nuclear and cytoplasmic substrates can be activated by p42/p44^MAPK in vivo. However, the functional targets of p42/p44^MAPK signaling depend on the precise location of p42/p44^MAPK within different subcellular compartments of brain regions. These results indicate unique functional pathways of p42/p44^MAPK-mediated signal transduction within different brain regions in vivo.     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.     

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Introduction  

Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.