Gold surface supported spherical liposome-gold nano-particle nano-composite for label free DNA sensing.

Immobilization of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) liposome-gold nano-particle (DOPE-AuNP) nano-composite covalently on 3-mercaptopropionic acid (MPA) on gold surface is demonstrated for the first time for electrochemical label free DNA sensing. Spherical nature of the DOPE on the MPA monolayer is confirmed by the appearance of sigmoidal voltammetric profile, characteristic behavior of linear diffusion, for the MPA-DOPE in presence of [Fe(CN)(6)](3-/4-) and [Ru(NH(3))(6)](3+) redox probes. The DOPE liposome vesicle fusion is prevented by electroless deposition of AuNP on the hydrophilic amine head groups of the DOPE. Immobilization of single stranded DNA (ssDNA) is made via simple gold-thiol linkage for DNA hybridization sensing in the presence of [Fe(CN)(6)](3-/4-). The sensor discriminates the hybridized (complementary target hybridized), un-hybridized (non-complementary target hybridized) and single base mismatch target hybridized surfaces sensitively and selectively without signal amplification. The lowest target DNA concentration detected is 0.1×10(-12)M. Cyclic voltammetry (CV), electrochemical impedance (EIS), differential pulse voltammetry (DPV) and quartz crystal microbalance (QCM) techniques are used for DNA sensing on DOPE-AuNP nano-composite. Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), Atomic Force Microscopy (AFM), Dynamic Light Scattering (DLS) and Ultraviolet-Visible (UV) spectroscopic techniques are used to understand the interactions between the DOPE, AuNP and ssDNA. The results indicate the presence of an intact and well defined spherical DOPE-AuNP nano-composite on the gold surface. The method could be applied for fabrication of the surface based liposome-AuNP-DNA composite for cell transfection studies at reduced reagents and costs.

Gold nano particle decorated graphene core first generation PAMAM dendrimer for label free electrochemical DNA hybridization sensing.

A novel first generation (G1) poly(amidoamine) dendrimer (PAMAM) with graphene core (GG1PAMAM) was synthesized for the first time. Single layer of GG1PAMAM was immobilized covalently on mercaptopropionic acid (MPA) monolayer on Au transducer. This allows cost effective and easy deposition of single layer graphene on the Au transducer surface than the advanced vacuum techniques used in the literature. Au nano particles (17.5 nm) then decorated the GG1PAMAM and used for electrochemical DNA hybridization sensing. The sensor discriminates selectively and sensitively the complementary double stranded DNA (dsDNA, hybridized), non-complementary DNA (ssDNA, un-hybridized) and single nucleotide polymorphism (SNP) surfaces. Interactions of the MPA, GG1PAMAM and the Au nano particles were characterized by Ultra Violet (UV), Fourier Transform Infrared (FTIR), Raman spectroscopy (RS), Thermo gravimetric analysis (TGA), Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Cyclic Voltmetric (CV), Impedance spectroscopy (IS) and Differntial Pulse Voltammetry (DPV) techniques. The sensor showed linear range 1×10(-6) to 1×10(-12) M with lowest detection limit 1 pM which is 1000 times lower than G1PAMAM without graphene core.

Ternary mixed monolayers for simultaneous DNA orientation control and surface passivation for label free DNA hybridization electrochemical sensing.

Target recognizing ternary mixed monolayer is constructed by simple sequential adsorption of thiol caped single stranded DNA (HS-ssDNA), 6-mercapto-1-hexanol (MCH) and 3-mercaptopropionic acid (MPA) diluents (abbreviated as HS-ssDNA/MPA/MCH) and applied for detection of DNA hybridization sequence by electrochemical method. The method is more reliable and reproducible on both conventional wire electrode and 100 nm scale gold-coated silicon chips. Detection limit of 10 pM is observed constantly on all types of electrodes. This ternary layer approach provides 60-80% discrimination effect between the hybridized and un-hybridized surfaces compared to the binary mixed monolayers HS-ssDNA/MCH and HS-ssDNA/MPA that showed 20-30% only. Characterization by impedance spectroscopy, X-ray photoelectron spectroscopy and surface Fourier Transform Infrared (FT-IR) techniques reveals 'head-to-head' anisotropic hydrogen bonding between MPA and MCH diluents that controls the HS-ssDNA orientation and enhancing the electrostatic blockade for K3[Fe(CN)6]. This is the first report characterizing the ternary layer for DNA molecular affinity sensing. Results provided unprecedented insight onto the label free electrochemical sensing and understanding of the complex sensing mechanism to develop sensors that is more reliable.

