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PubMed1999

Restoration of normal bone development by human homologue of collagen type II (COL2A1) gene in Col2a1 null mice.

Development of the vertebrate skeleton is a highly complex process in which collagen type II plays a vital role in the formation of long bones via endochondral ossification. Collagen type II, which is encoded by a single COL2A1/ Col2a1 gene, is the most abundant structural protein in the cartilage matrix, where it undergoes complex interactions with several other proteins. The sequence of mature collagen type II chains, each with about 1,100 amino acids, is conserved between different mammalian species. There are 37 amino acid positions that are different between mouse and human collagen type II. Previously, we have demonstrated that transgenic mice, in which Col2a1 gene is knocked out, exhibit a lethal phenotype due to the absence of endochondral bone formation. To investigate whether the biological role of collagen type II is conserved between the species, human COL2A1 gene was expressed in Col2a1 null mice by crossing with transgenic mice in which human COL2A1 gene was integrated. The collagen type II from human gene rescued the lethal phenotype in null mice, indicating that the biological function of collagen type II is conserved between human and mouse. The animals exhibited normal endochondral bone formation and a normal growth plate in tibio-tarsal joint. Chondrocytes isolated from the cartilage of these mice secreted human protein, suggesting that the animals incorporated heterologous protein to form cartilage which is essentially "humanized." The animals reached puberty and produced normal progeny. A completely normal phenotype in newborns indicates that human COL2A1 gene is expressed properly both temporally and spatially. These animals may be useful to generate models to study the effect of COL2A1 mutations on skeletal development in humans by introducing mutated gene constructs either into embryos or by crossing with transgenic animals with COL2A1 mutations.

Animals↗

PubMed1998

Identification of pollutants in a municipal well using high resolution mass spectrometry.

An elevated incidence of childhood cancer was observed near a contaminated site. Trace amounts of several isomeric compounds were detected by gas chromatography/mass spectrometry (GC/MS) in a concentrated extract of municipal well water. No matching library mass spectra were found and Fourier transform IR and NMR analyses were not feasible due to the low concentration of the compounds. Mass peak profiling from selected-ion-recording data (MPPSIRD) provided the sensitivity and scan speed necessary to acquire mass peak profiles at mass resolutions of 10,000 to 20,000 for the molecular ion (M+) and 10 fragment ions as capillary GC peaks eluted. Using a profile generation model (PGM), the elemental composition of the molecular ion was determined from the exact masses and abundances of the M, M + 1 and M + 2 profiles. Fragment ion compositions were determined from their exact masses based on the elements in the molecular ion. Exact mass differences between the molecular and fragment ions corresponded to unique combinations of atoms for the neutral losses. Consequent reduction of the number of possible structures for the fragment ions simplified mass spectral interpretation. After inspecting library mass spectra for smaller molecules, isomeric structures were hypothesized with cyano and alkylcyano groups attached to tetralin. A literature search found such isomers produced by an industrial polymer synthesis. Three isomers in a standard form polymerization of styrene and acrylonitrile provided the same mass spectra and GC retention times as isomers in the extract.

Acrylonitrile↗

PubMed1998

Optimising fed-batch production of recombinant proteins using the baculovirus expression vector system.

Fed-batch culture can offer significant improvement in recombinant protein production compared to batch culture in the baculovirus expression vector system (BEVS), as shown by Nguyen et al. (1993) and Bedard et al. (1994) among others. However, a thorough analysis of fed-batch culture to determine its limits in improving recombinant protein production over batch culture has yet to be performed. In this work, this issue is addressed by the optimisation of single-addition fed-batch culture. This type of fed-batch culture involves the manual addition of a multi-component nutrient feed to batch culture before infection with the baculovirus. The nutrient feed consists of yeastolate ultrafiltrate, lipids, amino acids, vitamins, trace elements, and glucose, which were added to batch cultures of Spodoptera frugiperda (Sf9) cells before infection with a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) expressing beta-galactosidase (beta-Gal). The fed-batch production of beta-Gal was optimised using response surface methods (RSM). The optimisation was performed in two stages, starting with a screening procedure to determine the most important variables and ending with a central-composite experiment to obtain a response surface model of volumetric beta-Gal production. The predicted optimum volumetric yield of beta-Gal in fed-batch culture was 2.4-fold that of the best yields in batch culture. This result was confirmed by a statistical analysis of the best fed-batch and batch data (with average beta-Gal yields of 1.2 and 0.5 g/L, respectively) obtained from this laboratory. The response surface model generated can be used to design a more economical fed-batch operation, in which nutrient feed volumes are minimised while maintaining acceptable improvements in beta-Gal yield.

