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Inhibition of protein kinase C by alcohols and anaesthetics.

Despite almost a century of research, the mechanism of anaesthesia remains obscure and there is still no agreement on the location of the site(s) of action. Because the potencies of general anaesthetics increase in proportion to their solubility in olive oil, this led to a consensus that the site is within the cell membrane. This led to theories that lipid bilayer perturbation was the primary event, which was then transmitted to a membrane protein. But at the concentrations used clinically, such perturbations are small. A plausible site would be in or on ion channels at the synapse, where a number of modulatory effects have been described. A possible location for such a site would be at the protein-lipid interface. We report here that anaesthetics inhibit protein kinase C, a key component in signal transduction. The potency is a linear function of the octanol-water partition coefficient (the Meyer-Overton rule of anaesthesia). The effect was obtained in a lipid-free assay, implicating a hydrophobic site in the protein, supporting the contention that a (membrane) protein may be a target for anaesthetic interactions. In a lipid-dependent assay, a potential role of lipids in the protein-site model was demonstrated. The inhibition was absent in the isolated catalytic domain, suggesting that the site of inhibition is on the regulatory subunit, which is unique to protein kinase C.

Alcohols↗

Identification of the cysteine residues in the amino-terminal extracellular domain of the human Ca(2+) receptor critical for dimerization. Implications for function of monomeric Ca(2+) receptor.

We analyzed the effect of substituting serine for each of the 19 cysteine residues within the amino-terminal extracellular domain of the human Ca(2+) receptor on cell surface expression and receptor dimerization. C129S, C131S, C437S, C449S, and C482S were similar to wild type receptor; the other 14 cysteine to serine mutants were retained intracellularly. Four of these, C60S, C101S, C358S and C395S, were unable to dimerize. A C129S/C131S double mutant failed to dimerize but was unique in that the monomeric form expressed at the cell surface. Substitution of a cysteine for serine 132 within the C129S/C131S mutant restored receptor dimerization. Mutation of residues Cys-129, Cys-131, and Ser-132, singly and in various combinations caused a left shift in Ca(2+) response compared with wild type receptor. These results identify cysteines 129 and 131 as critical in formation of intermolecular disulfide bond(s) responsible for receptor dimerization. In a "venus flytrap" model of the receptor extracellular domain, Cys-129 and Cys-131 are located within a region protruding from one lobe of the flytrap. We suggest that this region represents a dimer interface for the receptor and that mutation of residues within the interface causes important changes in Ca(2+) response of the receptor.

Amino Acid Sequence↗

HAGR: the Human Ageing Genomic Resources.

The Human Ageing Genomic Resources (HAGR) is a collection of online resources for studying the biology of human ageing. HAGR features two main databases: GenAge and AnAge. GenAge is a curated database of genes related to human ageing. Entries were primarily selected based on genetic perturbations in animal models and human diseases as well as an extensive literature review. Each entry includes a variety of automated and manually curated information, including, where available, protein-protein interactions, the relevant literature, and a description of the gene and how it relates to human ageing. The goal of GenAge is to provide the most complete and comprehensive database of genes related to human ageing on the Internet as well as render an overview of the genetics of human ageing. AnAge is an integrative database describing the ageing process in several organisms and featuring, if available, maximum life span, taxonomy, developmental schedules and metabolic rate, making AnAge a unique resource for the comparative biology of ageing. Associated with the databases are data-mining tools and software designed to investigate the role of genes and proteins in the human ageing process as well as analyse ageing across different taxa. HAGR is freely available to the academic community at http://genomics.senescence.info.

Aging↗

The implantable cardioverter defibrillator: modern implantation techniques and their impact on outcomes.

The implantable cardioverter defibrillator has revolutionized the management of lethal ventricular arrhythmias in susceptible patients. In its second decade of existence, the implantable cardioverter defibrillator has undergone significant technologic enhancements which have resulted in ease of implantation, lower mortality rates, and shorter hospital stays. The newer pectoral size devices have been successfully implanted in a variety of patients, using models from several device manufacturers. Improvements in lead technology have paralleled those of the device itself. These include the unique concept of "unipolar" defibrillation as well as the trend toward dual chamber lead systems. Results of these newer technologies are favorable: comparably low defibrillation thresholds have been reported with the newer lead configurations, with lower operative mortality. However, morbidity attached to earlier lead systems remains as high as 16%. It is anticipated that the results will further improve as shorter transvenous leads and better connector material become routinely available. Finally, the clinical outcomes in the early postoperative phase indicate fewer proarrhythmic effects leading to shorter hospital stays in patient equipped with the latest types of pectoral implants. Continued progress at the level of the patient-device interface is expected to result in every better patient acceptance and proliferation of implantable cardioverter defibrillator therapy.

