Young Tai Kim

Immunological studies of aging: Normal B-cell repertoire in aged mice: Studies at a clonal level

As previously reported, old mice produce lower avidity plaque-forming cells (PFC) after immunization with 2,4,6-trinitrophenyl-Ficoll (TNP-F) than do young mice. However, if spleen cells from TNP-F-immunized old mice are incubated with hapten to elute auto-anti-idiotype antibody then high avidity PFC, comparable to those in young mice, are detected. To further evaluate the effect of age on the B-cell repertoire anti-2,4,6-trinitrophenyl-bovine gamma globulin (TNP-BGG) hybridomas were prepared from young (6 to 8 weeks old) and old (18 to 24 months old) mice which had been primed and boosted with TNP-BGG. The monoclonal antibodies (MoAbs) were TNP-specific. Spleens from old and young mice were comparable with respect to the incidence of immunoglobulin-secreting hybridomas obtained, the incidence of TNP-BGG-specific hybridomas obtained, and the isotype distribution of the anti-TNP-BGG hybridomas. The avidities for TNP-BGG of the IgG1 anti-TNP-BGG MoAbs obtained from old and young donors were also comparable. The overall results thus suggest that old and young mice have similar B-cell repertoires and that differences in the antibodies produced are due to regulatory influences.

The subcellular localization of glutamate dehydrogenase (GDH): is GDH a marker for mitochondria in brain?

Glutamate dehydrogenase (GDH, EC 1.4.1.2) has long been used as a marker for mitochondria in brain and other tissues, despite reports indicating that GDH is also present in nuclei of liver and dorsal root ganglia. To examine whether GDH can be used as a marker to differentiate between mitochondria and nuclei in the brain, we have measured GDH by enzymatic activity and on immunoblots in rat brain mitochondria and nuclei which were highly enriched by density-gradient centrifugation methods. The activity of GDH was enriched in the nuclear fraction as well as in the mitochondrial faction, while the activities of other “mitochondrial” enzymes (fumarase, NAD-isocitrate dehydrogenase and pyruvate dehydrogenase complex) were enriched only in the mitochondrial fraction. Immunoblots using polyclonal antibodies against bovine liver GDH confirmed the presence of GDH in the rat brain nuclear and mitochondrial fractions. The GDH in these two subcellular fractions had a very similar molecular weight of 56,000 daltons. The mitochondrial and nuclear GDH differed, however, in their susceptibility to solubilization by detergents and salts. The mitochondrial GDH could be solubilized by extraction with low concentrations of detergents (0.1% Triton X-100 and 0.1% Lubrol PX), while the nuclear GDH could be solubizeded only by elevated concentrations of detergents (0.3% each) plus KCl (>150mM). Our results indicate that GDH is present in both nuclei and mitochondria in rat brain. The notion that GDH may serve as a marker for mitochondria needs to be re-evaluated.

Ribavirin pharmacodynamics in high‐risk patients for acquired immunodeficiency syndrome

Ribavirin was administered orally in escalating doses for 2 or 4 weeks to 15 symptom‐free, human immunodeficiency virus seropositive homosexual men with generalized lymphadenopathy. Reverse transcriptase activity was inhibited during therapy when steady‐state plasma concentrations were >6 µmol/L. These concentrations were achieved with 1200 or 2400 mg/day for 2 weeks or a loading dose of 2400 mg/day for 3 days followed by 600 mg/day for 4 weeks. Drug accumulation occurred at all doses. The elimination half‐life appeared to be approximately 2 weeks. Reversible adverse reactions, principally resulting in central nervous system symptoms and anemia, correlated with dose and duration of therapy. Immunologic enhancement of T‐lymphocyte—mediated mitogen‐induced responses was observed in the majority of patients who had reduction in reverse transcriptase activity. However, specific T4 + lymphocyte—mediated antigen‐induced responses increased to within the normal range in only three patients. Significant enhancement appeared to correlate with the severity of baseline antigen‐induced functional impairment. These data indicate that oral ribavirin can be given for at least 1 month with acceptable toxicity at doses that appear to inhibit human immunodeficiency virus replication.

A comparison of the Farr technique with equilibrium dialysis for measurement of antibody concentration and affinity

The Farr technique has been compared with equilibrium dialysis in regard to its ability to measure antibody concentration and affinity. An excellent qualitative agreement between the two techniques was obtained. Quantitatively, the Farr technique tended to underestimate antibody concentration and overestimate antibody affinity. It is likely that these observations are due to the failure of some low affinity antibodies to bind hapten under the conditions of the Farr assay.

Distribution of antibody affinities—II. Fractionation of antibody with respect to its hapten binding affinity☆

Abstract Antibodies from individual sera were fractionated on the basis of their binding affinity by use of a specific immunoadsorbent. The distribution of affinities in the original serum and in each of the series of 10 fractions derived from it was computed. The antibodies were highly heterogeneous and generally not distributed in a symmetrical manner. The distribution of affinities varied with different immunizing conditions. It is clear from the data presented that while values for ‘average affinity’ generally reflect accurately the overall binding properties of an antibody sample, they do not provide a complete picture of the actual distribution of affinities present. It was found that antibody samples exist which are indistinguishable with regard to ‘average affinity’ but differ significantly with respect to the actual distribution of the antibody affinities. Detailed analysis of the binding curves indicated that even animals forming mainly high affinity antibody had considerable amounts of very low affinity antibodies present in their serum.

