Thermodynamic dissection of the substrate-ribozyme interaction in the hammerhead ribozyme

The free energy of substrate binding to the hammerhead ribozyme was compared for 10 different hammerheads that differed in the length and sequence of their substrate recognition helices. These hammerheads were selected because neither ribozyme nor substrate oligonucleotide formed detectable alternate secondary structures. The observed free energies of binding varied from -8 to -24 kcal/mol and agreed very well with binding energies calculated from the nearest-neighbor free energies if a constant energetic penalty of DeltaG degreescore = +3.3 +/- 1 kcal/mol is used for the catalytic core. A set of substrates that contained a competing hairpin secondary structure showed weaker binding to the ribozyme by an amount consistent with the predicted free energy for hairpin formation. These thermodynamic conclusions permit the prediction of substrate binding affinities for ribozyme-substrate pairs of any helix length and sequence, and thus, should be very valuable for the rational design of ribozymes directed toward gene inactivation.     

Sandwich-type deoxyribonucleic acid hybridization assays based on enzyme amplified time-resolved fluorometry

We report microtiter well-based sandwich-type DNA hybridization assays using enzyme amplified time-resolved fluorometry of Tb3+ chelates. The target DNA was hybridized with two adjacent and non-overlapping oligonucleotide probes, one oligonucleotide serving as the capture probe and the other as the detection probe. Two ligand-specific binding protein pairs were used alternately for capture of the hybrids to the solid phase and detection; the biotin-streptavidin and the digoxigenin-anti-digoxigenin interaction. In both cases, alkaline phosphatase was used as a reporter molecule and diflunisal phosphate as a substrate. The catalytic hydrolysis of the substrate produces diflunisal which forms ternary fluorescent complex with Tb(3+)-EDTA. Furthermore, we studied the effect of the probe labeling method and the position of the label on the sensitivity of the assays. The data suggest that capture of the hybrids through biotin-streptavidin and detection via digoxigenin-anti-digoxigenin offer 2-3 times higher sensitivity than the reverse configuration. The highest sensitivity was achieved with enzymatic labeling of capture and detection probes at the 3' end. A signal-to-background ratio of 4 was achieved for 0.2 fmol of target DNA. The RSD were better than 4%.     

Human P2Y1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites

The molecular basis for recognition by human P2Y1 receptors of the novel, competitive antagonist 2'-deoxy-N6-methyladenosine 3', 5'-bisphosphate (MRS 2179) was probed using site-directed mutagenesis and molecular modeling. The potency of this antagonist was measured in mutant receptors in which key residues in the transmembrane helical domains (TMs) 3, 5, 6, and 7 were replaced by Ala or other amino acids. The capacity of MRS 2179 to block stimulation of phospholipase C promoted by 2-methylthioadenosine 5'-diphosphate (2-MeSADP) was lost in P2Y1 receptors having F226A, K280A, or Q307A mutations, indicating that these residues are critical for the binding of the antagonist molecule. Mutation of the residues His132, Thr222, and Tyr136 had an intermediate effect on the capacity of MRS 2179 to block the P2Y1 receptor. These positions therefore appear to have a modulatory role in recognition of this antagonist. F131A, H277A, T221A, R310K, or S317A mutant receptors exhibited an apparent affinity for MRS 2179 that was similar to that observed with the wild-type receptor. Thus, Phe131, Thr221, His277, and Ser317 are not essential for antagonist recognition. A computer-generated model of the human P2Y1 receptor was built and analyzed to help interpret these results. The model was derived through primary sequence comparison, secondary structure prediction, and three-dimensional homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. We have introduced a "cross-docking" procedure to obtain energetically refined 3D structures of the ligand-receptor complexes. Cross-docking simulates the reorganization of the native receptor structure induced by a ligand. A putative nucleotide binding site was localized and used to predict which residues are likely to be in proximity to agonists and antagonists. According to our model TM6 and TM7 are close to the adenine ring, TM3 and TM6 are close to the ribose moiety, and TM3, TM6, and TM7 are near the triphosphate chain.     

