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PubMed1999

Examination of the structure/function relationship in the exchangeable apolipoprotein, apolipophorin-III.

Exchangeable apolipoproteins are proteins that reversibly bind lipoprotein particles to facilitate their transport in vivo. The structure/function relationship of apolipophorin-III (apo-III), the only insect exchangeable apolipoprotein, has been investigated by examining the association of this protein with lipid vesicles. The importance of a conserved leucine residue, reported to be essential for apo-III binding to lipids, has been evaluated through site-directed mutagenesis. A unique cysteine replaces the conserved leucine at position 30 in recombinant apo-III (L30C protein). This substitution results in the covalent dimerization of the apo-III mutant via a disulfide bond. The cysteine mutation causes no difference in surface hydrophobicity of the L30C proteins when compared to the wild type apo-III. Wild type apo-III, L30C monomer, and L30C dimer associate with dimyristoylphosphatidylcholine (DMAC) vesicles in a similar manner, resulting in a reduction of turbidity of a phospholipid vesicle suspension. Analysis with transmission electron microscopy (TEM) reveals disk-like complexes identical to those previously reported with the wild type apo-III. Because the mutation of the conserved leucine seems to affect the solution behavior and surface hydrophobicity of apo-III, this residue is likely to be exposed to the aqueous environment. However, the similar behaviors of the wild type protein, the L30C monomer, and L30C dimer with respect to the binding of phospholipid vesicles suggest that this residue is not absolutely required for the protein binding to hydrophobic or amphiphilic interfaces.

Amino Acid Substitution↗

PubMed1985

Fluorescence emission spectroscopy of 1,4-dihydroxyphthalonitrile. A method for determining intracellular pH in cultured cells.

We have developed new methodology for measuring intracellular pH (pHi) in cultured cell monolayers and epithelia by analyzing the emission spectra of the trapped fluorescent pH probe, 1,4-dihydroxyphthalonitrile (1,4-DHPN). This compound is unique since both its acid and base forms possess different fluorescence emission characteristics that can be used to quantitate pHi. The fluorescence difference spectrum between an acid and alkaline solution of 1,4-DHPN has a maximum at 455 nm and a minimum at 512 nm. By determining the ratio of the intensity at these two wavelengths as a function of pH, a calibration curve was constructed. Since the two intensities are determined simultaneously, the measurement is independent of dye concentration, bleaching, and intensity fluctuation of the excitation source. Furthermore, analysis of the emission spectra permitted the detection of light scattering, binding effects, and chemical modification of the probe. A microspectrofluorometer was constructed to analyze low light level emission spectra from intracellular 1,4-DHPN. The instrument consists of a modified Leitz inverted microscope (E. Leitz, Inc., Rockleigh, NJ) with a Ploem illuminator adapted for broadband excitation and objective focusing capability. The emission spectra were collected by focusing the fluorescence from the cell onto the entrance slit of an imaging monochromator, which was scanned by a SIT camera interfaced with a computer. This permitted the acquisition of fluorescence emission spectra extending from 391-588 nm in approximately 33 ms. pHi measured in the cultured toad kidney epithelial cell line, A6, was 7.49 +/- 0.04 (n = 12) with an external pH of 7.6. A6 cells were found to regulate pHi in response to both acute acid and alkali loads and maintained pHi relatively constant over a wide range of external pH values. The technique described in this report overcomes several of the difficulties encountered with other fluorescent pH probes where excitation spectroscopy is required to monitor pH.

Animals↗

PubMed2001

GOBASE: the organelle genome database.