Arrhythmias, Cardiac↗

Clinical experience with a second-generation hospital-integrated picture archiving and communication system.

In a previous report we described a second-generation hospital-integrated picture archiving and communication system (HI-PACS) developed in-house. This HI-PACS had four unique features not found in other PAC systems. In this report, we will share some of our clinical experiences pertaining to these features during the past 12 months. We first describe the usage characteristics of two 2,000-line workstations (WSs), one in the in-patient and the second in the out-patient neuroradiology reading area. These two WSs can access neuro-images from 10 computed tomographic and magnetic resonance scanners located two medical centers through an asynchronous transfer mode network connection. The second unique feature of the system is an intensive care unit (ICU) server, which supports three WSs in the pediatric, medical surgery, and cardiac ICUs. The users' experiences and requests for refinement of the WSs are given. Another feature is physician desk-top access of PACS data. The HI-PACS provides a server connected to more than 100 Macintosh users for direct access of PACS data from their offices. The server's performance and user critiques are described. The last feature is a digital imaging and communication in medicine (DICOM) connection of the HI-PACS to a manufacturer's ultrasound PACS module. The authors then outline the interfacing process and summarize some of the difficulties encountered. Developing an in-house PACS has many advantages but also some drawbacks. Based on experience, the authors have formulated three axioms as a guide for in-house PACS development.

Computer Communication Networks↗

Structural plasticity of the Flp-Holliday junction complex.

The Flp recombinase, a member of the lambda integrase or tyrosine-based family of site-specific recombinases, is an interesting example of an enzyme whose catalytic activity is regulated by protein-protein contacts. It exhibits half-of-the-sites activity throughout its catalytic cycle. Flp is unique among these recombinases, in that it assembles each active site in trans through the interaction of two protein monomers within the catalytic tetramer, with isomerization of interacting pairs being essential to complete a full reaction. We report here the structure of a DNA-bound tetramer of Flpe, a variant of Flp that is more active at 37 degrees C than the wild-type recombinase. This new structure includes the first observation of a tyrosine recombinase with an invading 5'-OH poised to attack the covalent phosphotyrosine residue. Comparison with the previously determined Flp structure highlights differences in flexibility between the two types of protein-protein interfaces in the tetramer and better defines the range of conformations available to this remarkably flexible complex. These results suggest a steric occlusion model for enforcement of half-of-the-sites activity.

Bacterial Outer Membrane Proteins↗

Virtual reality as an adjunctive pain control during burn wound care in adolescent patients.

For daily burn wound care procedures, opioid analgesics alone are often inadequate. Since most burn patients experience severe to excruciating pain during wound care, analgesics that can be used in addition to opioids are needed. This case report provides the first evidence that entering an immersive virtual environment can serve as a powerful adjunctive, nonpharmacologic analgesic. Two patients received virtual reality (VR) to distract them from high levels of pain during wound care. The first was a 16-year-old male with a deep flash burn on his right leg requiring surgery and staple placement. On two occasions, the patient spent some of his wound care in VR, and some playing a video game. On a 100 mm scale, he provided sensory and affective pain ratings, anxiety and subjective estimates of time spent thinking about his pain during the procedure. For the first session of wound care, these scores decreased 80 mm, 80 mm, 58 mm, and 93 mm, respectively, during VR treatment compared with the video game control condition. For the second session involving staple removal, scores also decreased. The second patient was a 17-year-old male with 33.5% total body surface area deep flash burns on his face, neck, back, arms, hands and legs. He had difficulty tolerating wound care pain with traditional opioids alone and showed dramatic drops in pain ratings during VR compared to the video game (e.g. a 47 mm drop in pain intensity during wound care). We contend that VR is a uniquely attention-capturing medium capable of maximizing the amount of attention drawn away from the 'real world', allowing patients to tolerate painful procedures. These preliminary results suggest that immersive VR merits more attention as a potentially viable form of treatment for acute pain.

