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PubMed1997

The ability of human Schwann cell grafts to promote regeneration in the transected nude rat spinal cord.

Advances in the purification and expansion of Schwann cells (SCs) from adult human peripheral nerve, together with biomaterials development, have made the construction of unique grafts with defined properties possible. We have utilized PAN/PVC guidance channels to form solid human SC grafts which can be transplanted either with or without the channel. We studied the ability of grafts placed with and without channels to support regeneration and to influence functional recovery; characteristics of the graft and host/graft interface were also compared. The T9-T10 spinal cord of nude rats was resected and a graft was placed across the gap; methylprednisolone was delivered acutely to decrease secondary injury. Channels minimized the immigration of connective tissue into grafts but contributed to some necrotic tissue loss, especially in the distal spinal cord. Grafts without channels contained more myelinated axons (x = 2129 +/- 785) vs (x = 1442 +/- 514) and were larger in cross-sectional area ( x = 1.53 +/- 0.24 mm2) vs (x = 0.95 +/- 0.86 mm2). The interfaces formed between the host spinal cord and the grafts placed without channels were highly interdigitated and resembled CNS-PNS transition zones; chondroitin sulfate proteoglycans was deposited there. Whereas several neuronal populations including propriospinal, sensory, motoneuronal, and brainstem neurons regenerated into human SC grafts, only propriospinal and sensory neurons were observed to reenter the host spinal cord. Using combinations of anterograde and retrograde tracers, we observed regeneration of propriospinal neurons up to 2.6 mm beyond grafts. We estimate that 1% of the fibers that enter grafts reenter the host spinal cord by 45 days after grafting. Following retrograde tracing from the distal spinal cord, more labeled neurons were unexpectedly found in the region of the dextran amine anterograde tracer injection site where a marked inflammatory reaction had occurred. Animals with bridging grafts obtained modestly higher scores during open field [(x = 8.2 +/- 0.35) vs (x = 6.8 +/- 0.42), P = 0.02] and inclined plane testing (x = 38.6 +/- 0. 542) vs (x = 36.3 +/- 0.53), P = 0.006] than animals with similar grafts in distally capped channels. In summary, this study showed that in the nude rat given methylprednisolone in combination with human SC grafts, some regenerative growth occurred beyond the graft and a modest improvement in function was observed.

Adolescent↗

PubMed1999

Crystal structure of bullfrog M ferritin at 2.8 A resolution: analysis of subunit interactions and the binuclear metal center.

Ferritins concentrate and store iron as a mineral in all bacterial, plant, and animal cells. The two ferritin subunit types, H or M (fast) and L (slow), differ in rates of iron uptake and mineralization and assemble in vivo to form heteropolymeric protein shells made up of 24 subunits; H/L subunit ratios reflect cell specificity of H and L subunit gene expression. A diferric peroxo species that is the initial reaction product of Fe(II) in H-type ferritins, as well as in ribonucleotide reductase (R2) and methane monooxygenase hydroxylase (MMOH), has recently been characterized, exploiting the relatively high accumulation of the peroxo intermediate in frog H-subunit type recombinant ferritin with the M sequence. The stability of the diferric reaction centers in R2 and MMOH contrasts with the instability of diferric centers in ferritin, which are precursors of the ferric mineral. We have determined the crystal structure of the homopolymer of recombinant frog M ferritin in two crystal forms: P4(1)2(1)2, a = b = 170.0 A and c = 481.5 A; and P3(1)21, a = b = 210.8 A and c = 328.1 A. The structural model for the trigonal form was refined to a crystallographic R value of 19.0% (Rfree = 19.4%); the two structures have an r.m.s.d. of approximately 0.22 A for all C alpha atoms. Comparison with the previously determined crystal structure of frog L ferritin indicates that the subunit interface at the molecular twofold axes is most variable, which may relate to the presence of the ferroxidase site in H-type ferritin subunits. Two metal ions (Mg) from the crystallization buffer were found in the ferroxidase site of the M ferritin crystals and interact with Glu23, Glu58, His61, Glu103, Gln137 and, unique to the M subunit, Asp140. The data suggest that Gln137 and Asp140 are a vestige of the second GluxxHis site, resulting from single nucleotide mutations of Glu and His codons and giving rise to Ala140 or Ser140 present in other eukaryotic H-type ferritins, by additional single nucleotide mutations. The observation of the Gln137xxAsp140 site in the frog M ferritin accounts for both the instability of the diferric oxy complexes in ferritin compared to MMOH and R2 and the observed kinetic variability of the diferric peroxo species in different H-type ferritin sequences.