GOBASE (http://megasun.bch.umontreal.ca/gobase/) is a network-accessible biological database, which is unique in bringing together diverse biological data on organelles with taxonomically broad coverage, and in furnishing data that have been exhaustively verified and completed by experts. So far, we have focused on mitochondrial data: GOBASE contains all published nucleotide and protein sequences encoded by mitochondrial genomes, selected RNA secondary structures of mitochondria-encoded molecules, genetic maps of completely sequenced genomes, taxonomic information for all species whose sequences are present in the database and organismal descriptions of key protistan eukaryotes. All of these data have been integrated and organized in a formal database structure to allow sophisticated biological queries using terms that are inherent in biological concepts. Most importantly, data have been validated, completed, corrected and standardized, a prerequisite of meaningful analysis. In addition, where critical data are lacking, such as genetic maps and RNA secondary structures, they are generated by the GOBASE team and collaborators, and added to the database. The database is implemented in a relational database management system, but features an object-oriented view of the biological data through a Web/Genera-generated World Wide Web interface. Finally, we have developed software for database curation (i.e. data updates, validation and correction), which will be described in some detail in this paper.

Animals↗

PubMed2001

Oligomerization of uniquely folded mini-protein motifs: development of a homotrimeric betabetaalpha peptide.

The discovery of a discretely folded homotrimeric betabetaalpha motif (BBAT1) was recently reported (J. Am. Chem. Soc. 2001, 123, 1002-1003). Herein the design, synthesis, and analysis of a small library of peptides which led to the isolation of BBAT1 is described. betabetaalpha peptides based on the monomeric sequence of BBA5 (Folding Des. 1998, 120, 95-103) were synthesized to include the anthranilic acid/nitrotyrosine fluorescence quenching pair to rapidly detect interpeptide association. In the first generation of peptides synthesized, truncations in the loop region connecting the beta-hairpin to the alpha-helix revealed that a two-residue deletion in the loop promoted an interpeptide association as detected by fluorescence quenching. An additional library of 22 loop-truncated betabetaalpha peptides was subsequently synthesized to include a variety of sequence mutations in an effort to enhance the observed peptide-peptide binding. From the fluorescence quenching screen, peptide B2 was found to possess the strongest fluorescence-quenching response, indicative of a strong peptide-peptide association. Due the poor solubility of peptide B2, the S-methylated cysteine at position 9 in the loop was substituted with a glycine to generate peptide BBAT1 which possessed greatly improved water solubility and formed discrete trimers. The successful design of this oligomeric betabetaalpha structure will likely aid the design of more complex alpha-beta superstructures and further our understanding of the factors controlling protein-protein interactions at alpha-beta protein interfaces.

Amino Acid Motifs↗

PubMed2000

Multidimensional analysis of poly(ethylene glycols) by size exclusion chromatography and dynamic surface tension detection

Substantial improvements in a multidimensional dynamic surface tension detector (DSTD) are presented. Rapid, online calibration and measurement of the dynamic surface tension for high-performance liquid chromatography separations is achieved. Dynamic surface tension is determined by measuring the differential pressure across the liquid-air interface of repeatedly growing and detaching drops. Continuous surface tension measurement throughout the entire drop growth (50 ms to 2 s) is achieved, for each eluting drop, providing insight into the kinetic behavior of molecular orientation processes at the liquid-air interface. Three-dimensional data are obtained, with surface tension first converted to surface pressure, which is plotted as a function of elution time axis versus drop time axis. Two key innovations will be reported. First, a novel calibration procedure is described and implemented. Differential pressure signals from three drops (mobile phase, standard in mobile phase, and analyte in mobile phase) are utilized to make the dynamic surface tension measurement, thereby eliminating the need for optical imaging, and viscosity and hydrostatic pressure corrections, as required by other methods. Only pressure signals from one mobile-phase drop and one standard drop pressure signal are required, while the analyte drop pressure signal is measured along the chromatographic time axis. Second, corrections for drop elongation are not required, because the drops are precisely detached by an air burst actuation method in a regime were the surface tension forces significantly dominate gravitational forces. Drops that would fall with a volume of approximately 10 microL due to gravity are precisely and repeatedly detached earlier at a volume of 2 microL. The sensitivity and unique selectivity of the DSTD opens up new possibilities in the analysis of small molecular weight polymers of varying degrees of surface activity, as illustrated for the size-exclusion chromatography analyses of complex poly(ethylene glycol) (PEG) samples. Using partial least squares for data analysis, polydispersity of complex PEG samples is determined at a relative precision of approximately 1%.