Adolescent↗

Materials management information systems.

The hospital materials management function--ensuring that goods and services get from a source to an end user--encompasses many areas of the hospital and can significantly affect hospital costs. Performing this function in a manner that will keep costs down and ensure adequate cash flow requires effective management of a large amount of information from a variety of sources. To effectively coordinate such information, most hospitals have implemented some form of materials management information system (MMIS). These systems can be used to automate or facilitate functions such as purchasing, accounting, inventory management, and patient supply charges. In this study, we evaluated seven MMISs from seven vendors, focusing on the functional capabilities of each system and the quality of the service and support provided by the vendor. This Evaluation is intended to (1) assist hospitals purchasing an MMIS by educating materials managers about the capabilities, benefits, and limitations of MMISs and (2) educate clinical engineers and information system managers about the scope of materials management within a healthcare facility. Because software products cannot be evaluated in the same manner as most devices typically included in Health Devices Evaluations, our standard Evaluation protocol was not applicable for this technology. Instead, we based our ratings on our observations (e.g., during site visits), interviews we conducted with current users of each system, and information provided by the vendor (e.g., in response to a request for information [RFI]). We divided the Evaluation into the following sections: Section 1. Responsibilities and Information Requirements of Materials Management: Provides an overview of typical materials management functions and describes the capabilities, benefits, and limitations of MMISs. Also includes the supplementary article, "Inventory Cost and Reimbursement Issues" and the glossary, "Materials Management Terminology." Section 2. The MMIS Selection Process: Outlines steps to follow and describes factors to consider when selecting an MMIS. Also includes our Materials Management Process Evaluation and Needs Assessment Worksheet (which is also available online through ECRInet(TM)) and a list of suggested interview questions to be used when gathering user experience information for systems under consideration. Section 3A. MMIS Vendor Profiles: Presents information for the evaluated systems in a standardized, easy-to-compare format. Profiles include an Executive Summary describing our findings, a discussion of user comments, a listing of MMIS specifications, and information on the vendor's business background. Section 3B. Discussion of Vendor Profile Conclusions and Ratings: Presents our ratings and summarizes our rationale for all evaluated systems. Also includes a blank Vendor Profile Template to be used when gathering information on other vendors and systems. We found that, in general, all of the evaluated systems are able to meet most of the functional needs of a materials management department. However, we did uncover significant differences in the quality of service and support provided by each vendor, and our ratings reflect these differences: we rated two of the systems Acceptable--Preferred and four of the systems Acceptable. We have not yet rated the seventh system because our user experience information may not reflect the vendor's new ownership and management. When this vendor provides the references we requested, we will interview users and supply a rating. We caution readers against basing purchasing decisions solely on our ratings. Each hospital must consider the unique needs of its users and its overall strategic plans--a process that can be aided by using our Process Evaluation and Needs Assessment Worksheet. Our conclusions can then be used to narrow down the number of vendors under consideration...

Computer Security↗

Development of the choroid plexus.

Mammalian choroid plexuses develop at four sites in the roof of the neural tube shortly after its closure, in the order IVth, lateral, and IIIrd ventricles. Bone morphogenetic proteins and tropomyosin are involved in early specification of these sites and in early plexus growth. Four stages of lateral ventricular plexus development have been defined, based on human and sheep fetuses; these depend mainly on the appearance of epithelial cells and presence or absence of glycogen. Other plexuses and other species are probably similar, although marsupials may lack glycogen. Choroid plexuses form one of the blood-brain barrier interfaces that control the brain's internal environment. The mechanisms involved combine a structural diffusion restraint (tight junctions between the plexus epithelial cells) and specific exchange mechanisms. In this review, it is argued that barrier mechanisms in the developing brain are different in important respects from those in the adult brain, but these differences do not necessarily reflect immaturity of the system. Absence of a barrier mechanism or presence of one not found in the adult may be a specialisation that is appropriate for that stage of brain development. Emphasis is placed on determining which mechanisms are present in the immature brain and relating them to brain development. One mechanism unique to the developing brain transfers specific proteins from blood to cerebrospinal fluid (CSF), via tubulocisternal endoplasmic reticulum in plexus epithelial cells. This results in a high concentration of proteins in early CSF. These proteins do not penetrate into brain extracellular space because of "strap" junctions between adjacent neuroependymal cells, which disappear later in development, when the protein concentration in CSF is much lower. Functions of the proteins in early CSF are discussed in terms of generation of a "colloid" osmotic pressure that expands the ventricular system as the brain grows; the proteins may also act as specific carriers and growth factors in their own right. The pathway for low molecular weight compounds, which is much more permeable in the developing choroid plexuses, appears also to be a transcellular one, rather than paracellular via tight junctions. There is thus good evidence to support a novel view of the state of development and functional significance of barrier mechanisms in the immature brain. It grows in an environment that is different from that of the rest of the fetus/neonate and that is also different in some respects from that of the adult. But these differences reflect developmental specialisation rather than immaturity.