Mechanistic insight into the inactivation of carboxypeptidase A by alpha-benzyl-2-oxo-1,3-oxazolidine-4-acetic acid, a novel type of irreversible inhibitor for carboxypeptidase A with no stereospecificity.

On the basis of the active site topology and enzymic catalytic mechanism of carboxypeptidase A (CPA), a prototypical zinc-containing proteolytic enzyme, alpha-benzyl-2-oxo-1,3-oxazolidine-4-acetic acid (1), was designed as a novel type of mechanism-based inactivator of the enzyme. All four possible stereoisomers of the inhibitor were synthesized in an enantiomerically pure form starting with optically active aspartic acid, and their CPA inhibitory activities were evaluated to find that surprisingly all of the four stereoisomers inhibit CPA in a time dependent manner. The inhibited enzyme did not regain its enzymic activity upon dialysis. The inactivations were prevented by 2-benzylsuccinic acid, a competitive inhibitor that is known to bind the active site of the enzyme. These kinetic results strongly support that the inactivators attach covalently to the enzyme at the active site. The analysis of ESI mass spectral data of the inactivated CPA ascertained the conclusion from the kinetic results. The values of second-order inhibitory rate constants (k(obs)/[I](o)) fall in the range of 1.7-3.6 M(-1) min(-1). The lack of stereospecificity shown in the inactivation led us to propose that the ring cleavage occurs by the nucleophilic attack at the 2-position rather than at the 5-position and the ring opening takes place in an addition-elimination mechanism. The tetrahedral transition state that would be generated in this pathway is thought to be stabilized by the active site zinc ion, which was supported by the PM3 semiemprical calculations. In addition, alpha-benzyl-2-oxo-1,3-oxazolidine-5-acetic acid (18), a structural isomer of 1 was also found to inactivate CPA in an irreversible manner, reinforcing the nucleophilic addition-elimination mechanism. The present study demonstrates that the transition state for the inactivation pathway plays a critical role in determining stereochemistry of the inactivation.

Effect of ginsenosides, active components of ginseng, on capsaicin-induced pain-related behavior.

Our recent study demonstrated that ginsenosides had antinociceptive effects by reducing some types of pain-related behavior in mice (Yoon et al., 1998. Ginsenosides induce differential antinociception and inhibit substance P-induced nociceptive response in mice. Life Science 62, PL319-PL325). In the present study we further investigated whether ginsenosides produce antinociceptive effects through an action at central or peripheral site(s) and whether these effects are mediated by the opioid system. Intraperitoneally injected ginsenosides suppressed in a dose-dependent manner the pain-related behavior produced by capsaicin injection into the plantar surface of the hind paw; the ED(50) was 49 mg/kg [26-92 mg/kg, 95% confidence interval (C.I.)]. Intrathecally or intracerebroventricularly administered ginsenosides also suppressed the capsaicin-induced pain-related behavior in a dose-dependent manner; the ED(50)s were 1.72 mg/kg (0.8-3.72 mg/kg, 95% C.I.) and 1. 48 mg/kg (0.8-2.6 mg/kg, 95% C.I.), respectively. On the other hand, subcutaneously injected ginsenosides to the plantar surface prior to the capsaicin injection did not alter the pain-related behavior. Naloxone pretreatment was without effect in blocking the antinociceptive effect of intrathecally administered ginsenosides. Intraperitoneally injected ginsenosides also did not significantly affect the motor response of animals. These results suggest that ginsenosides produce antinociceptive effects through their action at the spinal and/or supraspinal site(s), not at nociceptors in the periphery. In addition, the results suggest that the antinociceptive effects are not mediated by opioid receptors.

Memory deficits after bilateral anterior fornix infarction.

The authors report a patient who suddenly developed memory loss without any other focal neurologic deficits. Neuropsychological testing showed severe anterograde verbal and visual memory deficits that improved gradually over several months, but not to the point of the premorbid state. Brain MRI with diffusion-weighted imaging performed 3 days after onset revealed acute infarction involving the bilateral fornices and the right genu of the corpus callosum. The authors suggest that acute fornix infarction may cause anterograde memory deficits.

