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At least 1,333 records · Page 74Linked to original sources

PubMed2003

Monitoring molecular beacon DNA probe hybridization at the single-molecule level.

We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.

Biosensing Techniques↗

PubMed2002

Domain arrangement of Der, a switch protein containing two GTPase domains.

The EngA subfamily of essential bacterial GTPases has a unique domain structure consisting of two adjacent GTPase domains (GD1 and GD2) and a C-terminal domain. The structure of Thermotoga maritima Der bound to GDP determined at 1.9 A resolution reveals a novel domain arrangement in which the GTPase domains pack at either side of the C-terminal domain. Unexpectedly, the C-terminal domain resembles a KH domain, missing the distinctive RNA recognition elements. Conserved motifs of the nucleotide binding site of GD1 are integral parts of the GD1-KH domain interface, suggesting the interactions between these two domains are directly influenced by the GTP/GDP cycling of the protein. In contrast, the GD2-KH domain interface is distal to the GDP binding site of GD2.

Bacterial Proteins↗

PubMed2006

Monolithic column HPLC separation of intact proteins analyzed by LC-MALDI using on-plate digestion: An approach to integrate protein separation and identification.

A method is developed to integrate a protein separation by monolithic capillary reversed-phase high-performance liquid chromatography to on-probe tryptic digestion for subsequent analyses by MALDI-TOF MS and MALDI-TOF/TOF MS. The method provides a means of directly interfacing separations to MALDI-MS, reducing the amount of time required for traditional procedures involving in-solution enzymatic digestion and sample cleanup prior to MALDI-MS analysis. When used with pI-based fractionation as a first dimension, it provides a means of analyzing complex mixtures of proteins with minimal sample handling and cleanup. The use of monolithic capillary columns sufficiently resolved intact proteins so that peptide mass fingerprinting analysis by MALDI-TOF MS resulted in the identification of close to 40 unique proteins from 120 ng of sample obtained from a prefractionated MCF10 cell line at pH 6.34, where the identifications of several of these proteins were also confirmed by intact MW and tandem mass spectrometric analysis. The reproducibility of this method has been demonstrated to be sufficient for the purpose of protein identifications. Experimental values of protein intact MW are obtained and compared to that expected for each protein identified.

Amino Acid Sequence↗

PubMed2002

Structure of the MUR1 GDP-mannose 4,6-dehydratase from Arabidopsis thaliana: implications for ligand binding and specificity.

GDP-D-mannose 4,6-dehydratase catalyzes the first step in the de novo synthesis of GDP-L-fucose, the activated form of L-fucose, which is a component of glycoconjugates in plants known to be important to the development and strength of stem tissues. We have determined the three-dimensional structure of the MUR1 dehydratase isoform from Arabidopsis thaliana complexed with its NADPH cofactor as well as with the ligands GDP and GDP-D-rhamnose. MUR1 is a member of the nucleoside-diphosphosugar modifying subclass of the short-chain dehydrogenase/reductase enzyme family, having homologous structures and a conserved catalytic triad of Lys, Tyr, and Ser/Thr residues. MUR1 is the first member of this subfamily to be observed as a tetramer, the interface of which reveals a close and intimate overlap of neighboring NADP(+)-binding sites. The GDP moiety of the substrate also binds in an unusual syn conformation. The protein-ligand interactions around the hexose moiety of the substrate support the importance of the conserved triad residues and an additional Glu side chain serving as a general base for catalysis. Phe and Arg side chains close to the hexose ring may serve to confer substrate specificity at the O2 position. In the MUR1/GDP-D-rhamnose complex, a single unique monomer within the protein tetramer that has an unoccupied substrate site highlights the conformational changes that accompany substrate binding and may suggest the existence of negative cooperativity in MUR1 function.

Arabidopsis Proteins↗

PubMed2004

Regulation of Bin1 SH3 domain binding by phosphoinositides.