Animals↗

PubMed1997

Investigating the numerical effects of ascertainment bias in linkage analysis: development of methods and preliminary results.

It is general practice to have nonsingle ascertainment of pedigrees for linkage studies, along with intrafamilial sampling that is dependent on who among the related individuals was initially ascertained (Proband dependent or PD sampling). Vieland and Hodge [1995; 1996] have shown that under these conditions, the likelihood used in calculating the lod score is not strictly correct and can produce asymptotically biased estimates of the recombination fraction, theta. However they speculated that this bias would be small in most applications. This paper presents preliminary work aimed at quantifying the numerical magnitude of the bias introduced by PD sampling and nonsingle ascertainment in linkage analysis. We considered five generating models where we varied the ascertainment procedure, intrafamilial sampling scheme, and the sample size for each model. In this limited initial set of simulations, asymptotic bias in theta appears to be trivial, while PD sampling procedures can increase the efficiency of theta. These preliminary results support the view that the advantages of unsystematic ascertainment may offset any small estimation bias that may arise.

Bias↗

PubMed1997

Signal generation in a non-EU country.

One of the most important objects of post-marketing surveillance is the early detection of serious, unknown and unexpected adverse events. As spontaneous reported adverse events vary considerably in their signal-generating value the Swiss Drug Monitoring Center SANZ implemented an early signal detection system in 1991. With a set of screening criteria the individual case reports are automatically checked by the system and read into a signal file. Subsequently these potential signals are processed to quantify the signal generating value of the particular case. According to the screening criteria 2072 (53%) of the reported cases were considered as potential signals. After validating and quantifying their signal value 27% (565) were drug-related and unlabelled, 8% (173) were also rated as serious. On the basis of these cases 114 labelling changes were made. Some of the most interesting cases are discussed. This computer-assisted early signal generation model in a small country with 7 million inhabitants has a high detectability of new, rare, serious and quality of life affecting adverse drug reactions (ADRs). These newly detected ADRs must be interpreted in cognizance of the limitations and restrictions of spontaneous reporting and do not allow pharmacoepidemiologic conclusions.

Journal Article↗

PubMed1999

Specific RNA Dinucleotide Cleavage by a Synthetic Calix

Large rate enhancements and considerable nucleobase specificity in the catalytic transesterification of RNA dinucleotides are observed with the calix[4]arene enzyme mimic 1-Zn(3) (the computer-generated model of the complex with GpG is shown). The higher activity toward GpG over ApA (factor of 160) originates from favorable substrate binding and cooperative catalytic action of all three Zn(II) ions. The heterotrinuclear metallo-phosphoesterase mimic 1-Zn(2)Cu is even more active.

Journal Article↗

PubMed2000

Extracellular lipid-mediated signaling in tumor-cell activation and pseudopod protrusion.

We have pioneered an in vitro pseudopod-generation model wherein suspended tumor cells are stimulated to form pseudopods into glass micropipettes in response to soluble collagen type IV (CIV). Pertussis toxin and removing intracellular calcium were found previously to be inhibitory to that process. We now extend those observations to dissect the roles of transmembrane calcium influx and circulating fatty acids on pseudopod extension. Removal of fatty acids from BSA in basal media resulted in abrogation of pseudopod formation, while reconstitution of free fatty acids restored cell pseudopod protrusion. We thus hypothesized that fatty acids may provide necessary pseudopod stimulatory signals. Addition of lysophosphatidic acid (LPA) to the fatty acid-free CIV solution or in an opposite pipette without CIV permitted approximately 50% pseudopod recovery in all pipette directions in a dose-dependent fashion. Thapsigargin (TG), an agent that releases internal calcium stores and causes opening of store-operated calcium channels, restored pseudopod protrusion up to 80% in CIV with fatty acid-free albumin. [Ca(2+)](i) release was non-additive when cells were stimulated by TG and LPA, suggesting overlapping [Ca(2+)](i) stores. The combination of TG and LPA in fatty acid-free albumin fully restored the pseudopod response to CIV. Addition of EGTA to chelate stimulatory media calcium blocked the pseudopod response to CIV in the presence of fatty acids. This indicates that pseudopod protrusion requires transmembrane calcium entry. Thus, extracellular lipids and calcium mobilization are required to complement CIV in pseudopod protrusion from suspended cells.

