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At least 433 records · Page 24Linked to original sources
PubMed2003

Bright spots: correlations of gray matter volume with IQ in a normal pediatric population.

The localization of brain areas related to cognitive functions has yet to be thoroughly explored in children. We therefore set out to apply volumetric, voxel-based, and structural connectivity analyses to magnetic resonance images from a large sample of healthy children. We could confirm a strong correlation of whole-brain gray matter volume and the individual intelligence quotient; however, this correlation only developed with age in our sample, in that it was not present in the younger children. With the application of an optimized protocol for voxel-based morphometry, the anterior cingulate was shown to be directly correlated with a measure of human intelligence. Furthermore, an analysis of structural connectivity identified gray matter volume in several distinct brain areas to be related to cognitive functions. The implications of our findings for normal development, pathological processes, and our understanding of cognition are discussed and related to previous findings.

Aging↗
PubMed2003

A voxel-based morphometric MRI study in female patients with borderline personality disorder.

Subtle prefrontal and limbic structural abnormalities have been reported in borderline personality disorder (BPD). In order to further validate the previously reported findings and to more precisely describe the nature of the structural change we performed a voxel-based morphometric (VBM) study in patients with BPD. Twenty female patients with BPD and 21 female healthy controls were investigated. High-resolution 3-D datasets were acquired and analyzed following an optimized protocol of VBM in the framework of statistical parametric mapping (SPM99). Gray matter volume loss was found in the left amygdala. No other differences in gray or white matter volume or density were found anywhere else in the brain. Our findings support the hypothesis that temporolimbic abnormalities play a role in the pathophysiology of BPD. Prefrontal structural alterations in BPD were not observed in this study.

Adult↗
PubMed1999

Target organ and time-course in the mutagenicity of five carcinogens in MutaMouse: a summary report of the second collaborative study of the transgenic mouse mutation assay by JEMS/MMS.

We studied five carcinogens for (a) organ-specific mutagenicity and expression time in the transgenic (TG) mouse mutation assay and (b) clastogenicity in the peripheral blood micronucleus assay in the same mice. Groups of mice were injected intraperitoneally (ip) with N-nitroso-di-n-propylamine (NDPA), propylnitrosourea (PNU), 7, 12-dimethylbenz[a]anthracene (DMBA), 4-nitroquinoline-1-oxide (4NQO), or procarbazine (PCZ); 4NQO was also administered orally. LacZ mutant frequencies (MF) of various organs, sampled 7, 14 and 28 days after treatment, were analyzed by galE positive selection. At least 5 organs were analyzed in each experiment. Bone marrow, liver, and testis were always analyzed, as were each chemical's target organs. All chemicals, except NDPA, induced micronuclei. All chemicals increased lacZ MF in all of their target organs for carcinogenesis and, to a lesser extent, in some non-target organs. That suggests that an organ that has a positive response to a chemical in the TG mouse mutation assay is likely to develop tumors on exposure to that chemical, but it does not always happen. The time-course of MF increases (7-28 days) differed among tissues. In general, time-dependent increase in MF occurred in organs with a low cell proliferation rate whereas no increase, or even a decrease, occurred in organs with a high proliferation rate. Our results demonstrated that the TG mouse mutation assay is effective for the detection of chemical mutagenesis in the target organs for carcinogenesis, and organ and time-course variations in chemical mutagenesis are important issues for the establishment of an optimal protocol for the assay.

4-Nitroquinoline-1-oxide↗
PubMed2003

Transduction and selection of human T cells with novel CD34/thymidine kinase chimeric suicide genes for the treatment of graft-versus-host disease.