Bin1/M-amphiphysin-II is an amphiphysin-II isoform highly expressed in transverse tubules of adult striated muscle and is implicated in their biogenesis. Bin1 contains a basic unique amino-acid sequence, Exon10, which interacts with certain phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), to localize to membranes. Here we found that Exon10 also binds to the src homology 3 (SH3) domain of Bin1 itself, and hence blocks the binding of the SH3 domain to its canonical PxxP ligands, including dynamin. This blockage was released by addition of PI(4,5)P(2) in vitro or in cells overexpressing phosphatidylinositol 4-phosphate 5-kinase. The Exon10-binding interface of the Bin1 SH3 domain largely overlapped with its PxxP-binding interface. We also show that the PLCdelta pleckstrin homology domain, another PI(4,5)P(2)-binding module, cannot substitute for Exon10 in Bin1 function in transverse tubule formation, and suggest the importance of the dual biochemical properties of Exon10 in myogenesis. Our results exemplify a novel mechanism of SH3 domain regulation, and suggest that the SH3-mediated protein-protein interactions of Bin1 are regulated by Exon10 so that it may only occur when Bin1 localizes to certain submembrane areas.

Adaptor Proteins, Signal Transducing↗

PubMed1999

X-ray crystal structure of human dopamine sulfotransferase, SULT1A3. Molecular modeling and quantitative structure-activity relationship analysis demonstrate a molecular basis for sulfotransferase substrate specificity.

Humans are one of the few species that produce large amounts of catecholamine sulfates, and they have evolved a specific sulfotransferase, SULT1A3 (M-PST), to catalyze the formation of these conjugates. An orthologous protein has yet to be found in other species. To further our understanding of the molecular basis for the unique substrate selectivity of this enzyme, we have solved the crystal structure of human SULT1A3, complexed with 3'-phosphoadenosine 5'-phosphate (PAP), at 2.5 A resolution and carried out quantitative structure-activity relationship (QSAR) analysis with a series of phenols and catechols. SULT1A3 adopts a similar fold to mouse estrogen sulfotransferase, with a central five-stranded beta-sheet surrounded by alpha-helices. SULT1A3 is a dimer in solution but crystallized with a monomer in the asymmetric unit of the cell, although dimer interfaces were formed by interaction across crystallographic 2-fold axes. QSAR analysis revealed that the enzyme is highly selective for catechols, and catecholamines in particular, and that hydrogen bonding groups and lipophilicity (cLogD) strongly influenced K(m). We also investigated further the role of Glu(146) in SULT1A3 using site-directed mutagenesis and showed that it plays a key role not only in defining selectivity for dopamine but also in preventing many phenolic xenobiotics from binding to the enzyme.

Alanine↗

PubMed1993

Structural relationship between epidermal lipid lamellae, lamellar bodies and desmosomes in human epidermis: an ultrastructural study.

The water permeability of the stratum corneum (SC) appears to be regulated primarily by the lamellar arrangement of lipid bilayers between the corneocytes. A significant body of evidence already exists, suggesting that the specific structural organization of these intercellular lipid lamellae is responsible for the very low water permeability of the intact skin and that these lipid-rich structures may also influence the process of desquamation in the SC. In this electron microscopic study the structure of the intercellular domains at different levels within the SC of normal skin from 18 healthy subjects has been evaluated with a special fixation protocol utilizing acrolein vapour as primary fixation, followed by a modified ruthenium tetroxide (RuO4)-post-fixation technique. This procedure permitted an insight into the process of post-secretory extracellular processing of the lamellar body (LB)-derived lipids into lamellar lipid bilayers. This transformation takes place in unique saccular invaginations of the intercellular domains, which indent the underlying stratum granulosum (SG) cells. In this specialized environment LB lipids are first processed into broad sheets before they become part of the typical lamellar lipid structure of the SC. Furthermore, in the process of lipid maturation distinct differences between inner and outer parts of the SC emerge, in particular an increase in both the number of the lamellae per intercellular space, and their order of arrangement. Moreover, distinct structural relationships between desmosomes (at the SG/SC interface and lower SC) and desmosomal remnants (at the stratum disjunctum) on the one hand, and lipid layers on the other, have been demonstrated, pointing to an important functional interaction of these components in normal human skin.

Adult↗

PubMed1989

Isokinetic dynamometry. Applications and limitations.

