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C V Harding

Publications and source records attributed to C V Harding.

33 records · Page 2Linked to original sources
PubMed1989

Antigen processing and intracellular Ia. Possible roles of endocytosis and protein synthesis in Ia function.

Anti-I-A mAb and monovalent Fab fragments were used to explore the cellular distribution and endocytosis of I-A in peritoneal exudate cells (PEC) and TA3 B lymphoma-hybridoma cells. TA3 cells contained 1.6 x 10(5) I-A sites/cell, 22 to 35% of which were intracellular. This intracellular pool was cycloheximide resistant. PEC contained 1.8 x 10(5) I-A sites/cell, 25 to 40% of which were intracellular. Upon adherence, however, the intracellular pool of I-A in PEC dropped to 2 to 11% of the total cellular I-A. Ag processing by TA3 cells was unaffected 3 h after abrogation of protein synthesis with cycloheximide, suggesting that newly synthesized I-A is not necessary for Ag processing in TA3 cells (post-synthetic processing and transport of I-A to the plasma membrane were complete by 2 h in TA3 cells with or without cycloheximide, as assessed by sequential immunoprecipitation of surface and intracellular I-A). In adherent PEC, however, cycloheximide markedly inhibited Ag processing, suggesting depletion of factors necessary for Ag processing. Ag processing may involve binding of processed Ag peptides to intracellular Ia derived to varying degrees from both endocytosis and new biosynthesis. To explore the possibility of I-A recycling, I-A endocytosis was demonstrated using mAb and monovalent Fab probes; internalization occurred within 5 min and peaked by 10 to 15 min with 15 to 35% of bound antibody in an intracellular compartment, resistant to an acid wash. Subcellular density gradient fractionation demonstrated that I-A and transferrin were processed exclusively in an endosomal fraction of relatively light density, whereas ligands of the mannose receptor were processed in light endosomes and in a distinct, denser population of endosomes, and accumulated in lysosomes. Thus, I-A appears to be internalized into a specific population of endosomes that may play a central role in Ag processing.

Animals↗
PubMed1989

Turnover of Ia-peptide complexes is facilitated in viable antigen-presenting cells: biosynthetic turnover of Ia vs. peptide exchange.

Macrophages and B cells process antigens to produce antigenic peptides that associate with class II major histocompatibility complex molecules (e.g., Ia molecules); these Ia-peptide complexes are recognized by CD4+ T lymphocytes. Processing of the antigen hen egg white lysozyme was inhibited by cycloheximide in peritoneal exudate cells (PECs, largely macrophages), but not in TA3 B-lymphoma cells. The uptake and metabolism of hen egg white lysozyme was largely intact in cycloheximide-treated PECs, implicating a blockade in other steps in the formation of Ia-peptide complexes. Turnover of Ia-peptide complexes was markedly enhanced in viable antigen-presenting cells (TA3 and PEC) as compared to such complexes studied on fixed cells or in isolated preparations of Ia and peptide. In B cells the half-life of Ia-peptide complexes was much shorter than the half-life of the Ia molecules, implying turnover of Ia-peptide complexes by dissociatin and peptide exchange. In PECs, the dissociation of Ia-peptide complexes was more limited; the enhanced Ia-peptide turnover in viable PECs reflected in part biosynthetic turnover of Ia molecules. Specific mechanisms may exist in TA3 cells to facilitate exchange of peptides bound to Ia, allowing recycling of Ia to present another antigenic peptide; such Ia recycling would explain the ability of these cells to process and present antigen in the absence of Ia synthesis.

Animals↗
PubMed1989

Elemental and structural studies of the rat galactose cataract.

A series of rat galactose lenses, from 1 to 20 days on the 50% galactose diet, were frozen in the whole eye, and fractured from pole to pole in the frozen state. Lyophilized half-lenses were prepared for analysis by energy dispersive spectrometry (EDS). Following elemental analysis, some specimens were embedded and sectioned for histological studies. Elemental X-ray maps, and/or profiles, were made for K, Na, Cl, Ca, P, and S. As early as two days on the galactose diet, a crescent-shaped region ("streak") of Cl, Na, and Ca gain, and K loss develops near the equatorial surface. Between this region and the equatorial surface are the nucleated differentiating fiber cells which maintain low Cl, Na, and Ca, and high K (viable equatorial zone, VEZ). With time the "streak" expands anteriorly, centrally and posteriorly, eventually (by 20 days) including most of the lens. The VEZ, including the epithelium, however, is non-reactive to the galactose diet, which is deleterious to the fully differentiated fiber cells. Eventually, the VEZ undergoes a characteristic morphological change, apparently due in part to changes in its physical environment.