Clinical trials evaluating the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) suicide gene therapy system for the control of graft-versus-host disease (GVHD) have been limited by low transduction efficiencies and inefficient selection procedures. In this study, we designed and evaluated a novel chimeric suicide gene consisting of the extracellular and transmembrane domains of human CD34 and full-length HSV-tk (DeltaCD34-tk). High-efficiency transfer of DeltaCD34-tk to primary human T cells was accomplished after a single exposure to VSV-G-pseudotyped, Moloney murine leukemia virus-based retrovirus 48 h after activation of human PBMCs with anti-CD3 and anti-CD28 antibodies immobilized on magnetic beads. Using an optimized 5-day transduction and selection procedure, transduction efficiencies averaged 71%, with isolation purities greater than 95% and yields exceeding 90%. The immunoselected T cells were selectively eliminated by GCV (IC(50) approximately 3 nM), maintained a normal subset composition, exhibited a polyclonal TCR Vbeta family repertoire, and contained 5 or 6 vector copies per transduced cell when optimally transduced. No increase in GCV sensitivity was observed upon incorporation of highly active mutant HSV-tk enzymes into the DeltaCD34-tk suicide gene. T cells modified with the DeltaCD34-tk gene using the optimized protocol should improve the overall efficacy of the HSV-tk/GCV suicide gene therapy method of GVHD control.

Animals↗
PubMed2001

Laser-assisted microdissection of membrane-mounted paraffin sections for polymerase chain reaction analysis: identification of cell populations using immunohistochemistry and in situ hybridization.

Laser microbeam microdissection (LMM) is an increasingly important method for obtaining pure cell samples for genetic and proteomic analysis. Immunohistochemistry (IHC) and in situ hybridization (ISH) are useful techniques for targeting specific cell populations for microdissection but are difficult to apply with the tissue support membranes often used during LMM. Using detection of cytokeratins and Epstein-Barr virus gene products in head and neck carcinoma as a model, we describe optimized protocols for membrane and section preparation and for low temperature antigen retrieval that allow IHC and ISH to be used reliably on membrane mounted paraffin tissue sections. Visualization of cellular targets was markedly improved by staining and this could be further improved using a variety of optical media before microdissection. Tissue fragments thus stained were suitable for subsequent polymerase chain reaction analysis of extracted DNA using standard techniques. These IHC and ISH procedures are generally applicable and will be useful for detecting a wide range of antigens and nucleic acids in paraffin sections in conjunction with LMM.

Head and Neck Neoplasms↗
PubMed2001

Demonstration of downregulation of alpha-smooth muscle actin in interferon-gamma-treated myofibroblast by a novel cell-capture enzyme immunoassay.

We developed a simple method for determining the relative amount of alpha-smooth muscle actin (alphaSMA) produced in fibroblasts. The principle of the method is based on an enzyme immunoassay (EIA) for alphaSMA in microcultured fibroblasts. The optimized protocol of the assay is as follows. Human fibroblasts were cultured with transforming growth factor beta1 (TGFbeta1) in a microtiter plate and directly immobilized on the plate. The alphaSMA produced was labeled and subjected to indirect enzyme immunoassay using alkaline phosphatase, and optical density was measured. Semiquantitativeness was confirmed using various numbers of cells in which alphaSMA production was induced by treatment with TGFbeta1. The assay simply demonstrated that interferon-gamma (INF-gamma) inhibited the production of alphaSMA in an established cell line and that in primary cultured cells originated from the contractile nodule. Since the assay is simple and semi-quantitative, it is useful for elucidating the mechanism of contractile diseases and screening a large number of substances that have an inhibitory effect on the change in activity of myofibroblasts.

Actins↗
PubMed2003

Simple immunoaffinity method to purify recombinant hepatitis B surface antigen secreted by transfected mammalian cells.

Purification of recombinant hepatitis B surface antigen (recHBsAg) produced in a stable Chinese hamster ovary (CHO) cell line was evaluated using Linx Affinity Purification System (Invitrogen, USA). To purify HBsAg secreted by this cell line, a murine monoclonal antibody (MAbAH1) raised against native HBsAg was used. The purified AH1MAb was conjugated with phenyldiboronic acid (PDBA) and immobilized on the immunoaffinity chromatographic support. Using an optimized protocol the affinity column was able to purify recHBsAg from supernatant of mammalian cells cultures with more than 80% purity. This method showed to be simple and quicker than the current ultracentrifugation methods. The method is also efficient and economical in obtaining purified recHBsAg.

Animals↗
PubMed2000

A miniaturized DNA amplifier: its application in traditional Chinese medicine.

