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At least 1,225 records · Page 68Linked to original sources

PubMed2006

Organotypic (raft) culture of biopsy-derived upper respiratory epithelium.

OBJECTIVE: The objective of this study was to derive a reliable technique for culturing biopsy-derived upper respiratory epithelium in a system that supports epithelial differentiation and simulates the normal epithelial life cycle. STUDY DESIGN: The authors conducted a prospective study of modification and development of an in vitro tissue culture method. METHODS: Thirty biopsy specimens from 16 individuals with recurrent respiratory papillomatosis and chronic tonsillitis, pretreated to prevent bacterial and fungal overgrowth, were digested with trypsin to create a supernatant of individual cells. The cells were plated and incubated. At 14 to 16 days, the resulting colonies were placed on a wire cloth raft and fed through diffusion from the underlying culture medium in an air-liquid interface. RESULTS: Eight specimens were successfully cultured for an average of over 32 days. The longest duration of sustained growth was 60 days. Low-risk human papillomavirus specimen-based cultures reproduced infection in cultured squamous epithelium with corresponding histopathologic features indicating a high level of stratification and differentiation. CONCLUSIONS: Unlike commercially available cell lines, biopsy-derived material is predisposed to contamination, and successful in vitro culture and experimentation creates many unique challenges. An organotypic culture system, capable of reproducing the differentiation-dependent replication cycle of human papillomavirus, may be used for culturing biopsy-derived specimens for a variety of studies.

Biopsy↗

PubMed2004

Neuroendocrinology of acute immunity.

Bacterial endotoxins such as Escherichia coli lipopolysaccharide (LPS) bind to specific Toll-like receptors in fixed and circulating immunocompetent cells and activate the sympathetic and pituitary-adrenal system through similar receptors in cells that form the blood-brain interface. The latter, in turn, lead to the formation, within the brain, of proinflammatory cytokines including interleukin (IL)-1alpha, IL-1beta, IL-6, tumor necrosis factor alpha (TNF-alpha), and one or more antinflammatory cytokines including tumor growth factor beta (TGF-beta), and IL-10. Although the full panoply of central cytokines can be activated after systemic exposure, direct introduction of IL-1beta into the brain exerts a unique pattern of peripheral immune responses attributable to the special properties of reactive cells within the brain and to the "reservoir" function of the cerebrospinal fluid compartment. De Simoni et al. were the first to show that the intracerebroventricular (icv) injection of IL-1beta in rats induced a dramatic increase in the concentration of circulating IL-6 that was much greater and more prolonged than that induced by intravenous bolus injection of the same dose of cytokine. The work reported in this paper shows that the "De Simoni paradox" is mainly due to the prolonged release of the injected IL-1beta from brain to blood: a large proportion is not degraded (86%), transport out is both active and passive, and the initial exposure to IL-1beta sensitizes peripheral responses to the continuing exposure to the cytokine. Although IL-6, TNF-alpha and LPS are passively transferred from brain to blood (as shown by radioiodine-labeled tracer studies) peripheral cytokine responses to central injection differ from responses to IL-1beta. Peripheral responses to central TNF-alpha are similar to those after peripheral administration because TNF-alpha has no peripheral sensitizing effect. Peripheral response to central LPS exposure is much less than that which occurs when an amount identical with that transferred from brain to blood is administered peripherally. This is due to the fact that intracerebral injection of LPS suppresses response to peripherally administered endotoxin. Since the effect can still be demonstrated in adrenalectomized animals maintained on a constant dose of corticosterone, the brain has the capacity by as yet undefined mechanisms to suppress peripheral inflammatory responses.

Brain↗

PubMed2006

UniHI: an entry gate to the human protein interactome.

Systematic mapping of protein-protein interactions has become a central task of functional genomics. To map the human interactome, several strategies have recently been pursued. The generated interaction datasets are valuable resources for scientists in biology and medicine. However, comparison reveals limited overlap between different interaction networks. This divergence obstructs usability, as researchers have to interrogate numerous heterogeneous datasets to identify potential interaction partners for proteins of interest. To facilitate direct access through a single entry gate, we have started to integrate currently available human protein interaction data in an easily accessible online database. It is called UniHI (Unified Human Interactome) and is available at http://www.mdc-berlin.de/unihi. At present, it is based on 10 major interaction maps derived by computational and experimental methods. It includes more than 150,000 distinct interactions between more than 17 000 unique human proteins. UniHI provides researchers with a flexible integrated tool for finding and using comprehensive information about the human interactome.

