George N. Abraham

A technique for the removal of pyroglutamic acid from the amino terminus of proteins using calf liver pyroglutamate amino peptidase

Abstract An improved technique has been developed which can quickly and easily unblock proteins and peptides containing pyrrolidone carboxylic acid (PCA) or pyroglutamic acid in the amino terminal position utilizing commercially available pyroglutamate amino peptidase isolated from calf liver. The successful application of the technique to five immunoglobulin polypeptide chains including two heavy chains and three light chains is demonstrated. Description of the deblocking procedure under stabilizing enzyme conditions and biochemical properties of the enzyme are given.

Aging and T-cell-mediated immunity

Changes in the T-lymphocyte compartment represent the most critical component of immunological aging. Recent studies have demonstrated that the age-related decline in T-cell-mediated immunity is a multifactorial phenomenon affecting T-cell subset composition as well as several proximal events such as protein tyrosine phosphorylation, generation of second messengers, calcium mobilization and translocation of protein kinase C, and distal events such as lymphocyte proliferation and cytokine production of the T-cell activation pathway. Age-related T-cell immune deficiency is preceded by thymic involution and is influenced by several intrinsic as well as extrinsic factors. Further, the role of monocytes and macrophages in T-cell activation changes with advancing age. This brief review will summarize the current knowledge of the cellular as well as molecular aspects of immunodeficiency of T cells due to aging, some of the paradoxes of aging as related to T-cell-mediated immunity, and possible factors which contribute to this paradox. Finally, experimental approaches will be suggested that might resolve these controversies and that might provide insights into the diverse and complex mechanisms that contribute to immunodeficiency of T cells. Ultimately these studies may suggest possible therapeutic interventions to enhance immune function in the elderly.

Proteolytic processing of histone H3 in chromatin: a physiologically regulated event in tetrahymena micronuclei

Micronuclei of Tetrahymena thermophila contain two electrophoretically distinct forms of histone H3. The slower migrating micronuclear species, H3S, is indistinguishable from the macronuclear H3 by electrophoretic analyses in three gel systems and by partial proteolytic peptide mapping. The faster species, H3F, is unique to micronuclei. Pulse-chase experiments with radioactive amino acids show that H3S is a precursor to H3F. We present evidence that the in vivo processing of H3S into H3F requires cell growth and/or division and may occur regularly each generation at a specific point in the cell cycle. The processing event must occur after H3F is deposited on micronuclear chromatin, since both H3S and H3F can be isolated from sucrose gradient-purified mononucleosomes (Allis, Glover and Gorovsky, 1979). Partial proteolytic peptide mapping coupled with 3H-N-ethylmaleimide labeling suggest that the processing event involves a proteolytic cleavage from the amino terminal end of H3F. Automated sequence analyses of 14C-lysine-labeled macronuclear H3 together with either 3H-lysine-labeled H3S or H3F demonstrated that H3F is derived from H3S by a proteolytic cleavage which removes six residues from the amino terminus. These observations represent the first demonstration of a physiologically regulated proteolytic processing event in histone metabolism.

Hepatitis B immunization of healthy elderly adults: relationship between naïve CD4+ T cells and primary immune response and evaluation of GM-CSF as an adjuvant.

The efficacy of granulocyte–macrophage colony-stimulating factor (GM-CSF) to enhance the primary immune response to hepatitis B vaccine was studied in healthy elderly with young volunteers included as controls in this double-blind, placebo-controlled trial of GM-CSF as an immune adjuvant. Na¨ıve T-helper cells (CD4+CD45RA+) were determined at baseline. Forty-five healthy elderly (average age, 74 years) and 37 healthy young controls (average age, 28 years) were randomized. Hepatitis B vaccine was administered at 0, 1, and 6 months. GM-CSF as a single injection of either 80 μg or 250 μg with the first and second doses of hepatitis B vaccine. In this trial GM-CSF did not enhance antibody responses. However, the antibody responses were dramatically different between these two groups: 35/35 young developed a protective titer versus 19/45 elderly (P < 0.0001). In addition, the mean logarithm of anti-hepatitis B antibody level in the 35 young who completed the study was 3.17 (log mIU/ml) but only 2.21 in the 19 elderly responders (P < 0.0001). Na¨ıve T-helper cells differed significantly between the two groups: the mean percentage of CD4+CD45RA+ T cells was 47.9% versus 35.0% (P < 0.0001) in the young and elderly volunteers respectively. Na¨ıve T cells also differed significantly between elderly who did or did not respond to HBV (39.9% vs. 31.7%, P = 0.039). Using linear regression, age, and percent na¨ıve, CD4 T cells were determined to significantly influence the anti-hepatitis B antibody response, but sex and dose of GM-CSF did not. For a two-parameter model: logarithm of antibody titer = (−0.038 × age in years) + (0.031 × % na¨ıve CD4T cells) + 2.68; adjusted r2 = 0.605 and P < 0.0001. However, age had a larger effect than na¨ıve CD4 T cells, i.e., in comparing young and elderly groups the log antibody titer decreased by 1.73 due to the increase in age but only 0.40 due to the decrease in na¨ıve CD4 T cells. Thus, there was a large effect of age that could not be explained by the quantitative change in the na¨ıve T-helper cells.