Animals↗
PubMed1988

Mechanisms of antigen processing.

Using MAb and monovalent Fab probes and saponin permeabilization we have demonstrated that PEC and TA3 B lymphoma-hybridoma cells contain a significant intracellular pool of Ia. At least in TA3 cells, this intracellular pool was independent of protein synthesis. In PEC, adherence caused redistribution of Ia with disappearance of the intracellular pool. Endocytosis of Ia occurred in both TA3 and PEC, and internalized Ia reached a plateau level corresponding in size to the total intracellular Ia pool revealed by saponin treatment. These results suggest that intracellular Ia is largely in a recycling pool derived from the plasma membrane by endocytosis. Subcellular fractionation studies suggest that Ia processing occurs in endosomes similar to those involved in transferrin processing. Antigen processing by TA3 cells was found to be unaffected by cycloheximide. In contrast, antigen processing by adherent PEC was markedly inhibited by cycloheximide, despite the fact that they maintained surface Ia and were still capable of presenting antigen peptides. This suggests that an important intracellular Ia processing step or antigen processing step was blocked in these cells. Adherent PEC may contain less recycling Ia, making protein synthesis the major source for intracellular Ia and the availability of intracellular Ia sensitive to cycloheximide. Alternatively, the inhibition of antigen processing by cycloheximide in PEC may reflect depletion of enzymes or other factors involved in antigen processing. Proteins and polysaccharides may interfere with the events that result in the formation of an immunogenic Ia-peptide complex. We had previously documented that peptides compete for the binding site of Ia molecules. We discussed here a second form of interference by polysaccharides and microbial products. These materials did not compete or interfere with the binding and presentation of processed peptides by Ia. Rather, their presence inside the macrophage inhibited MHC-dependent presentation of immunogenic proteins by inhibiting intracellular steps in antigen processing. This intracellular interference with antigen presentation can be of major importance in the presentation of complex mixtures of protein and carbohydrates.

Antigen-Presenting Cells↗
PubMed1986

Structure and distribution of gap junctions in lens epithelium and fiber cells.

We report a comparative study of gap junctions in lens epithelia of frog, rabbit, rat and human, using a "double mounting" method for freeze-fracture electron microscopy. The gap junctions on the narrow sides of hexagonal cortical fiber cells of various species were also studied with the same technique. Gap junctions were commonly present between epithelial cells of the entire undifferentiated epithelium, between fiber cells on both wide and narrow sides, and between epithelial cells and fiber cells. Structural diversity of gap junctions, based on connexon arrangements, was evident in lens epithelia among the four species studied. Gap junctions with random arrays of connexons were found predominantly in frog lens epithelium, while the crystalline and striated configurations were mainly observed in the epithelia of human and rat, and of rabbit, respectively. On the other hand, there was no structural variation of gap junctions observed on either wide or narrow sides of lens fiber cells from any species studied. Only the random-type gap junction was found. However, the distribution of gap junctions was unique on the narrow sides. There was a single row of junctional plaques along the middle of the narrow sides, whereas the wide sides showed an uneven distribution pattern. The gap junctions between epithelial cells and fiber cells had a random packing of connexons.

Adolescent↗
PubMed1976

The nuclear envelope in the crystalline lens fiber cell.

Rabbit lenses which have been fixed, dehydrated, and dried by a critical-point drying method, can be fractured through the cytoplasm of the differentiating lens fibers, exposing the cell nuclei. The fracture, under these conditions, causes a complete separation of the two membranes of the nuclear envelope from one another, thus exposing entire membrane surfaces (those which line the perinuclear space). These surfaces are not seen in their entirety in typical freeze-fracture or freeze-etch preparations, and consequently have not been described previously. The exposed membrane surfaces which line the perinuclear space have numerous convex structures of approximately 1,000 A, and some larger more irregularly shaped structures. These appear to be fragments of the nuclear pore complexes. Differences in these structures between young fibers and those nearing completion of differentiation is suggested.

Animals↗