Computer Graphics↗

PubMed1989

Neural prosthetic interfaces with the nervous system.

Once damaged, the adult mammalian nervous system is capable of little functional regeneration. Thus, clinical disorders such as deafness, blindness and paralysis have been treated primarily by substitution rather than correction (e.g. teletype for telephone, braille for print, wheelchair instead of walking). However, recent advances in the technology of miniature electronic implants and in the basic understanding of sensory and motor functions have made it possible to build neural prosthetic devices that work by exchanging information directly between computing devices and neurons. Such interfaces are already permitting thousands of otherwise deaf patients to hear sounds directly; some have appreciable speech comprehension without visual cues. There is active research on restoring many types of sensory, motor and autonomic function. Two interesting synergies have emerged. The first stems from the recognition that the biophysical processes involved in stimulating and recording from neurons are universal; thus, technical advances have broad implications in many clinical areas. The second stems from the unique opportunity that such prostheses present to conduct neurophysiological and psychophysical studies directly in conscious human subjects.

Cochlea↗

PubMed2006

The alpha-helix of the second chromodomain of the 43 kDa subunit of the chloroplast signal recognition particle facilitates binding to the 54 kDa subunit.

Chloroplasts of higher plants contain a unique signal recognition particle (cpSRP) that consists of two proteins, cpSRP54 and cpSRP43. CpSRP43 is composed of a four ankyrin repeat domain and three functionally distinct chromodomains (CDs). In this report we confirm previously published data that the second chromodomain (CD2) provides the primary binding site for cpSRP54. However, quantitative binding analysis demonstrates that cpSRP54 binds to CD2 significantly less efficiently than it binds to full-length cpSRP43. Further analysis of the binding interface of cpSRP by mutagenesis studies and a pepscan approach demonstrates that the C-terminal alpha-helix of CD2 facilitates binding to cpSRP54.

Amino Acid Sequence↗

PubMed1999

Structure of CD94 reveals a novel C-type lectin fold: implications for the NK cell-associated CD94/NKG2 receptors.

The crystal structure of the extracellular domain of CD94, a component of the CD94/NKG2 NK cell receptor, has been determined to 2.6 A resolution, revealing a unique variation of the C-type lectin fold. In this variation, the second alpha helix, corresponding to residues 102-112, is replaced by a loop, the putative carbohydrate-binding site is significantly altered, and the Ca2+-binding site appears nonfunctional. This structure may serve as a prototype for other NK cell receptors such as Ly-49, NKR-P1, and CD69. The CD94 dimer observed in the crystal has an extensive hydrophobic interface that stabilizes the loop conformation of residues 102-112. The formation of this dimer reveals a putative ligand-binding region for HLA-E and suggests how NKG2 interacts with CD94.

Amino Acid Sequence↗

PubMed2006

Kinetically controlled catalytic formation of zinc oxide thin films at low temperature.

We developed a unique method to produce ZnO thin films by kinetically controlled catalytic hydrolysis of a molecular precursor at low temperature, operating in conjunction with the vectorial control of crystal growth. Using a system in which the diffusion of a volatile catalyst into a solution of molecular precursor of the metal oxide limits the rate of hydrolysis and establishes a gradient of catalyst concentration, we investigated the nucleation of textured nanoparticles at the gas-liquid interface and characterized their subsequent growth. Use of this slow diffusion method combined with prediction of molecular species using a partial charge model enables a higher level of organizational control than obtained in other low-temperature synthesis methods, without the use of organic molecules. Various metal oxides and their morphologies and chemical compositions can be tailored for specific applications using this relatively simple approach.

Catalysis↗

PubMed1998

Epibatidine binds with unique site and state selectivity to muscle nicotinic acetylcholine receptors.

