The efficiency of hypoxanthine excision by alkyladenine DNA glycosylase is altered by changes in nearest neighbor bases

Alkyladenine DNA glycosylase (AAG) excises a structurally diverse group of damaged purines including hypoxanthine, 1,N(6)-ethenoadenine, 3-methyladenine, and 7-methylguanine from DNA to initiate base excision repair at these sites. Excision occurs in an enzyme.DNA complex in which the damaged base is flipped out of the DNA helix into the enzyme active site. To determine whether local DNA sequence could affect the overall efficiency of excision of hypoxanthine from DNA, single-turnover kinetics of excision, AAG.DNA binding, and melting temperatures were measured for DNA substrates that differed in the base pairs immediately 5' and 3' to hypoxanthine. When Hx was flanked by a 5'G and a 3'C, the efficiency of excision was reduced dramatically in comparison to a duplex containing a 5'T and 3'A. The reduction in excision efficiency was largely due to a decrease in binding affinity of AAG for DNA. The overall effect of GC versus TA nearest neighbors was to magnify the difference in the efficiencies of excision of Hx from pairs with thymine and difluorotoluene from a factor of 5 to a factor of about 100. In general, DNA substrates that were more stable as indicated by higher melting temperatures gave reduced efficiencies of excision of Hx. These results are discussed in terms of a model in which the relative stabilities of base-flipped versus unflipped complexes contribute the overall efficiency of excision and substrate specificity of AAG.     

R120G alphaB-crystallin promotes the unfolding of reduced alpha-lactalbumin and is inherently unstable

alpha-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, alphaB-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92-95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G alphaB-crystallin to stabilize the partially folded, molten globule state of alpha-lactalbumin was significantly reduced in comparison with wild-type alphaB-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced alpha-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G alphaB-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G alphaB-crystallin exists as a larger oligomer than wild-type alphaB-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of alphaB-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G alphaB-crystallin with human disease states.     

Expression of insulin-like growth factor-1 and insulin-like growth factor-1 receptors in EL4 lymphoma cells overexpressing growth hormone

Almost all of the previous studies with growth hormone (GH) have been done with exogenously supplied GH and, therefore, involve actions of the hormone through its receptor. However, the actions of endogenous or lymphocyte GH are still unclear. In a previous study, we showed that overexpression of GH (GHo) in a lymphoid cell line resulted in protection of the cells to apoptosis mediated by nitric oxide (NO). In the present study, we show that the protection from apoptosis could be transferred to control cells with culture fluids obtained from GHo cells and blocked by antibodies to the insulin-like growth factor-1 (IGF-1) or antibodies to the IGF-1-receptor (IGF-1R). Northern and Western blot analysis detected significantly higher levels of IGF-1 in cells overexpressing GH. An increase in the expression of the IGF-1R in GHo cells was also detected by Western blot analysis, (125)I-IGF-1 binding and analysis of IGF-1R promoter luciferase constructs. Transfection of GHo cells with a dominant negative IGF-1R mutant construct blocked the generation of NO and activation of Akt seen in GHo cells compared to vector alone control EL4 cells. The results suggest that one of the consequences of the overexpression of GH, in cells lacking the GH receptor, is an increase in the expression of IGF-1 and the IGF-1R which mediate the protection of EL4 lymphoma cells from apoptosis.     

Genomic data for alternate production strategies. I. Identification of major contaminating species for Cobalt(+2) immobilized metal affinity chromatography

Recent advances in technology have allowed for the identification of complex protein mixtures in a rapid fashion. This report highlights the use of 2D gel electrophoresis, mass spectrometry, and database analysis to determine contaminating species of the Escherichia coli genome that are present during immobilized metal affinity chromatography (IMAC), highlighting Co(2+) as the affinity ligand. Four proteins (triosephosphate isomerase, alpha galactosidase, Hsp90, and glucosamine 6-phosphate synthase) constitute the majority of E. coli proteins that bind and potentially may coelute during chromatography. Results are discussed within the context of changes that when implemented could lead to an increase in IMAC efficiency, not by altering column conditions, but rather by changing the nature of the nuisance proteins that principally reduce column capacity and extend processing times. Such a study illustrates the use of proteome data to aid in bioprocess design.     