Structure analysis of trivalent glycoclusters by post-source decay matrix-assisted laser desorption/ionization mass spectrometry

Post-source decay (PSD) matrix-assisted laser desorption/ionization time-of-flight mass spectra were found to be useful for the structural elucidation of a series of tris [2-(glycosylthiourylene)ethyl]amines. The reported fragmentation behaviours of [M + H]+, [M + Na]+ and [M - H]- ions differ from each other significantly; however, they can be compared to tree pruning in every case. Whereas detailed structural information on unprotected glycoclusters is obtained from all PSD experiments, only the positive-ion mode can be used to gain relevant information about the acetylated glycoclusters.     

Uncertainty analysis methods for comparing predictive models and biomarkers: A case study of dietary methyl mercury exposure

Biologically based markers (biomarkers) are currently used to provide information on exposure, health effects, and individual susceptibility to chemical and radiological wastes. However, the development and validation of biomarkers are expensive and time consuming. To determine whether biomarker development and use offer potential improvements to risk models based on predictive relationships or assumed values, we explore the use of uncertainty analysis applied to exposure models for dietary methyl mercury intake. We compare exposure estimates based on self-reported fish intake and measured fish mercury concentrations with biomarker-based exposure estimates (i.e., hair or blood mercury concentrations) using a published data set covering 1 month of exposure. Such a comparison of exposure model predictions allowed estimation of bias and random error associated with each exposure model. From these analyses, both bias and random error were found to be important components of uncertainty regarding biomarker-based exposure estimates, while the diary-based exposure estimate was susceptible to bias. Application of the proposed methods to a simple case study demonstrates their utility in estimating the contribution of population variability and measurement error in specific applications of biomarkers to environmental exposure and risk assessment. Such analyses can guide risk analysts and managers in the appropriate validation, use, and interpretation of exposure biomarker information.     

Immunohistochemical analysis of transferrin receptor: regional and cellular distribution in the hypotransferrinemic (hpx) mouse brain

The hypotransferrinemic (hpx) mouse mutant produces <1% of the normal circulating level of transferrin (Tf). Heterozygote animals of this strain (hpx/+) have approximately 50% of normal plasma Tf levels. In this study we examine the cellular and regional distribution of Tf receptor (Tf-R) in the brain of wild type, hpx/+ and mutant (hpx/hpx) mice. Also, using slot-blot (immunoblot) analysis, we describe the relative amount of Tf-R in brain microvessels of hpx/+ animals compared with wild type. Tf-R was seen primarily in neurons throughout the brains of wild type, hpx/+ and hpx/hpx animals. Gray matter areas immunoreacted more robustly than white matter areas. Oligodendrocytes and third ventricle tanycytes, both of which we have previously described as iron-positive, did not immunoreact for Tf-R. Tf-R immunohistochemical reaction in wild type, hpx/+ and hpx/hpx brains appeared similar. Immunoblot analysis of isolated cortical microvessels from wild type and hpx/+ animals revealed no upregulation of Tf-R expression in hpx/+ (relative to normal) despite a 50% decrease in circulating Tf levels. These results indicate that Tf-R is primarily expressed by neurons and that half normal levels of Tf (hpx/+) or transferrin supplementation (hpx/hpx) are apparently sufficient for normal expression and distribution of Tf-R. Because of the lack of circulating Tf, but unaltered Tf-R expression, hpx mice could serve as a model for delivery of therapeutic agents via the Tf/Tf-R system.     