Modified diphenylamine reaction for increased sensitivity.

Abstract A simple organic extraction procedure utilizing amyl acetate is described which produces a four-fold increase in the sensitivity of the standard diphenylamine reaction. The procedure makes possible the detection of DNA at concentrations as low as 1.5 μg/ml solution, and is useful where turbidity of the aqueous phase is present.

Response of C-Reactive Protein and Serum Amyloid A to Influenza A Infection in Older Adults

Influenza epidemics are associated with significant morbidity and mortality in the elderly, with a substantial proportion of deaths due to cardiovascular events. Elevations of acute-phase proteins have been associated with an increased risk of atherosclerotic events. Therefore, serum amyloid A (SAA) and C-reactive protein (CRP) were measured during influenza illness and 4 weeks later in 7 young persons, 15 elderly outpatients, and 36 hospitalized adults. Striking elevations were seen in mean acute SAA and CRP levels in all groups, but hospitalized patients had the highest levels (SAA, 503 vs. 310 microg/mL [P=.006]; CRP, 120 vs. 34 microg/mL [P<.001]). The presence of dyspnea, wheezing, and fever was also associated with high CRP levels. Influenza infection is associated with significant elevations of SAA and CRP levels in elderly patients, especially those who require hospitalization. It is possible that direct effects of CRP may exacerbate preexisting atherosclerotic lesions and may help explain cardiovascular events associated with acute influenza.

Effect of age on mitogen induced protein tyrosine phosphorylation in human T cell and its subsets: down-regulation of tyrosine phosphorylation of ZAP-70

Several events of T cell activation have been reported to decline in humans with age. Since protein tyrosine phosphorylation is an early critical event of T cell activation, we performed a systematic analysis of the age-associated changes in the mitogen induced protein tyrosine phosphorylation of human T lymphocytes using SDS-PAGE and Western blotting techniques. Following stimulation with Con A and PHA, an identical pattern of protein tyrosine phosphorylation was observed in the lysates of T cells prepared from seven healthy young adults and eight healthy elderly human subjects. Five different high molecular mass proteins (75, 115, 120, 140 and 170 kDa) were consistently tyrosine phosphorylated in all of the donors from both age groups and peaked between 3 and 10 min. Tyrosine phosphorylation of the above substrates was observed in both CD4 and CD8 subsets. When compared for individual donors from both age groups, variations in the T cell response with regard to net tyrosine phosphorylation for all the substrates was observed. However, the mitogen induced level of tyrosine phosphorylation of only p75 was found to be significantly lower in unfractionated T cells as well as CD4 and CD8 subsets of older subjects than that of young subjects. Using immunoblotting, p75 was identified as ZAP-70, a member of the syk family of protein tyrosine kinases. Understanding of the biochemical basis of the reduced level of tyrosine phosphorylation of ZAP-70 will be helpful in delineating the molecular basis of age-associated impairment of T cell activation.

Size, shape, and hydration of a self-associating human IgG myeloma protein: axial asymmetry as a contributing factor in serum hyperviscosity.

Studies of sedimentation, diffusion, viscosity, and buoyant density have been carried out on a human IgG1-lambda myeloma protein (IgG-MIT) isolated from the serum of a patient with multiple myeloma and the hyperviscosity syndrome. In comparison with pooled normal IgG, IgG-MIT exhibits smaller sedimentation and diffusion coefficients, a larger intrinsic viscosity, and a larger frictional ratio. The preferential hydration of IgG-MIT in cesium chloride was found to be within the range of values typically observed for globular proteins. The data are consistent with prolate ellipsoid geometry, and suggest that the axial ratio of the IgG-MIT monomer is approximately 50% greater than that typically observed for IgG. The concentration dependencies of the hydrodynamic data for IgG-MIT confirm the previous finding of reversible, concentration-dependent self-association for this protein. IgG-MIT thus represents the first reported instance of an IgG paraprotein for which in vivo hyperviscosity effects appear attributable to a twofold mechanism involving geometric asymmetry and concentration-dependent polymerization. The results are discussed in terms of the significant heterogeneity in molecular dimensions which may exist among normal IgG proteins.

Structural studies of monoclonal human cryoprecipitable immunoglobulins

Abstract The light chain type, immunoglobulin class and when possible, heavy chain subclass of eleven monoclonal human cryoglobulins were correlated with the variable region subgroup of their light chains. The variable region subgroups were assigned by determining the primary amino acid sequence for the first 15 amino-terminal residues of these light chains. 5 5 IgM cryoglobulins which react with human IgG had light chains of the variable region-III kappa chain subgroup (vK-III). 4 4 IgG and 2 2 IgM cryoglobulins with undefined antibody specificity had both lambda and kappa light chains none of which were vK-III. The data support the concept that there is marked restriction of the IgM anti-IgG antibody response to the IgG auto-antigen.

Gene-modified cells for the treatment of cancer.

Gene therapy involves the insertion of a gene into an organism to treat a disease. Since its early development in the 1970s, gene therapy has expanded rapidly both in terms of the methods available and the number of candidate diseases for treatment. This report reviews gene therapy for cancer, including methodology, pre-clinical studies and experimental clinical trials.