Apoptosis and its role in human disease

In a landmark paper published over two decades ago, Kerr et al. proposed the term apoptosis "for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations". In the ensuing years, this natural cell death process was studied at the basic science level, primarily with a view to understanding its roles in cancer and in the development and maintenance of the immune system. More recently, however, evidence has suggested a role for the failure of normal apoptosis control in many of the major diseases of the industrialized world. Though complex, apoptosis appears amenable to therapeutic intervention. The range of modern pharmaceutical strategies available to treat such disregulated gene-directed processes offers promise for advances in the control of cancer, immune system and neurodegenerative disorders, heart disease, and perhaps even the aging process itself.     

Natural ligands of the B cell adhesion molecule CD22 beta can be masked by 9-O-acetylation of sialic acids

CD22 beta is a B cell-restricted phosphoprotein expressed on the surface of mature resting B cells. It mediates interactions with other cells partly or exclusively via recognition of alpha 2-6-linked sialic acids on glycoconjugates. The sialylated N-linked oligosaccharides recognized best by CD22 beta are common to many glycoproteins, suggesting that additional regulatory mechanisms may exist. Since the exocyclic side chain of sialic acid is required for recognition, we explored the effects of a naturally occurring modification of the side chain, 9-O-acetylation. Semisynthetic N-linked oligosaccharides terminating with 9-O-acetylated, alpha 2-6-linked sialic acids showed markedly reduced binding to CD22 beta relative to their non-O-acetylated counterparts. Murine lymphoid cells were probed for natural CD22 beta ligands that might be O-acetylated using recombinant soluble forms of CD22 beta (CD22 beta Rg) and influenza C esterase (CHE-Fc, which specifically removes 9-O-acetyl esters from sialic acids). By flow cytometry analysis, CD22 beta Rg binding to splenic B cells and a subset of T cells was increased by pretreatment with CHE-Fc, indicating that some potential CD22 beta ligands are naturally "masked" by 9-O-acetylation. Unmasking of these CD22 beta ligands by removal of 9-O-acetyl esters from intact splenocytes substantially increases their CD22 beta-dependent adhesion in an in vitro adhesion assay. Probing of murine lymphoid tissue sections by CD22 beta Rg and CHE-Fc treatment demonstrates regionally restricted and differentially expressed patterns of distribution between masked and unmasked ligands. For example, lymph node-associated follicular B cells express high levels of CD22 beta ligands, none of which are masked by 9-O-acetylation. In contrast, the ligands on lymph node-associated dendritic cells are almost completely masked by 9-O-acetylation, suggesting that masking may regulate interactions between CD22 beta-positive B cells and dendritic cells. In the thymus, only medullary cells express CD22 beta ligands, and a significant portion of these are masked by 9-O-acetylation, particularly at the cortical-medullary junction. Thus, 9-O-acetylation of sialic acids on immune cells is in a position to negatively regulate CD22 beta adhesion events in a manner depending on both cell type and tissue localization.     

Isolation and characterization of a monoclonal anti-quadruplex DNA antibody from autoimmune "viable motheaten" mice

A cell line that produces an autoantibody specific for DNA quadruplex structures has been isolated and cloned from a hybridoma library derived from 3-month-old nonimmunized autoimmune, immunodeficient "viable motheaten" mice. This antibody has been tested extensively in vitro and found to bind specifically to DNA quadruplex structures formed by two biologically relevant sequence motifs. Scatchard and nonlinear regression analyses using both one- and two-site models were used to derive association constants for the antibody-DNA binding reactions. In both cases, quadruplexes had higher association constants than triplex and duplex molecules. The anti-quadruplex antibody binds to the quadruplex formed by the promoter-region-derived oligonucleotide d(CGCG4GCG) (Ka = 3.3 x 10(6) M-1), and has enhanced affinity for telomere-derived quadruplexes formed by the oligonucleotides d(TG4) and d(T2G4T2G4T2G4T2G4) (Ka = 5.38 x 10(6) and 1.66 x 10(7) M-1, respectively). The antibody binds both types of quadruplexes but has preferential affinity for the parallel four-stranded structure. In vitro radioimmunofilter binding experiments demonstrated that purified anti-DNA quadruplex antibodies from anti-quadruplex antibody-producing tissue culture supernatants have at least 10-fold higher affinity for quadruplexes than for triplex and duplex DNA structures of similar base composition and length. The antibody binds intramolecular DNA triplexes formed by d(G4T3G4T3C4) and d(C4T3G4T3G4), and the duplex d(CGCGCGCGCG)2 with an affinities of 6. 76 x 10(5), 5.59 x 10(5), and 8.26 x 10(5) M-1, respectively. Competition experiments showed that melted quadruplexes are not effective competitors for antibody binding when compared to native structures, confirming that the quadruplex is bound structure-specifically. To our knowledge, this is the first immunological reagent known to specifically recognize quadruplex structures. Subsequent sequence analysis demonstrates homologies between the antibody complementarity determining regions and sequences from Myb family telomere binding proteins, which are hypothesized to control cell aging via telomeric DNA interactions. The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.     