Human cytochrome P4502B6: interindividual hepatic expression, substrate specificity, and role in procarcinogen activation

In situ hybridization was combined with serotonin (5-hydroxytryptamine, 5-HT) or tyrosine hydroxylase immunocytochemistry and with Fluoro-Gold retrograde labeling of bulbo-spinal pathways in order to investigate the expression of GAP-43 mRNA in monoamine cell groups of the adult rat brain stem. Consistent with previous reports, GAP-43 mRNA was observed in serotonin and dopamine cell groups in the pons. In addition, GAP-43 expressing cells were observed in all the major monoamine cell groups in the medulla. Thus the B1, B2 and B3 serotonin cell groups all showed high GAP-43 expression in all contained many GAP-43 expressing serotonin cells with spinal cord projections. The A1, A2, A5 and A6 noradrenaline cell groups also showed high GAP-43 expression, although cells with spinal cord projections were largely restricted to the A5 group and A6 subcoeruleus region. In all areas, GAP-43 expressing cells with spinal cord projections were also observed which were not serotonergic or noradrenergic.     

Are Canadian Inuit at increased genetic risk for coronary heart disease?

The Keewatin Inuit of the Northwest Territories of Canada have a very low age-adjusted mortality rate from coronary heart disease. We hypothesized that this apparent protection from disease has a genetic basis. We determined the prevalence of the disease-associated alleles of five candidate genes for atherosclerosis-related phenotypes. Surprisingly, four of the five alleles studied, namely AGT T235, FABP2 T54, PON R192 and APOE E4, were significantly more frequent in a sample of 175 Keewatin Inuit than among a representative control sample of whites living in the region. The high frequencies of these disease-associated alleles suggests either that they have no relationship with disease susceptibility in the Inuit, or that some unmeasured genetic and/or environmental factors mitigate disease susceptibility that is associated with these alleles. This highlights the difficulty in extrapolating findings from one population to another. Also, very modest genotype-phenotype associations were observed between APOE genotype (P = 0.016) and plasma low-density lipoprotein cholesterol concentration and between FABP2 genotype and plasma 2-h postprandial, glucose concentration (P = 0.048). The relationship between APOE alleles and plasma low-density lipoprotein cholesterol was the same as has been previously reported in many study samples. However, the relationship between FABP2 alleles and plasma 2-h postprandial glucose concentrations was the opposite to that reported in other studies. This suggests that differences in environment, such as the type of fatty acid consumed, interacts with functional differences in gene products involved in candidate metabolic pathways to produce phenotypic differences.     

Attachment loss with postmenopausal age and smoking

To determine whether postmenopausal bone loss and factors associated with osteoporosis affect tooth retention, we examined vertebral and proximal femoral (postcranial) bone mineral density in relation to tooth loss and attachment loss in a cross-sectional study of 135 postmenopausal women (age range 41-70 yr). Women had at least 10 teeth and no evidence of moderate or severe periodontal disease. Full-mouth attachment loss measurements were made using a pressure-sensitive probe, and bone density was determined by dual-energy X-ray absorptiometry. Attachment loss was correlated with tooth loss (number of remaining teeth, radiologically determined), but not with vertebral or proximal femur bone density. Multivariate analysis showed current smoking (p = 0.01), years since menopause (p = 0.02) and the interaction of age and current smoking (p < 0.01), to be statistically significant predictors of attachment loss in our study population.     

Novel pharmacologic strategies in the treatment of experimental traumatic brain injury: 1998

The mechanisms underlying secondary or delayed cell death following traumatic brain injury are poorly understood. Recent evidence from experimental models suggests that widespread neuronal loss is progressive and continues in selectively vulnerable brain regions for months to years after the initial insult. The mechanisms underlying delayed cell death are believed to result, in part, from the release or activation of endogenous "autodestructive" pathways induced by the traumatic injury. The development of sophisticated neurochemical, histopathological and molecular techniques to study animal models of TBI have enabled researchers to begin to explore the cellular and genomic pathways that mediate cell damage and death. This new knowledge has stimulated the development of novel therapeutic agents designed to modify gene expression, synthesis, release, receptor or functional activity of these pathological factors with subsequent attenuation of cellular damage and improvement in behavioral function. This article represents a compendium of recent studies suggesting that modification of post-traumatic neurochemical and cellular events with targeted pharmacotherapy can promote functional recovery following traumatic injury to the central nervous system.