Isokinetic contraction is the muscular contraction that accompanies constant velocity limb movements around a joint. The velocity of movement is maintained constant by a special dynamometer. The resistance of the dynamometer is equal to the muscular forces applied throughout the range of movement. This method allows the measurement of the muscular forces in dynamic conditions and provides optimal loading of the muscles. However, during movements in the vertical plane, the torque registered by the dynamometer is the resultant torque produced by the muscular and gravitational forces. The error depends on the angular position and the torque potential of the tested muscle group. Several methods have been developed for the correction of gravitational errors in isokinetic data. The torque output also contains artefacts that are associated with the inertial forces during acceleration and deceleration periods before the development of the constant preset angular velocity. For an accurate assessment of muscle function, only constant velocity data should be analysed. The most frequently used isokinetic parameters are the maximum torque and the angular position where it was recorded, the torque output at different angular velocities of movement, the torque ratio of reciprocal muscle groups and the torque output during repeated contractions. The unique features of isokinetic dynamometry are optimal loading of the muscles in dynamic conditions and constant preselected velocity of movement. These features provide safety in the rehabilitation of patients with muscular and ligamentous injuries. Isokinetic dynamometry has also been used for the training of various muscle groups in order to improve the muscular performance in dynamic conditions. The movement velocity of different activities can be simulated during training in order to improve the training effect. Data acquisition and analysis have been improved by using computer systems interfaced to isokinetic dynamometers. Recently developed computer systems provide correction for gravitational and inertial errors, accurate computation of isokinetic parameters and real-time display of the torque output.

Biomechanical Phenomena↗

PubMed2004

Select ionotropic glutamate AMPA/kainate receptors are expressed at the astrocyte-vessel interface.

Ionotropic AMPA/kainate (KA) glutamate receptors are important for vasculature-astrocyte interaction in the central nervous system (CNS). To date, little is known about the expression of AMPA/KA receptors in the perivascular environment. Using double labeling immunohistochemistry on mouse and rat spinal cord sections, we show first evidence that perivascular astrocytic processes express specifically GluR4 (AMPA) and GluR5 (KA) subunits in both species. In contrast, astrocytes display GluR2/3 and GluR6/7 immunoreactivity throughout the cell but not limited to vascular profiles. Interestingly, KA2 immunoreactivity appears perivascular only in rats but not in mice. No perivascular labeling for any of the probed subunits was found in peripheral sciatic nerve. These data suggest that, in both mice and rats, few and select AMPA/KA subunits are uniquely positioned to mediate the effects of glutamate on astrocyte-vessel interactions.

Animals↗

PubMed2006

Human decidual NK cells: unique phenotype and functional properties -- a review.

Human decidual NK cells are massively recruited at the site of embryonic implantation (decidua basalis). They differ in many ways from their peripheral blood NK cell counterparts in terms of gene expression, phenotype and functionality. The major subpopulation of decidual NK cells is CD56(bright) whereas the minor subset is CD56(dim), contrasting with the peripheral blood NK cells whose major subpopulation is CD56(dim). Decidual NK cell cytolytic function is much reduced despite the presence of several activating receptors and the essential machinery required for lysis. Decidual NK cells produce a number of cytokines that are not normally secreted by peripheral blood NK cells. Human decidual NK cell potential functions at the maternal-fetal interface are not yet clearly established but several hypotheses are being evaluated, including control of extravillous invasion, control of uterine vascular remodeling, and local anti-viral activity.

Cell Movement↗

PubMed2004

[Weibel-Palade bodies: unique secretory organelles within endothelial cells].

Vascular endothelial cells contain typical elongated vesicles, known as Weibel-Palade bodies. These organelles serve as a storage compartment for a variety of proteins that play a part in controlling vascular homeostasis, including von Willebrand factor, endothelin, P-selectin and interleukin-8. Upon activation of endothelial cells, Weibel-Palade bodies are translocated to the periphery of the cell and there fuse with the plasma membrane to release their contents into the blood circulation and subendothelial connective tissue. This process provides an adequate means by which endothelial cells can actively participate in controlling the arrest of bleeding upon vascular damage or modulate inflammatory reactions and other physiological and pathophysiological processes at the blood-tissue interface. Weibel-Palade bodies may also move to the nucleus of the cell, thus escaping secretion. This phenomenon may play a part in controlling stimulus-induced exocytosis.