Ligand binding sites in fetal (alpha2betagammadelta) and adult (alpha2betadeltaepsilon) muscle acetylcholine receptors are formed by alphadelta, alphagamma, or alphaepsilon subunit pairs. Each type of binding site shows unique ligand selectivity due to different contributions by the delta, gamma, or epsilon subunits. The present study compares epibatidine and carbamylcholine binding in terms of their site and state selectivities for muscle receptors expressed in human embryonic kidney 293 cells. Measurements of binding to alphagamma, alphadelta, and alphaepsilon intracellular complexes reveal opposite site selectivities between epibatidine and carbamylcholine; for epibatidine the rank order of affinities is alphaepsilon > alphagamma > alphadelta, whereas for carbamylcholine the rank order is alphadelta congruent with alphaepsilon > alphagamma. Because the relative affinities of intracellular complexes resemble those of receptors in the closed activable state, the results suggest that epibatidine binds with unique site selectivity in activating the muscle receptor. Measurements of binding at equilibrium show that both enantiomers of epibatidine bind to adult and fetal receptors with shallow but monophasic binding curves. However, when receptors are fully desensitized, epibatidine binds in a biphasic manner, with dissociation constants of the two components differing by more than 170-fold. Studies of subunit-omitted receptors (alpha2betadelta2, alpha2betagamma2, and alpha2betaepsilon2) reveal that in the desensitized state, the alphadelta interface forms the low affinity epibatidine site, whereas the alphagamma and alphaepsilon interfaces form high affinity sites. In contrast to epibatidine, carbamylcholine shows little site selectivity for desensitized fetal or adult receptors. Thus epibatidine is a potentially valuable probe of acetylcholine receptor binding site structure and of elements that confer state-dependent selectivities of the binding sites.

Adult↗

PubMed2004

Transmembrane domain helix packing stabilizes integrin alphaIIbbeta3 in the low affinity state.

Regulated changes in the affinity of integrin adhesion receptors ("activation") play an important role in numerous biological functions including hemostasis, the immune response, and cell migration. Physiological integrin activation is the result of conformational changes in the extracellular domain initiated by the binding of cytoplasmic proteins to integrin cytoplasmic domains. The conformational changes in the extracellular domain are likely caused by disruption of intersubunit interactions between the alpha and beta transmembrane (TM) and cytoplasmic domains. Here, we reasoned that mutation of residues contributing to alpha/beta interactions that stabilize the low affinity state should lead to integrin activation. Thus, we subjected the entire intracellular domain of the beta3 integrin subunit to unbiased random mutagenesis and selected it for activated mutants. 25 unique activating mutations were identified in the TM and membrane-proximal cytoplasmic domain. In contrast, no activating mutations were identified in the more distal cytoplasmic tail, suggesting that this region is dispensable for the maintenance of the inactive state. Among the 13 novel TM domain mutations that lead to integrin activation were several informative point mutations that, in combination with computational modeling, suggested the existence of a specific TM helix-helix packing interface that maintains the low affinity state. The interactions predicted by the model were used to identify additional activating mutations in both the alpha and beta TM domains. Therefore, we propose that helical packing of the alpha and beta TM domains forms a clasp that regulates integrin activation.

Amino Acid Sequence↗

PubMedDate not supplied

A microchip glucose sensor.

A major problem in development of a glucose sensor for use in an implantable artificial pancreas is the lack of reproducibility in signals from sensor to sensor. Each glucose sensor fabricated with currently used methods has a unique response to varying levels of glucose concentration and thus needs to be individually calibrated before use. We have adapted microchip manufacturing techniques for the fabrication of electrochemically based glucose sensors with standardized and reproducible function. Scanning electron microscopic study of the resulting electrode surfaces shows them to be smooth and featureless at all levels of magnification. X-ray diffraction analysis of the electrodes indicates preferential exposure of the [1,1,1] crystal interface. Cyclic voltammetry evaluation of initial sensor response to varying glucose concentrations shows excellent sensor to sensor reproducibility for all sensors made with the same underlayment. Sensors made with titanium underlayment appear to be more differentiated and thus more sensitive to variations in glucose concentration than are sensors with chromium underlayment. Although the initial response of microchip glucose sensors appears to be standardized and reproducible, additional development of an appropriate electrical insulation material is required before long-term study of signal stability is feasible.