Animals↗

Ordered growth of neurons on diamond.

Diamond has a number of unique properties that make it an attractive electronic and bio-electronic material. Here we show the ordered growth of mammalian neurons, the principal electrogenic cells of the nervous system, on diamond. Proteins were specifically patterned on diamond surfaces by micro-contact printing. Mouse cortical neurons were then cultured on these substrates. Neuron adhesion and outgrowth was specific for those areas of the diamond that had been stamped with laminin, resulting in ordered growth of high resolution. Neurons survived in culture for the duration of the experiment, and laminin patterns were stable for at least 1 week in culture. The relative biocompatibility of diamond and the suitability of neuron interfacing with the hydrogen surface conductivity layer make this an interesting model for the formation of defined neuronal networks and for implants.

Adsorption↗

Secondary structure in lung surfactant SP-B peptides: IR and CD studies of bulk and monolayer phases.

Pulmonary surfactant protein SP-B is known to facilitate adsorption and spreading of surfactant components across the air/water interface. This property appears essential for in vivo function in the alveolar subphase and at the air/alveolar surface. Three peptides with amino acid sequences based on SP-B containing predicted alpha-helical regions (SP-B(1--20), SP-B(9--36A), SP-B(40--60A)) have been synthesized to probe structure-function relationships and protein-lipid interaction in bulk phase and monolayer environments. IR and CD studies are reported along with traditional surface pressure-molecular area (pi-A) isotherms and IR reflection-absorption spectroscopy (IRRAS) investigations conducted at the air/water interface. In bulk phase, helix-promoting environments (methanol and aqueous dispersions of lipid vesicles), SP-B(1--20) and SP-B(9--36A) contained significant amounts of alpha-helical structure, whereas varying degrees of alpha-helix, random coil, and beta-sheet were observed in aqueous solutions and monolayers. The most striking behavior was observed for SP-B(9--36A), which displayed reversible surface pressure-induced beta-sheet formation. Bulk phase lipid melting curves and monolayer experiments with peptide-lipid mixtures showed subtle differences in the degree of bulk phase interaction and substantial differences in peptide surface activity. The uniqueness of IRRAS is emphasized as the importance of evaluating secondary structure in both bulk phase and monolayer environments for lung surfactant peptide mimics is demonstrated.

Amino Acid Sequence↗

Osteopontin expression in gestational trophoblastic diseases: correlation with expression of the adhesion molecule, CEACAM1.