Calcium↗

PubMed1995

Stability of the WAIS-R and WISC-III factor structure using cross-validation of covariance structures.

The stability of the Verbal Comprehension, Perceptual Organization, and Freedom from Distractibility factors of the WAIS-R and WISC-III was tested using cross-validation of covariance structure models, a methodology that employs unrestricted and restricted factor analyses. Stability was indicated if the goodness of fit of restricted models generated from unrestricted factor patterns did not degrade when applied to other age groups. If goodness of fit degraded inconsistently, the factor structure of one or both instruments was unreliable. If goodness of fit changed in some systematic fashion, an argument for true intellectual changes could be made. Results indicated consistency across age groups. Therefore, it was concluded that the three-factor structure is robust and reliable across age groups and instruments.

Adolescent↗

PubMed1996

P2U purinoceptors: cDNA cloning, signal transduction mechanisms and structure-function analysis.

The cloning of a P2U purinoceptor cDNA has made it possible to use molecular biological approaches to investigate P2U purinoceptor function. Expression of recombinant P2U purinoceptors in mammalian cells lacking endogenous P2U purinoceptors has enabled us to characterize the receptor protein and its downstream effectors, and has allowed a partial analysis of the role of certain amino acid residues in ligand binding. These approaches have placed the pharmacological classification of the P2U purinoceptor on a firm molecular footing and have generated model systems that can be used to investigate receptor-ligand binding, regulation and signal transduction.

Amino Acid Sequence↗

PubMed2006

Mild-onset presentation of Canavan's disease associated with novel G212A point mutation in aspartoacylase gene.

We describe two sisters with a mild-onset variant of Canavan's disease who presented at age 50 and 19 months with developmental delay but without macrocephaly, hypotonia, spasticity, or seizures. Remarkably, both patients had age-appropriate head control, gross motor development, and muscle tone. There were very mild deficits in fine motor skills, coordination, and gait. Both sisters had a history of strabismus, but otherwise vision was normal. The older child showed evidence of mild cognitive and social impairment, whereas language and behavior were normal for age in the infant. Both patients were found to be compound heterozygotes for C914A (A305E) and G212A (R71H) mutations in ASPA. Like all other known ASPA mutations, this previously unknown G212A mutation appears to have low absolute enzyme activity. Nevertheless, it is associated in these patients with an extremely benign phenotype that is highly atypical of Canavan's disease. Biochemical and clinical data were evaluated using a generalized linear mixed model generated from 25 other subjects with Canavan's disease. There were statistically significant differences in brain chemistry and clinical evaluations, supporting a distinct variant of Canavan's disease. Future studies of ASPA enzyme structure and gene regulation in these subjects could lead to a better understanding of Canavan's pathophysiology and improvements in ASPA gene therapy.

Adult↗

PubMed2004

Rapid high resolution three dimensional reconstruction of embryos with episcopic fluorescence image capture.