Amino Acid Sequence↗

PubMed1997

Protein purification in multicompartment electrolyzers with isoelectric membranes.

Preparative purification of proteins under isoelectric conditions is reviewed, with particular regard to novel equipment, a multicompartment electrolyzer with isoelectric membranes, which can capture any desired protein into an isoelectric trap as the sole, ultra-pure component. This novel machine is based on the Immobiline chemistry, i.e. the novel generation of non-amphoteric buffers, based on the chemistry of acrylamides, which can be insolubilized onto polyacrylamide supports. After a description of the instrument and of its performance, a number of protein purification protocols are described, leading to truly homogeneous (by the most stringent criterion of surface charge) protein fractions. Such a high charge purity has been found to be often a fundamental prerequisite for the growth of protein crystals. Interfacing the electrolyzer with mass spectrometry has permitted the decoding of the structure of minor components generated from a parental molecule, especially ones having a higher pI. It was found that these species were often generated either by proteolytic cleavage or by the formation of a trisulphide bridge between two Cys residues. A unique application of the electrolyzer is finally described: its use as an immobilized enzyme reactor under an electric field. The performance of this reaction is outstanding, in that the kinetic parameters of the immobilized enzyme are identical to those of a free enzyme form.

Amino Acid Sequence↗

PubMed2006

Delivering octadecylphosphonic acid self-assembled monolayers on a Si wafer and other oxide surfaces.

We describe a simple experimental approach for delivering self-assembled monolayers (SAMs) of octadecylphosphonic acid (OPA) on many oxide surfaces using a nonpolar medium with a dielectric constant around 4 (e.g., trichloroethylene). This approach readily results in the formation of full-coverage OPA SAMs on a wide variety of oxide surfaces including cleaved mica, Si wafer, quartz, and aluminum. Especially, the availability of delivering full-coverage OPA SAM on a Si wafer is unique, as no OPA SAMs at all could be formed on a Si wafer when using a polar OPA solution. The reason a nonpolar solvent is superior lies in the very fact that the hydrophilic OPA headgroup tends to escape from the nonpolar solution and is thus enriched at the medium-air interface. It is these OPA headgroups seeking a hydrophilic surface that make possible the well-controlled OPA monolayer on an oxide surface.

Journal Article↗

PubMed2002

Identification of a ubiquitin-protein ligase subunit within the CCR4-NOT transcription repressor complex.

The RING finger protein CNOT4 is a component of the CCR4-NOT complex. This complex is implicated in repression of RNA polymerase II transcription. Here we demonstrate that CNOT4 functions as a ubiquitin-protein ligase (E3). We show that the unique C4C4 RING domain of CNOT4 interacts with a subset of ubiquitin-conjugating enzymes (E2s). Using NMR spectroscopy, we detail the interaction of CNOT4 with UbcH5B and characterize RING residues that are critical for this interaction. CNOT4 acts as a potent E3 ligase in vitro. Mutations that destabilize the E2-E3 interface abolish this activity. Based on these results, we present a model of how E3 ligase function within the CCR4-NOT complex relates to transcriptional regulation.

Amino Acid Sequence↗

PubMed2003

EXCAVATOR: a computer program for efficiently mining gene expression data.

Massive amounts of gene expression data are generated using microarrays for functional studies of genes and gene expression data clustering is a useful tool for studying the functional relationship among genes in a biological process. We have developed a computer package EXCAVATOR for clustering gene expression profiles based on our new framework for representing gene expression data as a minimum spanning tree. EXCAVATOR uses a number of rigorous and efficient clustering algorithms. This program has a number of unique features, including capabilities for: (i) data- constrained clustering; (ii) identification of genes with similar expression profiles to pre-specified seed genes; (iii) cluster identification from a noisy background; (iv) computational comparison between different clustering results of the same data set. EXCAVATOR can be run from a Unix/Linux/DOS shell, from a Java interface or from a Web server. The clustering results can be visualized as colored figures and 2-dimensional plots. Moreover, EXCAVATOR provides a wide range of options for data formats, distance measures, objective functions, clustering algorithms, methods to choose number of clusters, etc. The effectiveness of EXCAVATOR has been demonstrated on several experimental data sets. Its performance compares favorably against the popular K-means clustering method in terms of clustering quality and computing time.

Algorithms↗

PubMed1994

Computer-based videotape analysis of trauma resuscitations for quality assurance and clinical research.