Biomedical Engineering↗

PubMed2004

Concerns about ceramics in THA.

Currently available ceramic materials are superior to those used originally in total hip arthroplasty, which should translate into a much lower complication rate than what has been reported previously. In spite of this, a number of concerns remain. The ceramic-on-ceramic articulation is not immune to wear and surface damage. Conditions associated with ceramic wear include vertical cup position, femoral neck impingement, and femoral head separation. A unique pattern of stripe wear has been described as something that results from microseparation during gait. Catastrophic failure, although rare, continues to be a concern, and not all fractures can be predicted by proof testing. Revisions needed because of ceramic fractures can be extensive, and the results of the revision procedures can be compromised by the presence of highly abrasive particulate debris that is retained. Other concerns include the generation of debris from modular interfaces, neck damage and debris generation from impingement of some designs, inability to use a ceramic head a second time on a metal trunnion, and the dramatic loss of head and liner options intraoperatively. Although ceramics show great promise as a lower wear articulation, manufacturing and design modifications and improvements will continue in an attempt to address the substantial concerns that persist.

Ceramics↗

PubMed1995

The interaction between lipoamide dehydrogenase and the peripheral-component-binding domain from the Azotobacter vinelandii pyruvate dehydrogenase complex.

The sensitivity of lipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase E3) from Azotobacter vinelandii to inhibition by NADH requires measurement of the activity in the initial phase of the reaction. Stopped-flow turnover experiments show that kcat is 830 s-1 compared with 420 s-1 found in standard steady-state experiments. Mutations at the si-side of the flavin prosthetic group that cause severe inhibition by NADH were studied. Tyr16 was replaced by phenylalanine and serine, which causes the loss of two intersubunit H-bonds. [F16]E3 shows only 5.7% of wild-type activity in the standard assay procedure, but analyzed by stopped-flow the activity is 70% of the wild-type enzyme. The NADH-->Cl2Ind (dichloroindophenol) activity was normal or slightly increased. The inhibition by NADH is competitive with respect to NAD+, Ki = 50 microM. Spectral analysis show that electrons readily pass over from the disulfide to the FAD, indicating an increase in the redox potential of the flavin. It is concluded that subunit interaction plays an important role in the protection of the enzyme against over-reduction by decreasing the redox potential of the flavin. The interaction of wild-type or mutant enzymes with the core component of the pyruvate (E2p) or oxoglutarate (E2o) dehydrogenase multienzyme complex relieves the inhibition to a large extent. In the mutant enzymes, the mechanism of inhibition changes from competitive to the mixed-type inhibition observed for the wild-type enzyme. The stabilizing effect of E2 on [F16]E3 was used as an assay to analyze the stoichiometry of interaction of E3 with E2p as well as E2o. 1 mol E2p monomer was sufficient to saturate 1 mol E3 dimer with a Kd of about 1 nM. Similarly, 1 mol E2o saturated the E3 dimer with a Kd of 30 nM. From these experiments it is concluded that the E3-binding domain of E2 interacts with the subunit interface of E3 near the dyad axis, thus preventing sterically the interaction with a second molecule of the binding domain. This mode of interaction, which causes asymmetry in the complex, explains the stabilization against over-reduction by tightening the subunit interaction. Subgene cloning of the E2p component of the pyruvate dehydrogenase complex is described in order to obtain a complex between the lipoamide dehydrogenase component (E3) and the binding domain of E2p. A unique restriction site in the DNA encoding the flexible linker between the third lipoyl domain and the binding domain combined with timed digestion with exonuclease Bal31 was used to create a set of deletion mutants in the N-terminal region of the binding-catalytic didomain, fused to six N-terminal amino acids from beta-galactosidase. The expressed proteins, selected for E2p activity, were analyzed for binding of E3 and E1p. The shortest fusion protein containing a functional binding domain was expressed and purified. [F16]E3 was combined with this fusion protein in a stoichiometric ratio and the resulting complex was subjected to limited proteolysis to remove the catalytic domain. The resulting [F16]E3-binding domain preparation was purified to homogeneity.