Endothelial Cells↗

PubMed1992

Basis of unique red cell membrane properties in hereditary ovalocytosis.

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56----Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.

Adult↗

PubMed1990

Endothelial cell transformation in primary biliary cirrhosis: a morphological and biochemical study.

There is increasing interest in the changes of the endothelial lining of the hepatic sinusoids during the development of chronic liver disease. In this study we looked for evidence of hepatic sinusoidal endothelial cell transformation and basement membrane production in patients with primary biliary cirrhosis. Morphological transformation to vascular-type endothelial cells, as evidenced by the development VIII-related antigen, was seen at the interface between portal tracts or fibrous septae and hepatic parenchyma; the most marked changes were observed in patients with established cirrhosis. Increased immunohistochemical staining for the basement membrane components type IV collagen and laminin was also found in a similar distribution. Raised serum levels of hyaluronic acid, a glycosaminoglycan metabolized by normal hepatic endothelial cells, were found in most patients and correlated strongly with advancing histological stage. Furthermore, significant positive correlations were found between serum hyaluronic acid and serum levels of laminin and type IV collagen. The unique structure of the normal endothelial lining of the hepatic sinusoids is important in the maintenance of hepatic function. Our data show that significant changes in endothelial cell structure and function occur in primary biliary cirrhosis and appear to be a contributing factor to the progression of the disease. Further studies are needed to determine the extent and importance of these changes in other forms of chronic liver disease.

Endothelium↗

PubMed2003

Graphic-based musculoskeletal model for biomechanical analyses and animation.

The ability to combine physiology and engineering analyses with computer sciences has opened the door to the possibility of creating the 'Virtual Human' reality. This paper presents a broad foundation for a full-featured biomechanical simulator for the human musculoskeletal system physiology. This simulation technology unites the expertise in biomechanical analysis and graphic modeling to investigate joint and connective tissue mechanics at the structural level and to visualize the results in both static and animated forms together with the model. Adaptable anatomical models including prosthetic implants and fracture fixation devices and a robust computational infrastructure for static, kinematic, kinetic, and stress analyses under varying boundary and loading conditions are incorporated on a common platform, the VIMS (Virtual Interactive Musculoskeletal System). Within this software system, a manageable database containing long bone dimensions, connective tissue material properties and a library of skeletal joint system functional activities and loading conditions are also available and they can easily be modified, updated and expanded. Application software is also available to allow end-users to perform biomechanical analyses interactively. This paper details the design, capabilities, and features of the VIMS development at Johns Hopkins University, an effort possible only through academic and commercial collaborations. Examples using these models and the computational algorithms in a virtual laboratory environment are used to demonstrate the utility of this unique database and simulation technology. This integrated system will impact on medical education, basic research, device development and application, and clinical patient care related to musculoskeletal diseases, trauma, and rehabilitation.

Algorithms↗

PubMed2003

An infrared reflection-absorption spectroscopy study of the secondary structure in (KL4)4K, a therapeutic agent for respiratory distress syndrome, in aqueous monolayers with phospholipids.

KLLLLKLLLLKLLLLKLLLLK (KL(4)) has been suggested to mimic some aspects of the pulmonary surfactant protein SP-B and has been tested clinically as a therapeutic agent for respiratory distress syndrome in premature infants [Cochrane, C. G., and Revak, S. D. (1991) Science 254, 566-568]. It is of obvious interest to understand the mechanism of KL(4) function as a guide for design of improved therapeutic agents. Attenuated total reflection (ATR) IR measurements have indicated that KL(4) is predominantly alpha-helical with a transmembrane orientation in lipid multilayers (1), a geometry quite different from the originally proposed peripheral membrane lipid interaction. However, the lipid multilayer model required for ATR may not be the best experimental paradigm to mimic the in vivo function of KL(4). In the current experiments, IR reflection-absorption spectroscopy (IRRAS) was used to evaluate peptide secondary structure in monolayers at the air/water interface, the physical state that best approximates the alveolar lining. In contrast to the ATR-IR results, KL(4) (2.5-5 mol %) films with either DPPC or DPPC/DPPG (7/3 mol ratio) adopted an antiparallel beta-sheet structure at all surface pressures studied > or =5 mN/m, including pressures physiologically relevant for lung function (40-72 mN/m). In contrast, in DPPG/KL(4) films, the dominant conformation was the alpha-helix over the entire pressure range, a possible consequence of enhanced electrostatic interactions. IRRAS has thus provided unique molecular structure information and insight into KL(4)/lipid interaction in a physiologically relevant state. A structural model is proposed for the response of the peptide to surface pressure changes.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2002

Furfural-cysteine model reaction in food grade nonionic oil/water microemulsions for selective flavor formation.