Journal Article↗

PubMed2003

Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage.

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.

Animals↗

PubMed2006

Automated classification of alternative splicing and transcriptional initiation and construction of visual database of classified patterns.

MOTIVATION: Large-scale detection and classification of alternative splicing and transcriptional initiation (ASTI) is the first step towards detailed studies of the functional implication and mechanisms of these phenomena. RESULTS: We have developed an algorithm that classifies all observed units of ASTI into an extendable set of distinct types (e.g. cassette type) by converting a collection of alignments between a genomic DNA sequence and cDNA sequences into binary description. This description system can uniquely and compactly encode not only typical patterns but also any rare patterns that are usually collectively assigned to 'others.' More than 150 distinct ASTI types were found when this system was applied to genome-wide detection of ASTI units in human and five other eukaryotes. AVAILABILITY: The data detected by this system are available through ASTRA (http://alterna.cbrc.jp/), a database equipped with a Java-based browser that can interactively reorganize the order of displayed splicing patterns on demand.

Algorithms↗

PubMed2006

StellaBase: the Nematostella vectensis Genomics Database.

StellaBase, the Nematostella vectensis Genomics Database, is a web-based resource that will facilitate desktop and bench-top studies of the starlet sea anemone. Nematostella is an emerging model organism that has already proven useful for addressing fundamental questions in developmental evolution and evolutionary genomics. StellaBase allows users to query the assembled Nematostella genome, a confirmed gene library, and a predicted genome using both keyword and homology based search functions. Data provided by these searches will elucidate gene family evolution in early animals. Unique research tools, including a Nematostella genetic stock library, a primer library, a literature repository and a gene expression library will provide support to the burgeoning Nematostella research community. The development of StellaBase accompanies significant upgrades to CnidBase, the Cnidarian Evolutionary Genomics Database. With the completion of the first sequenced cnidarian genome, genome comparison tools have been added to CnidBase. In addition, StellaBase provides a framework for the integration of additional species-specific databases into CnidBase. StellaBase is available at http://www.stellabase.org.

Animals↗

PubMed2000

The ENN project. ENN exploitation plan.

The objective of the ENN-European Neurologic Network project is to improve knowledge and treatment of sleep disorders, headache and epilepsy. The means to obtain this objective shall be certain software to be distributed to the appropriate users in the medical field and the collection of relevant cases, in order to set up a neurological database. It is intended that the distribution of the above mentioned software and access to the database, will be able to finance research projects in the neurological field. The outcome of the EU funded project have been six prototypes, which need further work in order to establish a system of compatible and linked products. A particular emphasis of this work should be put on making the products as process oriented as possible. At the time being there are already products available in the market, which would be competing with particular ENN prototypes, but there is no set of compatible and linked products, which would be comparable with the intended set of ENN neurological tools. Such set of tools therefore could be a unique selling proposition. Intellectual property rights and legal implications have to be taken into consideration for the marketing of the ENN products. It has to be made sure, that no third party can assert violation of its IPRs and that, a protection of the products can be attained by appropriate application for IPRs. In the legal field in particular the prescriptions of data protection legislation have to be observed e.g. by obtaining the written consent of patients, whose cases are reported. The marketing concept should be set up as a short-term, middle-term, long-term strategy. The short-term strategy should concentrate on carrying out a market validation study at European level and simultaneously the development from prototype to products. The middle-term strategy should be directed towards the market introduction of the ENN products in Europe. The long-term strategy should comprise marketing of the products in all potential markets worldwide. It is very important to create an ENN brand, which means a common label, a common layout and a common user interface.