The human placenta is a complex tissue with multiple endocrine and nutritional functions and a unique capacity for rapid proliferation but tightly controlled invasion, differentiating it from malignant tumors. Osteopontin (OPN) is a glycoprotein of the extracellular matrix, which has been shown to mediate cellular migration and invasion and to contribute to tumorigenesis in several types of cancers. OPN also could be implicated in regulating implantation and placentation by promoting cellular migration and invasion in a placenta-specific fashion. We could demonstrate the expression pattern of OPN in the normal human placenta in which it is localized in the extravillous (intermediate) trophoblast and the villous cytotrophoblast. CEACAM1 is an adhesion molecule, which we have recently found to be expressed at the maternal-fetal interface of the normal placenta with a localization to the extravillous (invasive) trophoblast and in gestational trophoblastic disease (GTD) and also to be potentially implicated in trophoblast invasion and tumorigenesis. Both OPN and CEACAM1 have been shown to interact with integrin beta3. The purpose of this study was to investigate the expression pattern of OPN in GTD and to correlate it with the expression of CEACAM1. To analyze the expression of OPN, we performed immunohistochemistry on a total of 27 cases of GTD, including 21 hydatidiform moles and 6 choriocarcinomas, which had previously been characterized with respect to their CEACAM1 expression. Hydatidiform moles showed a positivity for OPN in villous cytotrophoblast and in the trophoblast proliferations on the villous surface. The strongest OPN expression could be observed in the choriocarcinomas with a heterogenous OPN expression pattern. CEACAM1 had shown similar results and was found to be expressed in choriocarcinoma. The expression pattern of osteopontin in gestational trophoblastic diseases indicates that it might play a role in the pathogenesis of GTD (possibly as a functional complex with CEACAM1 and integrin beta3) and might be useful as an additional diagnostic marker for such lesions.

Antigens, CD↗

NrdH-redoxin of Corynebacterium ammoniagenes forms a domain-swapped dimer.

NrdH-redoxins constitute a family of small redox proteins, which contain a conserved CXXC sequence motif, and are characterized by a glutaredoxin-like amino acid sequence but a thioredoxin-like activity profile. Here we report the structure of Corynebacterium ammoniagenes NrdH at 2.7 A resolution, determined by molecular replacement using E. coli NrdH as model. The structure is the first example of a domain-swapped dimer from the thioredoxin family. The domain-swapped structure is formed by an inter-chain two-stranded anti-parallel beta-sheet and is stabilized by electrostatic interactions at the dimer interface. Size exclusion chromatography, and MALDI-ESI experiments revealed however, that the protein exists as a monomer in solution. Similar to E. coli NrdH-redoxin and thioredoxin, C. ammoniagenes NrdH-redoxin has a wide hydrophobic pocket at the surface that could be involved in binding to thioredoxin reductase. However, the loop between alpha2 and beta3, which is complementary to a crevice in the reductase in the thioredoxin-thioredoxin reductase complex, is the hinge for formation of the swapped dimer in C. ammoniagenes NrdH-redoxin. C. ammoniagenes NrdH-redoxin has the highly conserved sequence motif W61-S-G-F-R-P-[DE]67 which is unique to the NrdH-redoxins and which determines the orientation of helix alpha3. An extended hydrogen-bond network, similar to that in E. coli NrdH-redoxin, determines the conformation of the loop formed by the conserved motif.

Amino Acid Sequence↗

Studies on the role of methionine in porcine pancreatic phospholipase A2.

The unique methionine-15 residue located at the N-terminal site of iso- or beta-phospholipase A2 from porcine pancrease has been specifically carboxymethylated with iodoacetic acid. The modification results in a complete inactivation of the enzymatic activity toward micellar and monomeric substrates. Spectroscopic measurements reveled that the carboxymethylated protein still binds Ca2+ and monomeric substrates with comparable affinities as the native enzyeme. The active site histidine-54 residue in the modified enzyme shows a reactivity toward the active site-directed irreversible inhibitor p-bromophenacylbromide which is identical to that of the native enzyme. The alkylated protein, however, has lost its ability to bind to lipid-water interfaces. Although circular dichroic spectra of the carboxymethylated enzyme display some changes in the tertiary structure as compared with the native enzyme, the alpha-helix content remains rather constant. It is concluded that carboxymethylation of methionine-15 destroys the interface recognition site but has only limited influence on the active site of the molecule. Therefore, it seems that methionine-15 is not involved in the catalytic events but that this residue is part of the interface recognition site which embraces the N-terminal hydrophobic part of the enzyme: Ala-Leu-Trp-Gln-Phe-Arg-Ser-Met.

Animals↗

Pro-1 of macrophage migration inhibitory factor functions as a catalytic base in the phenylpyruvate tautomerase activity.