PURPOSE: To automate time (and motion) studies of major trauma resuscitations. TECHNIQUES: We modified an existing time-motion study technique based on time-encoded videotaping for medical imaging workstation human factors analysis. Videotapes were recorded of the trauma room during normal clinical activity using an unobtrusive mounted camera. The videotapes are displayed through a customized interface using a Macintosh-based display system. Within this platform, the time-motion study module allows flexible task definition, multiple concordant task assignment, and various summary result presentations. We established an expert panel of two traumatologists, two emergency radiologists, and two time-motion study experts. The expert panel prospectively and uniquely defined important recognizable procedural and cognitive tasks and personnel (MD, RN, EMT, RT, etc.) involved in trauma resuscitation based on pilot recordings and collective experience. These task functions were used to define a menu in the time-motion analysis software. During retrospective videotape review, the beginning and ending times of each task performed were recorded by electronically highlighting each defined individual and task. The summary results can be displayed in list, tabular, or graphic form by individuals, personnel classifications, or tasks.

Clinical Protocols↗

PubMed2005

Pulmonary dendritic cells.

Dendritic cells (DCs) are leukocytes that are emerging as chief orchestrators of immune responses. The crucial task of DCs is the continuous surveillance of antigen-exposed sites throughout the body, and their unique responsibility is to decide whether to present sampled antigen in an immunogenic or tolerogenic way. Any misstep can either lead to a flawed immune defense or to allergy, even autoimmunity. It comes as no surprise that the lungs become increasingly the subject of DC-related investigations, as they represent a vast interface between the body and the outer world. This constitutes an enormous challenge for the immune system: "firing up" immune responses inappropriately could have devastating results for the fragile gas exchange structures. Evidence accumulates that DCs play a pivotal role in maintaining the delicate balance between tolerance and active immune response in our respiratory system. The exponentially growing body of DC-related publications is a big challenge. This article aims to provide researchers and clinicians with an up-to-date view on DC biology and its relevance to pulmonary medicine. A developing trend in the field of DCs is the shift from fundamental immunologic research toward exciting clinical insights and applications. For the pulmonary clinician, this heralds the dawn of promising therapies in various domains such as infections, allergy, and cancer.

Chemokines↗

PubMed2004

Science and technology integration for increased human potential and societal outcomes.

Unifying science based on the material unity of nature at the nanoscale provides a new foundation for knowledge, innovation, and integration of technology. Revolutionary and synergistic advances at the interfaces between previously separated fields of science, engineering and areas of relevance are ready to create nano-bio-info-cogno (NBIC) transforming tools. Developments in systems approach, mathematics, and computation in conjunction with NBIC allow us to understand the natural world and scientific research as closely coupled, complex, hierarchical entities. At this unique moment of scientific and technical achievement, improvement of human performance at individual and group levels, as well as development of suitable revolutionary products, becomes possible and these are primary goals for converging new technologies. NBIC addresses long-term advances in key areas of human activity, including working, learning, aging, group interaction, organizations, and human evolution ((Roco and Bainbridge, 2003)). Fundamentally new tools, technologies, and products will be integrated into individual and social human architecture. This introductory chapter of the Annals outlines research and education trends, funding activities, and the potential of development of revolutionary products and services.

Biological Evolution↗

PubMed2004

Microarray expression profiling reveals candidate genes for human uterine receptivity.

The endometrium undergoes cyclic changes in response to circulating ovarian steroid hormones as it prepares for implantation. This dynamic tissue is well suited to microarray expression profiling for elucidation of molecular players participating in the maturation of the endometrium and during the process of implantation. Recent advances in sequencing the human and mouse genomes and the availability of microarray technology and bioinformatic analyses have made elucidating these molecular participants and dialogs a reality. Analysis of the window of implantation, a temporal and spatially unique period in which the endometrium is receptive to embryonic implantation, has revealed numerous processes to be occurring simultaneously or sequentially. These include cell cycle regulation, angiogenesis, immune modulation of implantation, defense mechanisms put into place by antibacterial agents and detoxicants, secretion of unique products, transport of ions and water, growth factor actions, steroid hormone action and metabolism, and production of extracellular matrix proteins, unique cell surface glycoproteins, and a variety of transcription factors, to name a few. Several groups have recently conducted studies with human endometrium, and remarkable similarities exist with mouse. Also, many genes and gene families involved in the unique differentiation process of stromal cell decidualization are conserved. In addition, infertility associated with endometriosis is partly implantation-based, and gene profiling of such tissue during the window of implantation has revealed additional insight into mechanisms underlying infertility in this disorder. Global profiling of genes in the endometrium, decidua, and at the interface between the trophoblast and the decidua, has provided remarkable in sight into endometrial maturation and implantation.