Computer Communication Networks↗

PubMed2004

[Quality of community-based healthcare services].

The recent concern regarding the quality of healthcare services is partly due to the empowerment of health consumers, cost containment measures that may compromise quality, increased complexity of the medical practice and the accelerated growth of data on the magnitude and extent of quality problems. The framework of the delivery of health services, including quality parameters, differs fundamentally in the community as opposed to the hospital setting. In the community, the episode of care lacks geographic and temporal boundaries, and is divided among different facilities and caregivers. Hence, the healthcare systems lack control over the management of care. In the solo practice, the physician lacks the opportunity to discuss and share medical decisions with his/her peers and physician's reimbursement does not encourage him/her to invest time and effort in the provision of quality care. Furthermore, in the community setting, the patient is expected to take responsibility for compliance to the therapeutic regimen, a condition that may frequently interfere with regular life routines. Therefore, quality promotion should embody the "quality triangle" encompassing patients, caregivers, the healthcare system and appropriate interfaces. Ideally, the voice of the health consumer should be an integral consideration in the design of health policy, care should be patient-centered and physician reimbursement should reflect the quality of care provided. In addition, the design of the healthcare system information technology in supporting decision-making and training "quality leaders" to facilitate quality improvement programs. Consequently, it is pivotal to nurture agreement among policy-makers, patients and caregivers as to the essence of the dilemma: "What is quality in community care?" Meanwhile, we may suggest a primordial definition to community-based health care quality management: An ongoing multidisciplinary effort to identify and respond to the needs of patients, by providing systemic infrastructure that will support the caregivers and help in achieving better outcomes in the six basic dimensions of quality care-safe, timely, effective, efficient, equitable and patient-centered. In conclusion, in light of the medical activities conducted in the community, and the prevalent and ever-growing shortage of resources, there is a need to integrate efforts to develop and implement both unique tools and strategies to manage quality in community-based health services.

Community Health Services↗

PubMed2000

Three-dimensional computed tomography imaging in dentistry.

The authors have presented several 3-D CT imaging techniques that may be useful in dentistry. MPR and dental MPR provides cross-sectional images of the jaws and the dental arches, and its utility in implant treatment planning is widely recognized. SSD allows the clinician to visualize the surface of an anatomic structure that is selected by a threshold value. Volume rendering is a powerful and flexible three-dimensional imaging technique that can create various unique images. MIP may be useful in contrast-enhanced CT examinations, such as sialography. Model production and virtual reality three-dimensional imaging may become major techniques in the near future. Although the increased radiation dose to the patients has to be considered, future imaging demands will bring more access to 3-D CT imaging by dental patients and, with adequate three-dimensional rendering techniques, more accurate diagnosis and treatment planning.

Anatomy, Cross-Sectional↗

PubMed1994

Isolation and characterization of metallic wear debris from a dynamic intervertebral disc prosthesis.

A dynamic intervertebral disc prosthesis (DIDP) has been developed. It consists of a CoCrMo body and uses Ti6Al4V springs to replicate the mechanical function of the lumbar joint. Wear studies have been performed previously on the DIDP using two specialized simulators to test the wear properties of the moving parts of the disc prosthesis. A pin-in-slot simulator generates wear that would occur in the hinge-pin assembly of the prosthesis. A spring-in-pocket simulator approximates the conditions under which the springs would wear against the body of the prosthesis. The spring-pocket interface is responsible for the production of approximately 90% of the total wear occurring in the prosthesis, and is therefore the main focus of this study. Bovine serum with a preservative has been used as a lubricant in both simulators. The spring-in-pocket simulator compares the effects of two different manufacturing techniques of CoCrMo (HIPing and forging) on their wear characteristics against Ti6Al4V springs. Debris from the spring-in-pocket simulator has been isolated from the serum lubricant and characterized using scanning electron microscopy techniques. The morphology of the Ti6Al4V fragments is rough and irregularly shaped. The size of these fragments ranges from < 1 microns to > 30 microns. The forged CoCrMo alloy debris has an irregular polyhedral shape, with sizes in the same range as the spring fragments. The morphology of the HIPed CoCrMo debris is spherical with a size range < 5 microns to > 30 microns. Length and width measurements of micron-size particles were made with the particle measurements feature of the scanning electron microscope. Micron-size particles were found in all stations. This article provides a unique way to isolate and analyze debris from serum lubricants used in simulators.