The thermal reaction between cysteine and furfural was investigated at 65 degrees C in five-component food grade oil/water (O/W) microemulsions of R-(+)-limonene/ethanol, EtOH/water/propylene glycol, PG/Tween 60 as apart of a systematic study on the generation of aroma compounds by utilizing structured W/O and O/W fluids. The furfural-cysteine reaction led to the formation of unique aroma compounds such as 2-furfurylthiol (FFT), 2-(2-furanyl)thiazolidine (main reaction product), 2-(2-furanyl)thiazoline, and N-(2-mercaptovinyl)-2-(2-furanyl)thiazolidine. These products were determined and characterized by GC-MS. Enhancement in flavor formation is termed "microemulsion catalysis". The chemical reaction occurs preferably at the interfacial film, and therefore a pseudophase model was assumed to explain the enhanced flavor formation. The product internal composition is dictated by process conditions such as temperature, time, pH, and mainly the nature of the interface. Increasing water/PG ratio leads to a dramatic increase in the initial reaction rate (V(0)). V(0) increased linearly as a function of the aqueous phase content, which could be due to the increase in the interfacial concentration of furfural. Microemulsions offer a new reaction medium to produce selective aroma compounds and to optimize their formation.

Cyclohexenes↗

PubMed2001

Identification of a putative binding site for [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)thymine (TSAO) derivatives at the p51-p66 interface of HIV-1 reverse transcriptase.

A binding site for TSAO-m(3)T at the interface between the p66 and p51 subunits of HIV-1 reverse transcriptase (RT) and distinct from that of "classical" HIV-1 non-nucleoside inhibitors is proposed. The feasibility of the binding mode was assessed by carrying out nanosecond molecular dynamics simulations for the complexes of TSAO-m(3)T with reduced models of both the wild-type enzyme and a more sensitive R172A mutant. The molecular model is in agreement with a previous proposal, with known structure-activity and mutagenesis data for this unique class of inhibitors, and also with recent biochemical evidence indicating that TSAO analogues can affect enzyme dimerization. The relative importance of residues involved in dimer formation and TSAO-RT complex stabilization was assessed by a combination of surface area accessibility, molecular mechanics, and continuum electrostatics calculations. A structure-based modification introduced into the lead compound yielded a new derivative with improved antiviral activity.

Amino Acid Substitution↗

PubMed2006

Ultrafast hydration dynamics in the lipidic cubic phase: Discrete water structures in nanochannels.

We report here our studies of hydration dynamics of confined water in aqueous nanochannels (approximately 50 A) of the lipidic cubic phase. By systematically anchoring the hydrocarbon tails of a series of tryptophan-alkyl ester probes into the lipid bilayer, we mapped out with femtosecond resolution the profile of water motions across the nanochannel. Three distinct time scales were observed, revealing discrete channel water structures. The interfacial water at the lipid surface is well-ordered, and the relaxation dynamics occurs in approximately 100-150 ps. These dynamically rigid water molecules are crucial for global structural stability of lipid bilayers and for stabilization of anchored biomolecules in membranes. The adjacent water layers near the lipid interface are hydrogen-bonded networks and the dynamical relaxation takes 10-15 ps. This quasi-bound water motion, similar to the typical protein surface hydration relaxation, facilitates conformation flexibility for biological recognition and function. The water near the channel center is bulklike, and the dynamics is ultrafast in less than 1 ps. These water molecules freely transport biomolecules near the channel center. The corresponding orientational relaxation at these three typical locations is well correlated with the hydration dynamics and local dynamic rigidity. These results reveal unique water structures and dynamical motions in nanoconfinements, which is critical to the understanding of nanoscopic biological activities and nanomaterial properties.

Lipids↗