One of the overarching goals in developmental biology is the elucidation of mechanisms that elaborate form and function. To this end, an accurate morphological description of embryonic development is essential. However, visualizing dynamic changes in the three-dimensional (3D) structure of the developing embryo has been a "holy grail" in the field of developmental biology. The fundamental difficulties that have hindered all efforts in 3D reconstruction using two-dimensional (2D) image stacks revolve around the seemingly intractable problems of section registration and distortion. A remarkably simple solution has come about with the development of a new technique referred to as episcopic fluorescence image capture (EFIC). With EFIC imaging, tissue autofluorescence is used to image the block face prior to cutting each section. The 2D resolution obtained is close to that achieved by histology, and such 2D image stacks can be readily reconstructed in 3D. The 3D models generated provide fine structural details with resolution unmatched by 3D reconstructions obtained with any other imaging modalities. Given the perfect registration of EFIC image stacks, another important capability provided by EFIC is digital resectioning in any plane. This provides complete flexibility in the selection of optimal virtual sectioning planes for viewing different features in a specimen, and is invaluable for analyzing dynamic changes in tissue structure in the developing embryo. The capabilities provided by EFIC for rapid high resolution 3D reconstruction together with digital resectioning make this an unparalleled tool for characterizing morphogenetic events in the developing embryo. Although our review is focused on using EFIC for studying embryonic development, it is important to note that there is no intrinsic limitation on the size of the specimen that can be analyzed by EFIC imaging. Overall, EFIC should serve as an important imaging technique that will complement other 3D imaging modalities such as MRI and optical tomography. Given the feasibility of generating EFIC image stacks using cryoembedded or polyethylene glycol (PEG)-embedded specimens, there is the possibility that EFIC may be combined with 3D RNA or protein expression profiling. Together, such studies may help further elucidate the relationship between form and function.

Animals↗

PubMed2003

Actin-based motility: from molecules to movement.

Extensive progress has been made recently in understanding the mechanism by which cells move and extend protrusions using site-directed polymerization of actin in response to signalling. Insights into the molecular mechanism of production of force and movement by actin polymerization have been provided by a crosstalk between several disciplines, including biochemistry, biomimetic approaches and computational studies. This review focuses on the biochemical properties of the proteins involved in actin-based motility and shows how these properties are used to generate models of force production, how the predictions of different theoretical models are tested using a biochemically controlled reconstituted motility assay and how the changes in motility resulting from changes to the concentrations of components of the assay can help understand diverse aspects of the motile behavior of living cells.

Actin-Related Protein 2↗

PubMed2004

Atmospheric organic aerosol production by heterogeneous acid-catalyzed reactions.

Exploratory evidence from our laboratories shows that acidic surfaces on atmospheric aerosols lead to very real and potentially multifold increases in secondary organic aerosol (SOA) mass and build-up of stabilized nonvolatile organic matter as particles age. One possible explanation for these heterogeneous processes are the acid-catalyzed (e.g., H2SO4 and HNO3) reactions of atmospheric multifunctional organic species (e.g., multifunctional carbonyl compounds) that are accommodated onto the particle phase from the gas phase. Volatile organic hydrocarbons (VOCs) from biogenic sources (e.g., terpenoids) and anthropogenic sources (aromatics) are significant precursors for multifunctional organic species. The sulfur content of fossil fuels, which is released into the atmosphere as SO2, results in the formation of secondary inorganic acidic aerosols or indigenous acidic soot particles (e.g., diesel soot). The predominance of SOAs contributing to PM2.5 (particulate matter, that is, 2.5 microm or smaller than 2.5 microm), and the prevalence of sulfur in fossil fuels suggests that interactions between these sources could be considerable. This study outlines a systematic approach for exploring the fundamental chemistry of these particle-phase heterogeneous reactions. If acid-catalyzed heterogeneous reactions of SOA products are included in next-generation models, the predicted SOA formation will be much greater and have a much larger impact on climate-forcing effects than we now predict. The combined study of both organic and inorganic acids will also enable greater understanding of the adverse health effects in biological pulmonary organs exposed to particles.

Aerosols↗

PubMed2005

Conductivity properties of carrier ampholyte pH gradients in isoelectric focusing.

The conductivity properties of natural pH gradient created by carrier ampholytes were studied during the process of isoelectric focusing (IEF). IEF was performed in capillaries (10-30 mm long) or in microchips with the same channel length. A 10-30x reduction of the conductivity of the separation medium was observed during the establishment of pH gradient. Results obtained using different IEF voltages indicate that there is a nonlinear relationship between the conductivity of an established pH gradient and the applied electric field. Our theoretical analysis using a simplified model generated values that reasonably agree with the experimental data. In addition, we found that above a certain electric field ( approximately 300 V/cm), resolution does not increase with the applied voltage as predicated; we observed band-broadening and gel breakdown. The approach presented in this work can be used for optimization of the IEF separation and judicious selection of IEF conditions.