Alloys↗

PubMed2002

Concept mapping: a tool to bridge the disciplinary divide.

The American College of Women's Health Physicians has been exploring an on-line educational tool-concept mapping-to facilitate the development of an interdisciplinary and woman-centered women's health curriculum, and to implement The Women's Health Care Competencies for Medical Students. By using an on-line concept map of the menstrual cycle, we have built upon a standard piece of curricula that describes a unique aspect of female physiology and transformed it into a knowledge framework that builds capacity. The concept map highlights relationships between concepts and across disciplines, connecting the competencies to enable meaningful learning so that a learner can adapt their knowledge to multiple settings, incorporate new learning, and generate new knowledge to grow the interdisciplinary field of women's health. The on-line format allows access from multiple sites and courses, and allows the curricula to grow organically over time without upsetting current curricular design.

Artificial Intelligence↗

PubMed2005

Clustering short time series gene expression data.

MOTIVATION: Time series expression experiments are used to study a wide range of biological systems. More than 80% of all time series expression datasets are short (8 time points or fewer). These datasets present unique challenges. On account of the large number of genes profiled (often tens of thousands) and the small number of time points many patterns are expected to arise at random. Most clustering algorithms are unable to distinguish between real and random patterns. RESULTS: We present an algorithm specifically designed for clustering short time series expression data. Our algorithm works by assigning genes to a predefined set of model profiles that capture the potential distinct patterns that can be expected from the experiment. We discuss how to obtain such a set of profiles and how to determine the significance of each of these profiles. Significant profiles are retained for further analysis and can be combined to form clusters. We tested our method on both simulated and real biological data. Using immune response data we show that our algorithm can correctly detect the temporal profile of relevant functional categories. Using Gene Ontology analysis we show that our algorithm outperforms both general clustering algorithms and algorithms designed specifically for clustering time series gene expression data. AVAILABILITY: Information on obtaining a Java implementation with a graphical user interface (GUI) is available from http://www.cs.cmu.edu/~jernst/st/ SUPPLEMENTARY INFORMATION: Available at http://www.cs.cmu.edu/~jernst/st/

Algorithms↗

PubMed1994

Distinct binding determinants for 9-cis retinoic acid are located within AF-2 of retinoic acid receptor alpha.

Retinoids exert their physiological action by interacting with two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which regulate gene expression by forming transcriptionally active heterodimeric RAR/RXR or homodimeric RXR/RXR complexes on DNA. Retinoid receptor activity resides in several regions, including the DNA and ligand binding domains, a dimerization interface, and both a ligand-independent (AF-1) and a ligand-dependent (AF-2) transactivation function. While 9-cis retinoic acid (RA) alone is the cognate ligand for the RXRs, both 9-cis RA and all-trans RA (t-RA) compete for binding with high affinity to the RARs. This latter observation suggested to us that the two isomers may interact with a common binding site. Here we report that RAR alpha has two distinct but overlapping binding sites for 9-cis RA and t-RA. Truncation of a human RAR alpha to 419 amino acids yields a receptor that binds both t-RA and 9-cis RA with high affinity, but truncation to amino acid 404 yields a mutant receptor that binds only t-RA with high affinity. Remarkably, this region also defines a C-terminal boundary for AF-2, as addition of amino acids 405 to 419 restores receptor-mediated gene activity to a truncated human RAR alpha lacking this region. It is interesting to speculate that binding of retinoid stereoisomers to unique sites within an RAR may function with AF-2 to cause differential activation of retinoid-responsive gene pathways.