A Si-based miniaturized device for the polymerase chain reaction (PCR) has been developed. The device has Pt temperature sensors and heaters integrated on top of the reaction chamber for real-time accurate temperature sensing and control. Reaction temperature of the device is digitally controlled to achieve a temperature accuracy of +/-0.025 degrees C. The effects of PCR protocol optimization on the amplification performance of the surface-passivated chip reactor have been investigated in detail and quantitatively compared with that of the conventional thermal cycler. In this study, four traditional Chinese medicine genes including Fritillaria cirrhosa, Cartharmus tinctorius, Adenophora lobophilla, and Stephania tetrandra are used as model template. With appropriate chamber surface treatment (chlorotrimethylsilane/polyadenylic acid or SiO2 coatings), the device demonstrates amplification as efficient as that in the conventional thermal cycler at optimized MgCl2 concentration. The amplified DNA has concentration higher than 27 ng/microL, which is sufficient for subsequent on-chip analyses and detection. Experimental results reveal the importance of inclusion of BSA for an efficient amplification in the SiO2-passivated device and the excellent reusability of the device with a simple cleaning step.

DNA, Plant↗
PubMed1996

Bioluminescence hybridization assays using recombinant aequorin. Application to the detection of prostate-specific antigen mRNA.

We developed microtiter well-based bioluminescence hybridization assays using the photoprotein aequorin as a reporter molecule. The target DNA was hybridized simultaneously with a capture probe and a detection probe. The capture probe was immobilized on the wells through digoxigenin/anti-digoxigenin interaction. The detection probe was biotinylated. The hybrids were determined by using aequorin covalently attached to streptavidin or complexes of biotinylated aequorin with streptavidin. The luminescence was then measured in the presence of excess Ca2+. The optimized protocols showed linearity in the range from 5 amol to 10 fmol of target DNA. In combination with reverse transcriptase polymerase chain reaction, the proposed assay was applied to the detection of the mRNA for prostate-specific antigen (PSA). PSA mRNA from a single cell, in the presence of one million cells that do not express PSA, was detected with a signal-to-background ratio of 2.5. Typical CVs obtained were 6%.

Aequorin↗
PubMed2004

Photochromic biliproteins from the cyanobacterium Anabaena sp. PCC 7120: lyase activities, chromophore exchange, and photochromism in phytochrome AphA.

Photochromic biliproteins can be switched by light between two states, initiated by Z/E photoisomerization of the linear tetrapyrrole chromophore. The cyanobacterium Anabaena sp. PCC 7120 contains three genes coding for such biliproteins, two coding for phytochromes (aphA/B) and one for the alpha subunit of phycoerythrocyanin (pecA). (a) aphA was overexpressed in Escherichia coli with N-terminal His and S tags, and the protein was reconstituted by an optimized protocol with phycocyanobilin (PCB), to yield the photochromic chromoprotein, PCB-AphA, carrying the PCB chromophore. (b) AphA chromophorylation is autocatalytic such as in other phytochromes. (c) AphA chromophorylation is also possible by chromophore transfer from the PCB-carrying biliprotein, phycocyanin (CPC). The autocatalytic transfer is very slow, and it is enhanced more than 100-fold by catalysis of PCB:CpcA lyase and alpha-CPC as donor. (d) Through deletion mutations of aphA, a short sequence IQPHGV [amino acids (aa) 26-31] was found essential for the lyase activity of AphA, indicating an interaction of the N terminus with the chromophore-binding domain around cysteine 259. (e) A motif of at least 23 aa, starting with this sequence and located approximately 250 aa N terminal of the chromophore-binding cysteine, is proposed to relate to the lyase function in plant and most prokaryotic phytochromes. (f) Long-range interactions in AphA are further supported by blue-shifted absorptions (<or=12 nm) of both the Pr and Pfr forms of truncated chromoproteins.

Amino Acid Motifs↗
PubMedDate not supplied

High-speed optimization of inhibitors of the malarial proteases plasmepsin I and II.