Organ slices for the evaluation of human drug toxicity

Human organ slices, an in vitro model representing the multicellular and functional features of in vivo tissue, is a promising model for characterizing mechanisms of drug-induced organ injury and for identifying biomarkers of organ injury. Target organ injury is a significant clinical issue. In vitro models, which compare human and animal tissue to improve the extrapolation of animal in vivo studies for predicting human outcome, will contribute to improving drug candidate selection and to defining species susceptibilities in drug discovery and development programs. A critical aspect to the performance and outcome of human organ slice studies is the use of high quality tissue, and the use of culture conditions that support optimum organ slice survivability, in order to accurately reproduce mechanisms of organ injury in vitro. The attribute of organ slices possessing various cell types and interactions contributes to the overall biotransformation, inflammatory response and assessment of injury. Regional differences and changes in morphology can be readily evaluated by histology and special stains, similar to tissue obtained from in vivo studies. The liver is the major organ of which slice studies have been performed, however the utility of extra-hepatic derived slices, as well as co-cultures is increasing. Recent application of integrating gene expression, with human organ slice function and morphology demonstrate the increased potential of this model for defining the molecular and biochemical pathways leading to drug-induced tissue changes. By gaining a more detailed understanding of the mechanisms of drug-induced organ injury, and by correlating clinical measurements with drug-induced effects in the in vitro models, the vision of human in vitro models to identify more sensitive and discriminating markers of organ damage is attainable.     

Ca2+-sensing transgenic mice: postsynaptic signaling in smooth muscle

Genetically encoded signaling proteins provide remarkable opportunities to design and target the expression of molecules that can be used to report critical cellular events in vivo, thereby markedly extending the scope and physiological relevance of studies of cell function. Here we report the development of a transgenic mouse expressing such a reporter and its use to examine postsynaptic signaling in smooth muscle. The circularly permutated, Ca2+-sensing molecule G-CaMP (Nakai, J., Ohkura, M., and Imoto, K. (2001) Nat. Biotechnol. 19, 137-141) was expressed in vascular and non-vascular smooth muscle and functioned as a lineage-specific intracellular Ca2+ reporter. Detrusor tissue from these mice was used to identify two separate types of postsynaptic Ca2+ signals, mediated by distinct neurotransmitters. Intrinsic nerve stimulation evoked rapid, whole-cell Ca2+ transients, or "Ca2+ flashes," and slowly propagating Ca2+ waves. We show that Ca2+ flashes occur through P2X receptor stimulation and ryanodine receptor-mediated Ca2+ release, whereas Ca2+ waves arise from muscarinic receptor stimulation and inositol trisphosphate-mediated Ca2+ release. The distinct ionotropic and metabotropic postsynaptic Ca2+ signals are related at the level of Ca2+ release. Importantly, individual myocytes are capable of both postsynaptic responses, and a transition between Ca2+ -induced Ca2+ release and inositol trisphosphate waves occurs at higher synaptic inputs. Ca2+ signaling mice should provide significant advantages in the study of processive biological signaling.     

Cutting edge: tumor-specific CTL are constitutively cross-armed in draining lymph nodes and transiently disseminate to mediate tumor regression following systemic CD40 activation

The cross-arming of effector CTL in response to cross-presented tumor Ags is predicted to fail in the absence of CD40 stimulation. However, questions remain regarding the role of CD40 signaling and additional CD4+ T cell-derived signals in this process. To address this, we have analyzed the cross-arming of tumor-specific CTL effectors in vivo in a mouse model of established tumor and tumor regression following CD40 activation. We found that tumor-specific CTL were constitutively cross-armed in tumor-draining lymph nodes during tumor growth and that systemic CD40 activation did not alter CTL cross-arming in the tumor-draining lymph nodes. Rather, CD40 activation induced peripheral dissemination of tumor-specific CTL effectors that required continual CD40 stimulation to maintain peripheral CTL and tumor regression. These data indicate that CD40 activation enhances the peripheral survival of constitutively cross-armed CTL and that persistent CD4+ T cell signals are required for their long-term activity.     

Hypoallergenic variants of the major latex allergen Hev b 6.01 retaining human T lymphocyte reactivity

Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70-86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.