Amino Acid Sequence↗

PubMedDate not supplied

Virtual bronchoscopy for three--dimensional pulmonary image assessment: state of the art and future needs.

Virtual bronchoscopy is emerging as a useful approach for assessment of three-dimensional (3D) computed tomographic (CT) pulmonary images. A protocol for virtual bronchoscopic assessment of a 3D CT pulmonary image would have two main stages: (a) preprocessing of image data, which involves extracting objects of interest, defining paths through major airways, and preparing the extracted objects for 3D rendering; and (b) interactive image assessment, which involves use of graphics-based software tools such as surface-rendered views, projection images, virtual endoscopic views, tube views, oblique section images, measurement data, global two-dimensional section images, and cross-sectional views. Although a virtual bronchoscope offers a unique opportunity for exploration and quantitation, it cannot replace a real bronchoscope. Limitations of current virtual endoscopy systems include high cost, lack of visual aids beyond simulated endoscopic views, difficulty in performing interactive anatomic exploration, lack of quantitative information, use of surface rendering instead of volume rendering, and need for substantial off-line display computation. Future needs include development of fully integrated user-friendly virtual bronchoscopes, development of optimal CT protocols for generating artifact-free data sets, and improvements in automated preprocessing of 3D CT images.

Bronchoscopy↗

PubMed1990

Astrocytes induce neural microvascular endothelial cells to form capillary-like structures in vitro.

Astrocytes maintain a unique association with the central nervous system microvasculature and are thought to play a role in neural microvessel formation and differentiation. We investigated the influence of astroglial cells on neural microvascular endothelial differentiation in vitro. Using an astroglial-endothelial coculture system, rat brain astrocytes and C6 cells of astroglial lineage are shown to induce bovine retinal microvascular endothelial (BRE) cells to form capillary-like structures. Light microscopic evidence for endothelial reorganization began within 48 hours and was complete 72-96 hours following the addition of BRE cells to 1-day-old astroglial cultures. The extent of BRE reorganization was quantitated by computer-assisted analysis and shown to be dependent upon the density of both the BRE and C6 cells within the cocultures. Coculture conditions in which BRE cells were separated from C6 cells by porous membranes failed to generate this endothelial cell change. Likewise, C6-conditioned media and C6-endothelial coculture conditioned media did not induce BRE cell reorganization. Extracellular laminin within the C6-endothelial cocultures, identified by indirect immunofluorescence, was concentrated at the endothelial-astroglial interface of capillary-like structures consistent with incipient basement membrane formation. Astroglial cells accumulated adjacent to capillary-like structures suggesting the presence of bidirectional influences between the reorganized endothelial cells and astroglia. This is the first demonstration of astroglial induction of angiogenesis in vitro and these findings support a functional role for perivascular astrocytes in the vascularization of neural tissue such as retina and brain.

Animals↗

PubMed1988

Development of stereological analysis software for the calculation of volume, number and surface density.

COSAS, an acronym for COmputerized Stereological Analysis System, is software designed to facilitate the process of quantitative morphological analysis. It is a flexible, reliable, and easy to use software system which provides the biologist with a simple means for performing stereological analysis. It provides for estimation of volume and surface density and allows for direct calculation of diameter information from planimetric data. The latter capability is unique to COSAS and provides the ability to calculate longest and shortest diameters for convex profiles, enabling calculation of number density. Access to all intermediate calculations is possible, so the process of transformation of raw data into the stereological descriptors can be completely exposed. This serves two purposes: (1) it provides a means for tracing results; (2) the intermediate values can be utilized independently. This system was used to analyze peroxisomes in tissues from patients with Zellweger's disease and neonatal-onset adrenoleukodystrophy. The system was also used to facilitate an immunocytochemical analysis of cytochrome P450 topology in the endoplasmic reticulum membrane.

Algorithms↗

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