H] Tryptamine and 3H-water as diffusible internal standards for measuring brain extraction of radio-labeled substances following carotid injection

Urinary amino acid excretion was determined in 31 leukemic patients and 29 normal individuals by rapid gas chromatographic analysis of 16 amino acids as their N-acetyl-n-propyl esters. The leukemic patients were concurrently undergoing, or had recently completed, chemotherapy. It was found that aspartic acid, threonine, and serine were of significance in distinguishing between patients "on" therapy and those "off" therapy. Patients with advanced disease have the greatest aminoaciduria, although both the normal and leukemic populations have wide individual ranges. Within both populations, men excrete a greater variety and quantity of amino acids than women. It is concluded that analysis of urinary amino acids represents a history of complex metabolic events, which is potentially useful for evaluating patient response to chemotherapy in leukemia.     

Role of active site tyrosine residues in catalysis by human glutathione reductase

Tyr114 and Tyr197 are highly conserved residues in the active site of human glutathione reductase, Tyr114 in the glutathione disulfide (GSSG) binding site and Tyr197 in the NADPH site. Mutation of either residue has profound effects on catalysis. Y197S and Y114L have 17% and 14% the activity of the wild-type enzyme, respectively. Mutation of Tyr197, in the NADPH site, leads to a decrease in Km for GSSG, and mutation of Tyr114, in the GSSG site, leads to a decrease in Km for NADPH. This behavior is predicted for enzymes operating by a ping-pong mechanism where both half-reactions partially limit turnover. Titration of the wild-type enzyme or Y114L with NADPH proceeds in two phases, Eox to EH2 and EH2 to EH2-NADPH. In contrast, Y197S reacts monophasically, showing that excess NADPH fails to enhance the absorbance of the thiolate-FAD charge-transfer complex, the predominant EH2 form of glutathione reductase. The reductive half-reactions of the wild-type enzyme and of Y114L are similar; FAD reduction is fast (approximately 500 s-1 at 4 degreesC) and thiolate-FAD charge-transfer complex formation has a rate of 100 s-1. In Y197S, these rates are only 78 and 5 s-1, respectively. The oxidative half-reaction, the rate of reoxidation of EH2 by GSSG, of the wild-type enzyme is approximately 4-fold faster than that of Y114L. These results are consistent with Tyr197 serving as a gate in the binding of NADPH, and they indicate that Tyr114 assists the acid catalyst His467'.     

Effects of ibuprofen on airway vascular response to cotton smoke injury

We studied the effects of ibuprofen on bronchial blood flow and myocardial function after inhalation injury. Sheep (n = 12) were chronically instrumented with cardiovascular and pulmonary catheters. After 5 days of recovery period, baseline data were collected and the sheep were divided into two groups. Group S (n = 6) were insufflated with 48 breaths of cotton smoke; while group I (n = 6) were pretreated with ibuprofen (12mg/kg bolus followed by 3 mg/kg/h continuous infusion for 24 h) and challenged with the same dose of smoke. All the animals were studied for 24h. Bronchial blood flow increased significantly in both groups throughout the experimental period; while stroke volume as well as right and left ventricular stroke work indices of both groups were significantly decreased (group I worse than group S) in the second half of the experimental period. These data suggest that vasodilatory prostaglandins do not play a major role in the bronchial vascular response to smoke inhalation injury and myocardial depression seen post injury is worse in animals treated with ibuprofen.     