Animals↗

PubMed1990

Chitosan--as a biomaterial.

Chitosan [a (1----4) 2-amino-2-deoxy-beta-D-Glucan] is a unique polysaccharide derived from chitin. Several attempts have been made to use this biopolymer in biomedical field. The use of this material in the development of hemodialysis membranes, artificial skin, drug targetting and other applications are discussed. It appears, this novel biomolecule, biodegradable, and biocompatible, find applications in substituting or regenerating the blood/tissue interfaces. This polysaccharide having structural characteristics similar to glycosaminoglycans, seems to mimic their functional behaviour.

Animals↗

PubMed2003

A new concept for an integrated Healthcare Access Model.

The increase of population mobility has been promoting a crescent dispersion of patient clinical records in Healthcare Information Systems. In this scenario, it is mandatory that new services will be available for healthcare practitioners, namely web-based interfaces with strong control access mechanisms providing effective authentication and identification of persons, and the establishment of new access models to the disperse patient information. This paper proposes and describes a Healthcare Access Model that integrates a new set of functionalities coping with patient mobility and implements an innovative concept of a virtual unique Electronic Patient Record--EPR.

Access to Information↗

PubMed1991

Influence of different sources on the processing and biopharmaceutical properties of high-dose ibuprofen formulations.

It is known that depending on the manufacturing and synthetic processes, drugs may exist as different forms. As a result, physicochemical properties, compression characteristics, intrinsic dissolution and bioavailability may vary substantially. The purpose of this study was to investigate the effect of different sources of ibuprofen on the processing of tablets and on their properties. Another emphasis of this work was to rationalize one or several key characteristics of the raw material as directly related to wet granulation parameters and to the behavior of final tablets. Commercially available ibuprofen was obtained from different manufacturers and a preformulation program, including X-ray crystallography, differential scanning calorimetry, scanning electron microscopy, determination of particle size distribution and flowability, was performed to characterize the raw material. Granules were prepared with a planetary mixer and liquid requirements for the end point were obtained by monitoring power consumption. Tablets were manufactured on Stokes rotary and single punch instrumented presses. Data acquisition interfaces produced compression data for each formulation. Granules and final tablets were analyzed for hardness, dissolution profiles and content uniformity. Statistical evaluations using analysis of variance and multiple comparison procedures were performed on the results to determine the significance of the variability between independent parameters. The ibuprofen tested was found to be a unique polymorphic form with some differences in the external crystallinity. The particle size characteristics of the material also allowed a differentiation between sources and although there was no differences in dissolution patterns or content uniformity, particle size was found to account for 50% of the variability in tablet hardness. Two sources of ibuprofen with lower mean particle size showed significant variations in end point liquid requirements resulting in variable tablet crushing strength.

Biopharmaceutics↗

PubMed2005

Human-centered design of a distributed knowledge management system.

Many healthcare technology projects fail due to the lack of consideration of human issues, such as workflow, organizational change, and usability, during the design and implementation stages of a project's development process. Even when human issues are considered, the consideration is typically on designing better user interfaces. We argue that human-centered computing goes beyond a better user interface: it should include considerations of users, functions and tasks that are fundamental to human-centered computing. From this perspective, we integrated a previously developed human-centered methodology with a Project Design Lifecycle, and we applied this integration in the design of a complex distributed knowledge management system for the Biomedical Engineer (BME) domain in the Mission Control Center at NASA Johnson Space Center. We analyzed this complex system, identified its problems, generated systems requirements, and provided specifications of a replacement prototype for effective organizational memory and knowledge management. We demonstrated the value provided by our human-centered approach and described the unique properties, structures, and processes discovered using this methodology and how they contributed in the design of the prototype.

Artificial Intelligence↗

PubMed2005

DommiMOE: an implementation of ligand field molecular mechanics in the molecular operating environment.

The ligand field molecular mechanics (LFMM) model, which incorporates the ligand field stabilization energy (LFSE) directly into the potential energy expression of molecular mechanics (MM), has been implemented in the "chemically aware" molecular operating environment (MOE) software package. The new program, christened DommiMOE, is derived from our original in-house code that has been linked to MOE via its applications programming interface and a number of other routines written in MOE's native scientific vector language (SVL). DommiMOE automates the assignment of atom types and their associated parameters and popular force fields available in MOE such as MMFF94, AMBER, and CHARMM can be easily extended to provide a transition metal simulation capability. Some of the unique features of the LFMM are illustrated using MMFF94 and some simple [MCl)]2- and [Ni(NH3)n]2+ species. These studies also demonstrate how density functional theory calculations, especially on experimentally inaccessible systems, provide important data for designing improved LFMM parameters. DommiMOE treats Jahn-Teller distortions automatically, and can compute the relative energies of different spin states for Ni(II) complexes using a single set of LFMM parameters.