Macrophage migration inhibitory factor (MIF) is an important immunoregulatory molecule with a unique ability to suppress the anti-inflammatory effects of glucocorticoids. Although considered a cytokine, MIF possesses a three-dimensional structure and active site similar to those of 4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase. Moreover, a number of catalytic activities have been defined for MIF. To gain insight into the role of catalysis in the biological function of MIF, we have begun to characterize the catalytic activities in more detail. Here we report the crystal structure of MIF complexed with p-hydroxyphenylpyruvate, a substrate for the phenylpyruvate tautomerase activity of MIF. The three binding sites for p-hydroxyphenylpyruvate in the MIF trimer lie at the interface between two subunits. The substrate interacts with Pro-1, Lys-32, and Ile-64 from one subunit and Tyr-95 and Asn-97 from an adjacent subunit. Pro-1 is positioned to function as a catalytic base. There is no functional group that polarizes the alpha-carbonyl of the substrate to weaken the adjacent C-H bond. Mutation of Pro-1 to glycine substantially reduces the catalytic activity. The insertion of an alanine between Pro-1 and Met-2 essentially abolishes activity. Structural studies of these mutants define a source of the reduced activity and provide insight into the mechanism of the catalytic reaction.

Amino Acid Substitution↗

Cysteine-mediated cross-linking indicates that subunit C of the V-ATPase is in close proximity to subunits E and G of the V1 domain and subunit a of the V0 domain.

The vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible for ATP-dependent proton transport across both intracellular and plasma membranes. The V-ATPases are composed of a peripheral domain (V1) that hydrolyzes ATP and an integral domain (V0) that conducts protons. Dissociation of V1 and V0 is an important mechanism of controlling V-ATPase activity in vivo. The crystal structure of subunit C of the V-ATPase reveals two globular domains connected by a flexible linker (Drory, O., Frolow, F., and Nelson, N. (2004) EMBO Rep. 5, 1-5). Subunit C is unique in being released from both V1 and V0 upon in vivo dissociation. To localize subunit C within the V-ATPase complex, unique cysteine residues were introduced into 25 structurally defined sites within the yeast C subunit and used as sites of attachment of the photoactivated sulfhydryl reagent 4-(N-maleimido)benzophenone (MBP). Analysis of photocross-linked products by Western blot reveals that subunit E (part of V1) is in close proximity to both the head domain (residues 166-263) and foot domain (residues 1-151 and 287-392) of subunit C. By contrast, subunit G (also part of V1) shows cross-linking to only the head domain whereas subunit a (part of V0) shows cross-linking to only the foot domain. The localization of subunit C to the interface of the V1 and V0 domains is consistent with a role for this subunit in controlling assembly of the V-ATPase complex.

Adenosine Triphosphate↗

UniBLAST: a system to filter, cluster, and display BLAST results and assign unique gene annotation.

MOTIVATION: More and more often, a gene is epitomized by a large number of sequences in GenBank. This high redundancy makes it very difficult to identify a unique best match for a query sequence from its BLAST results. We developed a novel program UniBLAST that filters out uninformative hits, clusters the redundant hits, groups the hits by LocusLink, and graphically displays the results. We also implemented a scoring function in UniBLAST to assign a unique gene name to a query sequence. UniBLAST significantly increases the efficiency of gene annotation. AVAILABILITY: The program is available at http://south.genomics.org.cn/software/uniblast/index.html CONTACT: uniblast@genomics.org.cn; wei@nexusgenomics.com

Cluster Analysis↗

VNTRDB: a bacterial variable number tandem repeat locus database.

Variable number tandem repeat-PCR (VNTR-PCR) is a novel method developed for molecular typing of microorganisms. This method has proven useful in epidemiological studies in medical microbiology. Although hundreds of bacterial genomes have been sequenced, variable number tandem repeats (TRs) derived from comparative genome analyses are scarce. This may hamper their application to the surveillance of bacteria in molecular epidemiology. Here, we present a freely accessible variable number tandem repeat database (VNTRDB) that is intended to be a resource for helping in the discovery of putatively polymorphic tandem repeat loci and to aid with assay design by providing the flanking sequences that can be used in subsequent PCR primer design. In order to reveal possible polymorphism, each TR locus was obtained by comparing the sequences between different sets of bacterial genera, species or strains. Through this comparison, TRs which are unique to a genus can also be identified. Moreover, a visualization tool is provided to ensure that the copy number and locus length of repeats are correct. The VNTRDB is available at http://vntr.csie.ntu.edu.tw/.

Bacteria↗