Ampholyte Mixtures↗

PubMed1999

Genetic dissection of a complex trait.

A number of genetic and statistical tools were applied to various partitions of the simulated data to identify susceptibility loci, relevant environmental factors, and their interaction(s). The distribution of genotypes at D1G24 among affected children in the first population was found to differ significantly from Hardy-Weinberg expectation. Two transmission/disequilibrium tests identified the preferential transmission of allele 1 as the source of the disequilibrium. Simple contingency table analysis revealed a positive association between exposure to environmental factor E1 and disease phenotype. Multipoint linkage analyses on various subsets of the data identified three "signal" regions (in addition to the aforementioned D1G24) localized at D1G9-10, D3G45, and D5G38. The even numbered chromosomes appeared to be devoid of susceptibility loci. Further analyses of subsamples of affected sib pairs, selected according to their disease phenotype and their exposure to E1, clarified some linkage relationships, particularly for D3G45, thereby suggesting the presence of a specific gene x environment interaction. Logistic analysis designed to clarify the relationship between disease phenotype and two risk factors (E1 exposure and the presence of allele 1 at D1G24) in the first population, revealed a significantly negative interaction which, upon learning the details of the generating model, we now attribute to the presence of heterogeneity.

Environment↗

PubMed1999

A comparison of some allele-sharing based linkage analysis methods for detecting complex trait loci.

Using randomly selected sib pairs from a subset of the GAW11 simulated data in Problem 2, we compared the results of some linkage analysis methods based on allele sharing. One method was the Haseman-Elston test for a binary disease outcome (unaffected vs. mild or severe). The other methods, which analyzed the trinary ordered outcome unaffected/mild/severe were the Haseman-Elston test, an extended Haseman-Elston incorporating sib-pair sums, variance components analysis, and regression analysis. Our analysis was done without knowledge of the generating model.

Alleles↗

PubMed1999

Stratification techniques to explore genotype environment interactions.

Linkage analysis was performed on the GAW11 Problem 2 data set using stratification to explore the effects of the environmental risk factors and the differences between mild and severe phenotypes. Analysis of the four study populations stratified by the two risk factors identified regions on chromosomes 3 and 5 with significant evidence for linkage. Other loci were sought by removing families consistent with linkage to the chromosome 3 locus. Our studies identified a locus on chromosome 3 (markers 43-46) associated with the mild phenotype in the presence of risk factor 1 and with the severe phenotype independent of risk factor 1. This suggests that distinct allelic variants at the chromosome 3 locus may cause different forms of disease. The locus identified on chromosome 5 (markers 36-39) was linked to the severe phenotype, but exposure to factor 1 or 2 may have a protective effect. The regions on chromosomes 3 and 5 appeared to have independent roles in disease etiology. Evidence for two loci on chromosome 1 linked to the mild form was found. The methods successfully identified linkages and interaction consistent with the generating model.

Environment↗

PubMed2001

Preliminary implementation of new data mining techniques for the analysis of simulation data from Genetic Analysis Workshop 12: problem 2.

We introduce a new data mining method applicable to complex disease genetics. Our approach is suited to a broad spectrum of diseases, identifying the noteworthy sharing of combinations of alleles in unrelated affected individuals. Furthermore, this approach may be extended to comprise the common types of genotype data, including single-nucleotide polymorphisms, candidate-gene sequences, etc. Using a method derived from data-mining computer algorithms, we analyze a data set of unrelated affected individuals chosen from the simulated pedigrees of problem 2 of the Genetics Analysis Workshop 12. We observe that most marker subsets containing a flanking marker for each of six or seven of the disease-gene loci yield significant numbers of individuals manifesting substantially similar genotypes. However, initial attempts (blind to the generating model) to identify the predisposing loci have not been successful. Refining our methods so that such loci may routinely be found and validated is underway.

Algorithms↗