Amino Acid Sequence↗

PubMed2002

Biochemical and functional characterization of Rab27a mutations occurring in Griscelli syndrome patients.

Rab27a is a member of the Rab family of small GTPase proteins, and thus far is the first member to be associated with a human disease (ie, the Griscelli syndrome type 2). Mutations in the Rab27a gene cause pigment as well as cytotoxic granule transport defects, accounting for the partial albinism and severe immune disorder characteristics of this syndrome. So far, 3 Rab27a missense mutations have been identified. They open a unique opportunity to designate critical structural and functional residues of Rab proteins. We show here that the introduction of a proline residue in the alpha 4 (Ala152Pro) or beta 5 (Leu130Pro) loop, observed in 2 of these spontaneous mutants, dramatically affects both guanosine triphosphate (GTP) and guanosine diphosphate (GDP) nucleotide-binding activity of Rab27a, probably by disrupting protein folding. The third mutant, Trp73Gly, is located within an invariant hydrophobic triad at the switch interface, and was previously shown in active Rab3A to mediate rabphilin3A effector interaction. Trp73Gly is shown to display the same nucleotide-binding and GTPase characteristics as the constitutively active mutant Gln78Leu. However, in contrast to Gln78Leu, Trp73Gly mutant construct neither interacts with the Rab27a effector melanophilin nor modifies melanosome distribution and cytotoxic granule exocytosis. Substitutions introduced at the 73 position, including the leucine residue present in Ras, did not restore Rab27a protein functions. Taken together, our results characterize new critical residues of Rab proteins, and identify the Trp73 residue of Rab27a as a key position for interaction with the specific effectors of Rab27a, both in melanocytes and cytotoxic cells.

Albinism↗

PubMed1993

Cellular dynamics of growth in sheep and goat synepitheliochorial placentomes: an autoradiographic study.

This paper demonstrates that in sheep and goats the two definitive fetomaternal interface layers are developmentally related. The fetal trophectoderm consists of binucleate and uninucleate cells. The apical microvilli of the trophectoderm interdigitate with a layer consisting of syncytial plaques of limited area bounding the maternal connective tissue. Our previous histological ultrastructural and immunocytochemical work has indicated that throughout pregnancy the fetal binucleate cells migrate to and fuse with the uterine epithelium or its derivatives to form these syncytial plaques which constitute a persistent fetomaternal tissue unique to ruminants. This quantitative autoradiographic study of thymidine incorporation into sheep and goat placentas confirms the central role of the binucleate cell in placental growth, demonstrates that throughout pregnancy all binucleate cells migrate and indicates that most of the nuclei of the syncytial plaques, which appear to have a limited lifespan, derive from binucleate cell fusion.

Animals↗

PubMedDate not supplied

Bone response to titanium alloy implants placed in diabetic rats.

Although dental implants continue to provide consistent and predictable treatment options for most patients, some people with uncontrolled systemic disease may be denied implant treatment. Diabetes is one such disease. According to the U.S. Centers for Disease Control and Prevention, diabetes is a leading cause of blindness, kidney failure, and amputations of the lower extremities. These complications result from microvascular disturbances associated with diabetes. The effect of diabetes on the healing of titanium implants has not been well established. In this study of 32 rats, diabetes was induced in 16 animals by injection of streptozotocin (65 mg/kg); the remaining 16 animals served as controls. Titanium alloy implants were placed in the tibiae of all 32 rats using standard surgical techniques. Implants healed for 14 days. Blood samples were obtained for serum glucose, osteocalcin, and alkaline phosphatase analyses. Implants were retrieved and processed for histomorphometric analyses. Three quantities were measured using light microscopy, video capture, and computer analysis: percent osseointegration (i.e., linear bone interface), associated bone volume percent, and contact frequency. Diabetic animals demonstrated significantly less osseointegration than controls. However, bone volume percent in diabetic animals was about 4 times greater than controls. Biochemical analyses were mixed; diabetic animals demonstrated increased serum osteocalcin levels compared to controls but decreased alkaline phosphatase. Based on the results of this study, it was concluded that the bone response associated with titanium alloy implants in the tibiae of diabetic rats is uniquely different from controls.