Novel NMDA antagonists: replacement of the pyridinium ring of 6,11-ethanobenzo[b]quinolizinium cations with heteroisoquinolinium cations

Replacement of the pyridinium ring of 6,11-ethanobenzo[b]quinolizinium cations with thiazolium (4a and 4b) and N-methylimidazolium (4c and 4d) resulted in equipotent compounds in the [3H]TCP binding assay. The corresponding N-methyl-1,2,4-triazolium analogs were less potent in this assay. The thiazolium derivative 4b, with a Ki = 2.9 nM, is being evaluated as a possible neuroprotective N-methyl-D-aspartic acid (NMDA) antagonist.     

Parameter estimates for direct, maternal, and grandmaternal genetic effects for birth weight and weaning weight in Hereford cattle

Birth and weaning weights adjusted for age of dam from four lines of Hereford cattle were analyzed to determine the relationships among grandmaternal, maternal, and direct genetic effects. Three lines were selected for 1) weaning weight (WWL), 2) yearling weight (YWL), and 3) an index of yearling weight and muscle score (IXL). The fourth line was an unselected control line (CTL). Numbers of observations ranged from 1,699 (CTL) to 2,811 (WWL), and number of animals in the pedigree file ranged from 2,266 to 3,192. Two animal models were used to obtain estimates by REML using an average information method. Model 1 included random direct and maternal genetic, permanent maternal environmental, and residual environmental effects, and fixed sex x year effects. Model 2 additionally included random grandmaternal genetic and permanent grandmaternal environmental effects. For birth weight, Models 1 and 2 gave almost identical estimates for direct and maternal heritability, and for the fraction of variance that was due to maternal permanent environmental effects. Estimates for grandmaternal heritability could be obtained only for IXL (.03) and CTL (.01). For weaning weight, estimates for direct heritability were similar from both models. Estimates for maternal heritability from Model 1 were .18, .20, .13, and .20, and corresponding estimates from Model 2 were .34, .31, .13, and .34 for WWL, YWL, IXL, and CTL, respectively. For IXL, estimates for variances that were due to grandmaternal genetic and grandmaternal permanent environmental variances could not be obtained and were set to zero. Grandmaternal heritability estimates for WWL, YWL, and CTL were .05, .09, and .12. Estimates of correlations between direct and maternal genetic effects were -.13, -.44, -.11, and -.26 for WWL, YWL, IXL, and CTL. Estimates of correlations between direct and grandmaternal genetic effects were .21, .83, and .55, and those between maternal and grandmaternal genetic effects were -.99, -.84, and -.76 for WWL, YWL, and CTL, respectively. These results indicate that grandmaternal effects may be important for weaning weight and that maternal heritability may be underestimated if grandmaternal effects are not included in the model.     

Delayed cytogenetics effects of chronic irradiation of South Ural population

The aim of this study was to assess the referral pattern and the impact on long-term survival of postoperative radiotherapy in patients with adenocarcinoma of the endometrium stage I. This was a retrospective study performed in a regional cancer registry which covers a population of approximately 1,000,000 persons. All 724 patients registered between 1975 and 1992 in the Comprehensive Cancer Centre South, Eastern Section, The Netherlands, were analysed. All patients had received surgery as primary treatment which was performed in one of the seven community hospitals of the region. Radiotherapy was given in one regional department. All pathology reports were checked for data on tumour differentiation and myometrial invasion. Almost half the patients (45%) were referred for postoperative radiotherapy. The depth of myometrial invasion and the degree of tumour differentiation were the main factors (P < 0.0001) influencing referral for postoperative radiotherapy. The referral pattern varied between the different hospitals, but became more similar during 1985-1988, to diverge again in recent years. In patients younger than 60 years, the depth of myometrial invasion was significantly (P = 0.01) correlated with survival. In patients older than 60 years, tumour differentiation (P = 0.05) and age (P < 0.001) were correlated with survival, but not the depth of myometrial invasion. After adjustment for known prognostic factors, a survival benefit of postoperative radiotherapy could not be established. The studied group had an excess death rate over the normal Dutch female population. This excess death rate did not decrease during follow-up, as even after 10 years an excess death rate was found. A prospective randomised trial is ongoing in The Netherlands.