Studies on the Bovine Brain Pyruvate Dehydrogenase Complex Using the Antibodies Against Kidney Enzyme Complex

Abstract: Pyruvate dehydrogenase complex (PDHC) was purified from bovine kidney with a specific activity of 12–16 μmol of NADH or acetyl‐CoA formed/min/mg protein. The four peptides comprising its three catalytic components were separated by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Rabbit antibodies against this highly purified PDHC (anti‐PDHC) exhibited similar binding affinity to the phospho‐PDHC as it did to the PDHC antigen. To test whether there exist brain isozymes of PDHC differing from kidney enzyme, which has been extensively characterized, the PDHCs in bovine brain and kidney were compared using this anti‐PDHC. The PDHC activities in the brain and kidney mitochondrial extracts were inhibited to the same degree by varying amounts of anti‐PDHC. Brain PDHC was precipitated with the anti‐PDHC and resolved by SDS‐PAGE. The four brain PDHC peptides isolated immunochemically with anti‐PDHC had the same sizes as the kidney PDHC peptides. These PDHC peptides from kidney and brain were further compared by their peptide fragment patterns, which were generated by partial proteolysis with Staphylococcus aureus V8 protease or by CNBr and resolved by SDS‐PAGE. The peptide patterns generated with the former method indicated that the α and β peptides of the pyruvate dehydrogenase (E1) component and the peptide of dihydrolipoyl transacetylase (E2) component of kidney PDHC were very similar to the corresponding peptides immunologically isolated from brain. The peptide patterns generated with CNBr further confirmed that the βE1 and E2 peptides of kidney PDHC were similar to the corresponding peptides from brain.

Immunochemical Characterization of Pyruvate Dehydrogenase Complex in Rat Brain

Abstract: Pyruvate dehydrogenase complex (PDHC) in rat brain was studied immunochemically, using antibodies against the bovine kidney PDHC, by immunoblotting, immunoprecipitation, inhibition of enzyme activity, and enzyme‐linked immunoabsorbent assay (ELISA). The immunoblots showed that the antibodies bound strongly to the α peptide of the pyruvate dehydrogenase (E1) component, and to the dihydrolipoyl transacetylase (E2) and the dihydrolipoyl dehydrogenase (E3) components of PDHC. A similar immunoblotting pattern was observed in all eight brain regions examined. On immunoblotting of the subcellular fractions, these PDHC peptides were observed in mitochondria and synaptosomes but not in the postmitochondrial supernatants. This agrees with other evidence that brain PDHC is localized in the mitochondria. These results, together with those from sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the immunoprecipitin, also showed that the αE1, βE1, and E3 peptides of rat brain PDHC are very similar in sizes to those of the bovine kidney PDHC, being 42, 36, and 58 kD, respectively. The size of the E2 peptide, 66 kD, is different from that of bovine kidney E2, 73 kD. The relative abundance of PDHC protein in nonsynaptic mitochondria was compared by enzyme activity titration and ELISA. Both methods demonstrated that the amount of PDHC antigen in the mitochondria from cerebral cortex is greater than that in the olfactory bulb mitochondria. This is consistent with the results of the activity measurement. The ELISA also showed that the PDHCs in both mitochondrial populations are antigenically similar. Our results suggest that variations of PDHC activity among different brain regions represent quantitative variations in the amount of PDHC protein.

Mitochondrial Enzymes in Hereditary Ataxias

As a test of the hypothesis that mitochondrial abnormalities are common in patients with hereditary ataxias, the activities of two mitochondrial enzymes were studied in platelets from an unselected series of patients. For the group of ataxies, the activity of the pyruvate dehydrogenase complex (PDHC) was 68% of the control (P < 0.01) and that of glutamate dehydrogenase (GDH) was 81% of the control (P < 0.05). Of the ataxies studied, 30% had activities of either or both mitochondrial enzymes more than2 SD below the control mean. Immunoblots of PDHC revealed antibody cross-reacting material in platelets and fibroblasts very similar to those in human brain and appeared normal in platelets from patients with ataxias. Immunoblots of GDH showed a single antibody cross-reacting material in brain but at least two species in normal fibroblasts and platelets. The pathophysiology of hereditary ataxias may often involve mitochondrial damage associated with secondary decreases in the activities of mitohcondrial enzymes.

STUDIES ON THE CONTROL OF ANTIBODY SYNTHESIS

The effect of antigen dose on the kinetics of circulating antibody synthesis and on antibody affinity was studied in a haptenic system. High doses of antigen resulted, early in immunization, in higher concentrations of antibody followed later in the immune response by decreased serum levels of antibody as compared with lower doses of antigen. The affinity of the initial antibody synthesized was very similar over a wide antigen dose range. Subsequently, however, a rapid rise in affinity was seen in animals immunized with low doses of antigen, while relatively little change in affinity was seen in animals immunized with higher antigen doses. Suppression of active antibody formation by passive antiserum led to an increase in antibody affinity. The results are discussed in terms of the mechanisms involved in the selection of a population of cells to participate in the immune response and the mechanisms whereby antigen dose and circulating antibody function to control antibody synthesis.

A Quick Responsive Fluorogenic pH Probe for Ovarian Tumor Imaging

A novel cell-permeable compound, CypH-1, that is non-fluorescent at neutral pH, but fluoresces under mildly acidic conditions with a near infrared maximum emission wavelength was designed for the detection of tumors in the clinical setting. The potential of CypH-1 in ovarian cancer imaging was demonstrated using a murine model. The intraperitoneally administered CypH-1 results in a robust fluorescence signal of discrete neoplastic lesions with millimeter range resolution within few hours. Moreover, fluorescence signal is strikingly enhanced at peripheral regions of tumors at the microscopic level suggesting a sharp physiological difference at the tumor/normal tissue interface. This robust acid-activated imaging agent is expected to have significant impact in broad surgical and diagnostic applications.