Alkaline Phosphatase↗

PubMed2005

Limb regeneration in higher vertebrates: developing a roadmap.

We review what is known about amphibian limb regeneration from the prospective of developing strategies for the induction of regeneration in adult mammals. Prominent in urodele amphibian limb regeneration is the formation of a blastema of undifferentiated cells that goes on to reform the limb. The blastema shares many properties with the developing limb bud; thus, the outgrowth phase of regeneration can be thought of as cells going through development again, i.e., redevelopment. Getting to a redevelopment phase in mammals would be a major breakthrough given our extensive understanding of limb development. The formation of the blastema itself represents a transition phase in which limb cells respond to injury by dedifferentiating to become embryonic limb progenitor cells that can undergo redevelopment. During this phase, rapid wound closure is followed by the dedifferentiation of limb cells to form the blastema. Thus, the regeneration process can be divided into a wound-healing/dedifferentiation phase and a redevelopment phase, and we propose that the interface between the wound-healing response and gaining access to developmentally regulated programs (dedifferentiation) lies at the heart of the regeneration problem in mammals. In urodele amphibians, dedifferentiation can occur in all of the tissues of the limb; however, numerous studies lead us to focus on the epidermis, the dermis, and muscle as key regulators of regeneration. Among higher vertebrates, the digit tip in mammals, including humans, is regeneration-competent and offers a unique mammalian model for regeneration. Recent genetic studies in mice identify the Msx1 gene as playing a critical role in the injury response leading to digit tip regeneration. The results from regeneration studies ranging from amphibians to mammals can be integrated to develop a roadmap for mammalian regeneration that has as its focus understanding the phenomenon of dedifferentiation.

Animals↗

PubMed1997

Isoelectric focusing in immobilized pH gradients: recent analytical and preparative developments.

Isoelectric focusing in immobilized pH gradients (IPG), covering both analytical and preparative aspects, is here reviewed. An extensive introduction covers the development of the technique from its inception in 1982 to present day methodology, with particular emphasis on the development of computer programs able to calculate and optimize linear and nonlinear pH gradients, spanning as much as 9 pH units, from a mixture of as many as 10 different buffering ions and titrants. The unique resolving power of IPGs is illustrated with the resolution of fetal globin chains differing by an Ala/Gly substitution in residue 75, this bringing about a minute difference in pI value of only 0.001 pH units. IPG runs, performed under denaturing conditions, allow an excellent correlation between experimental and theoretical protein pIs, to the extent that outliers were found to be polypeptide chains which had undergone post-synthetic modifications. The IPG methodology allows easy interfacing with mass spectrometry, due to the fact that proteins eluted from an IPG gel are isoionic as well as isoelectric, and thus are not contaminated by any buffer ion. The review ends with an excursus on preparative aspects of IPGs: a novel apparatus, based on the principle of isoelectric, buffering membranes, allows pilot-scale purification of r-DNA proteins to extreme purity, with recovery in a liquid vein. Isoelectric membranes have a selectivity based on a continuous titration process, and thus act as isoelectric traps for individual protein species. This same preparative apparatus can be used as a novel immobilized enzyme reactor, with superior performance compared to conventional types of reactors.

Amino Acid Sequence↗

PubMed2005

The structural basis of myosin V processive movement as revealed by electron cryomicroscopy.

The processive motor myosin V has a relatively high affinity for actin in the presence of ATP and, thus, offers the unique opportunity to visualize some of the weaker, hitherto inaccessible, actin bound states of the ATPase cycle. Here, electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional (3D) reconstructions provide the atomic models of myosin V in both weak and strong actin bound states. One structure shows that ATP binding opens the long cleft dividing the actin binding region of the motor domain, thus destroying the strong binding actomyosin interface while rearranging loop 2 as a tether. Nucleotide analogs showed a second new state in which the lever arm points upward, in a prepower-stroke configuration (lever arm up) bound to actin before phosphate release. Our findings reveal how the structural elements of myosin V work together to allow myosin V to step along actin for multiple ATPase cycles without dissociating.

Actins↗