Journal Article↗

PubMed2006

X-ray crystallographic and steady state fluorescence characterization of the protein dynamics of yeast polyadenylate polymerase.

Polyadenylate polymerase (PAP) catalyzes the synthesis of poly(A) tails on the 3'-end of pre-mRNA. PAP is composed of three domains: an N-terminal nucleotide-binding domain (homologous to the palm domain of DNA and RNA polymerases), a middle domain (containing other conserved, catalytically important residues), and a unique C-terminal domain (involved in protein-protein interactions required for 3'-end formation). Previous X-ray crystallographic studies have shown that the domains are arranged in a V-shape such that they form a central cleft with the active site located at the base of the cleft at the interface between the N-terminal and middle domains. In the previous studies, the nucleotides were bound directly to the N-terminal domain and exhibited a conspicuous lack of adenine-specific interactions that would constitute nucleotide recognition. Furthermore, it was postulated that base-specific contacts with residues in the middle domain could occur either as a result of a change in the conformation of the nucleotide or domain movement. To address these issues and to better characterize the structural basis of substrate recognition and catalysis, we report two new crystal structures of yeast PAP. A comparison of these structures reveals that the N-terminal and C-terminal domains of PAP move independently as rigid bodies along two well defined axes of rotation. Modeling of the nucleotide into the most closed state allows us to deduce specific nucleotide interactions involving residues in the middle domain (K215, Y224 and N226) that are proposed to be involved in substrate binding and specificity. To further investigate the nature of PAP domain flexibility, 2-aminopurine labeled molecular probes were employed in steady state fluorescence and acrylamide quenching experiments. The results suggest that the closed domain conformation is stabilized upon recognition of the correct subtrate, MgATP, in an enzyme-substrate ternary complex. The implications of these results on the enzyme mechanism of PAP and the possible role for domain motion in an induced fit mechanism are discussed.

Binding Sites↗

PubMedDate not supplied

VRDD: applying virtual reality visualization to protein docking and design.

We have developed an interactive docking program called VRDD. It offers various modes of displaying molecules in an immersive, three-dimensional virtual reality (VR) environment. It allows a user to interactively perform molecular docking aided by automatic docking and side chain conformational search. Binding free energies are computed in real time, and the program enables the user to explore only clash-free orientations of a ligand. VRDD also supplies visual and auditory feedback during docking and side chain search, indicating the levels of atomic overlap and interaction energy. The stunning VR graphics immerse users in the scene and can maximally stimulate their design intuition. We have tested VRDD on three cases with increasing complexity: a nine-residue-long peptide bound to a major histocompatibility complex (MHC) molecule, barstar bound to barnase, and an antibody bound to a hemagglutinin. Without prior knowledge, combinations of hand-docking and automatic refinement led to accurate complex structures for the first two complexes. The third case, for which all automatic docking algorithms failed to identify the correct complex in a previous blind test, also failed for VRDD. Our results show that the combination of VR docking and automatic docking can make unique contributions to molecular modeling.

Antibodies↗

PubMed2005

The dentin-enamel junction and the fracture of human teeth.

The dentin-enamel junction (DEJ), which is the interfacial region between the dentin and outer enamel coating in teeth, is known for its unique biomechanical properties that provide a crack-arrest barrier for flaws formed in the brittle enamel1. In this work, we re-examine how cracks propagate in the proximity of the DEJ, and specifically quantify, using interfacial fracture mechanics, the fracture toughness of the DEJ region. Careful observation of crack penetration through the interface and the new estimate of the DEJ toughness ( approximately 5 to 10 times higher than enamel but approximately 75% lower than dentin) shed new light on the mechanism of crack arrest. We conclude that the critical role of this region, in preventing cracks formed in enamel from traversing the interface and causing catastrophic tooth fractures, is not associated with the crack-arrest capabilities of the interface itself; rather, cracks tend to penetrate the (optical) DEJ and arrest when they enter the tougher mantle dentin adjacent to the interface due to the development of crack-tip shielding from uncracked-ligament bridging.

Dental Enamel↗