Four focused libraries targeted for inhibition of the malarial proteases plasmepsin I and II were designed, synthesized, purified, and screened. Selected carboxylic acids and organometallic reactants with diverse physical properties were attached to the hydroxylethylamine scaffold in the P3 and P1' positions to furnish inhibitors with highly improved activity. The concept of controlled and sequential microwave heating was employed for rapid library generation. This combinatorial optimization protocol afforded plasmepsin inhibitors not only with K(i) values in the low nanomolar range, but also with high selectivity versus the human protease cathepsin D. With this class of inhibitory agents, modifications of the P1' substituents resulted in the largest impact on the plasmepsin/cathepsin D selectivity.

Animals↗
PubMed2002

Solid-phase synthesis of fullerene-peptides.

The solid-phase synthesis of peptides (SPPS) containing [60]fullerene-functionalized amino acids is reported. A new amino acid, fulleropyrrolidino-glutamic acid (Fgu), is used for the SPPS of a series of analogues of different length based on the natural Leu(5)-Enkephalin and on cationic antimicrobial peptides. These fullero-peptides were prepared on different solid supports to analyze the influence of the resin on the synthesis. Optimized protocols for the coupling and deprotection procedures were determined allowing the synthesis of highly pure peptides in sufficient quantities for evaluation of biological activities. In particular, to avoid side reactions of the fullerene moiety with bases and nucleophiles, the removal of the protecting groups was performed under inert conditions (nitrogen or argon in the dark). We have encountered serious problems with the recovery of the crude compounds, especially when Fgu was inserted in the proximity of the resin core as fullero-peptides tend to remain embedded inside the resin. Eventually, all of the fullero-peptides were easily purified, and the cationic peptides were tested for their antimicrobial activities. They displayed a specific activity against the Gram-positive bacterium S. aureus and also lysed erythrocytes. The availability of a fullero-amino acid easily useable in the SPPS of fullero-peptides may thus open the way to the synthesis of new types of biologically active oligomers.

Amino Acids↗
PubMed2003

Docking of protein-protein complexes on the basis of highly ambiguous intermolecular distance restraints derived from 1H/15N chemical shift mapping and backbone 15N-1H residual dipolar couplings using conjoined rigid body/torsion angle dynamics.

A simple and reliable method for docking protein-protein complexes using (1)H(N)/(15)N chemical shift mapping and backbone (15)N-(1)H residual dipolar couplings is presented and illustrated with three complexes (EIN-HPr, IIA(Glc)-HPr, and IIA(Mtl)-HPr) of known structure. The (1)H(N)/(15)N chemical shift mapping data are transformed into a set of highly ambiguous, intermolecular distance restraints (comprising between 400 and 3000 individual distances) with translational and some degree of orientational information content, while the dipolar couplings provide information on relative protein-protein orientation. The optimization protocol employs conjoined rigid body/torsion angle dynamics in simulated annealing calculations. The target function also comprises three nonbonded interactions terms: a van der Waals repulsion term to prevent atomic overlap, a radius of gyration term (E(rgyr)) to avoid expansion at the protein-protein interface, and a torsion angle database potential of mean force to bias interfacial side chain conformations toward physically allowed rotamers. For the EIN-HPr and IIA(Glc)-HPr complexes, all structures satisfying the experimental restraints (i.e., both the ambiguous intermolecular distance restraints and the dipolar couplings) converge to a single cluster with mean backbone coordinate accuracies of 0.7-1.5 A. For the IIA(Mtl)-HPr complex, twofold degeneracy remains, and the structures cluster into two distinct solutions differing by a 180 degrees rotation about the z axis of the alignment tensor. The correct and incorrect solutions which have mean backbone coordinate accuracies of approximately 0.5 and approximately 10.5 A, respectively, can readily be distinguished using a variety of criteria: (a) examination of the overall (1)H(N)/(15)N chemical shift perturbation map (because the incorrect cluster predicts the presence of residues at the interface that experience only minimal chemical shift perturbations; this information is readily incorporated into the calculations in the form of ambiguous intermolecular repulsion restraints); (b) back-calculation of dipolar couplings on the basis of molecular shape; or (c) the E(rgyr) distribution which, because of its global nature, directly reflects the interfacial packing quality. This methodology should be particularly useful for high throughput, NMR-based, structural proteomics.

Bacterial Proteins↗
PubMed2006

Two-phase flavonoid formation in developing strawberry (Fragaria x ananassa) fruit.

Flavonoids are important secondary metabolites in strawberry as they fulfill a wide variety of physiological functions. In addition, they are beneficial for human health. Previous studies have shown for selected enzymes from the flavonoid pathway that flavonoid biosynthesis shows two peaks during fruit development. We provide optimized protocols for the determination of the activities of the key flavonoid enzymes: phenylalanine ammonia lyase, chalcone synthase/chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, flavonol synthase, flavonoid 3-O-glucosyltransferase, and flavonoid 7-O-glucosyltransferase. Using these protocols we were able to demonstrate two distinct activity peaks during fruit ripening at early and late developmental stages for all enzymes with the exception of flavonol synthase. The first activity peak corresponds to the formation of flavanols, while the second peak is clearly related to anthocyanin and flavonol accumulation. The results indicate that flavonoid 3-O-glucosyltransferase activity is not essential for redirection from flavanol to anthocyanin formation in strawberry.

Acyltransferases↗
PubMed2001

Mechanistic investigations on the reaction between 1,2-dioxines and bulky stabilized phosphorus ylides: an efficient route to closely related cyclopropane stereoisomers.

The bulky stabilized ylides (2a-d) react with a range of 1,2-dioxines (1a-d) to afford the diversely functionalized cyclopropanes 7 in excellent yield and diastereomeric excess. This is in direct contrast to the situation when nonbulky ester ylides are utilized which results in a completely different cyclopropyl series. Through a combination of isolation, spectroscopic, temperature, and deuterium and additive effects studies, the mechanism of cyclopropane formation from this second pathway can be proposed. Importantly, enolate quenching of the intermediate 1-2lambda(5)-oxaphospholanes 4 prior to collapse results in an equilibrium mixture of intermediates 10 and 11 which have been fully characterized, and their formation is primarily a result of the steric bulk of the stabilized ester ylide. These intermediates (10/11) then collapse further and result in formation of the observed closely related cyclopropyl stereoisomers 7 and 8. Moreover, the addition of LiBr to these reactions allows for the control of which of the two possible cyclopropanation pathways will be dominant. Finally, optimal protocols that demonstrate the potential of this new cyclopropanation methodology for the ready construction of closely related cyclopropyl stereoisomers are presented.

Journal Article↗
PubMed2006

4,6-O-[1-cyano-2-(2-iodophenyl)ethylidene] acetals. improved second-generation acetals for the stereoselective formation of beta-D-mannopyranosides and regioselective reductive radical fragmentation to beta-D-rhamnopyranosides. scope and limitations.

The [1-cyano-2-(2-iodophenyl)]ethylidene group is introduced as an acetal-protecting group for carbohydrate thioglycoside donors. The group is easily introduced under mild conditions, over short reaction times, and in the presence of a wide variety of other protecting groups by the reaction of the 4,6-diol with triethyl (2-iodophenyl)orthoacetate and camphorsulfonic acid, followed by trimethylsilyl cyanide and boron trifluoride etherate. The new protecting group conveys strong beta-selectivity with thiomannoside donors and undergoes a tin-mediated radical fragmentation to provide high yields of the synthetically challenging beta-rhamnopyranosides. The method is also applicable to the glucopyranosides when high alpha-selectivity is observed in the coupling reaction and alpha-quinovosides are formed selectively in the radical fragmentation step. In the galactopyranoside series, beta-glycosides are formed selectively on coupling to donors protected by the new system, but the radical fragmentation is unselective and gives mixtures of the 4- and 6-deoxy products. Variable-temperature NMR studies for the glycosylation step, which helped define an optimal protocol, are described.

Acetals↗
PubMed2000

Improved procedure for the reductive acetylation of acyclic esters and a new synthesis of ethers.

An optimized protocol for the DIBALH reductive acetylation of acyclic esters and diesters is described. This reductive acetylation procedure allows a wide variety of esters to be converted into the corresponding alpha-acetoxy ethers in good to excellent yields. It was found that, under mild acidic conditions, many alpha-acetoxy ethers can be further reduced to the corresponding ethers. This net two-step ester deoxygenation is an attractive alternative to the classical Williamson synthesis for certain ethers.

Acetylation↗