A cytosolic catalase is needed to extend adult lifespan in C. elegans daf-C and clk-1 mutants.

The dauer larva is an alternative larval stage in Caenorhabditis elegans which allows animals to survive through periods of low food availability. Well-fed worms live for about three weeks, but dauer larvae can live for at least two months without affecting post-dauer lifespan. Mutations in daf-2 and age-1, which produce a dauer constitutive (Daf-C) phenotype, and in clk-1, which are believed to slow metabolism, markedly increase adult lifespan. Here we show that a ctl-1 mutation reduces adult lifespan in otherwise wild-type animals and eliminates the daf-c and clk-1-mediated extension of adult lifespan. ctl-1 encodes an unusual cytosolic catalase; a second gene, ctl-2, encodes a peroxisomal catalase. ctl-1 messenger RNA is increased in dauer larvae and adults with the daf-c mutations. We suggest that the ctl-1 catalase is needed during periods of starvation, as in the dauer larva, and that its misexpression in daf-c and clk-1 adults extends lifespan. Cytosolic catalase may have evolved to protect nematodes from oxidative damage produced during prolonged dormancy before reproductive maturity, or it may represent a general mechanism for permitting organisms to cope with the metabolic changes that accompany starvation.

Determination of the limited trypsinolysis pathways of tumor necrosis factor-alpha and its mutant by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is employed to directly analyze the limited trypsinolysis products of wild-type tumor necrosis factor-alpha (wtTNF-alpha) and its mutant, M3S. To determine the charge numbers of peaks of relatively small peptides in the ESI mass spectrum of a digest, a series of sodium-adduct ion peaks of each peptide are generated by adding a small quantity of NaCl to the digest before taking the spectrum. From the monitoring of the composition of proteolytic mixture as the incubation time is lengthened, it has been learned that the proteolysis of wtTNF-alpha by trypsin occurs sequentially: Arg2, Arg6, Arg32, Arg31, and Arg44, and that M3S is strongly resistant to the proteolysis. Since the cleavage sequence of wtTNF-alpha and the mutation-induced resistance of M3S are consistent with the structural features of the proteins, we can suggest a mutant more resistant to proteolysis than M3S, which has an additional point mutation, Ala35Leu or Ala35Ile.

High resolution crystal structure of a human tumor necrosis factor-alpha mutant with low systemic toxicity.

A human tumor necrosis factor-alpha (TNF-alpha) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two different crystal packings were determined crystallographically at 1.8 and 2.15-A resolution, respectively, to explain altered biological activities of the mutant. M3S contains four changes: a hydrophilic substitution of L29S, two hydrophobic substitutions of S52I and Y56F, and a deletion of the N-terminal seven amino acids that is disordered in the structure of wild-type TNF-alpha. Compared with wild-type TNF-alpha, it exhibits 11- and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic effect and in vivo systemic toxicity of M3S are 20 and 10 times lower, respectively. However, in a transplanted solid tumor mouse model, M3S suppresses tumor growth more efficiently than wild-type TNF-alpha. M3S is highly resistant to proteolysis by trypsin, and it exhibits increased thermal stability and a prolonged half-life in vivo. The L29S mutation causes substantial restructuring of the loop containing residues 29-36 into a rigid segment as a consequence of induced formation of intra- and intersubunit interactions, explaining the altered receptor binding affinity and thermal stability. A mass spectrometric analysis identified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop. The S52I and Y56F mutations do not induce a noticeable conformational change. The side chain of Phe56 projects into a hydrophobic cavity, while Ile52 is exposed to the bulk solvent. Ile52 should be involved in hydrophobic interactions with the receptors, since a mutant containing the same mutations as in M3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect of the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-type TNF-alpha. It is also expected that the 1.8-A resolution structure will serve as an accurate model for explaining the structure-function relationship of wild-type TNF-alpha and many TNF-alpha mutants reported previously and for the design of new TNF-alpha mutants.

CD99 (MIC2) regulates the LFA-1/ICAM-1-mediated adhesion of lymphocytes, and its gene encodes both positive and negative regulators of cellular adhesion.

Despite the fact that integrin-mediated lymphocyte adhesion is a crucial event for an appropriate immune response, little is known about the mechanisms that control the adhesion and deadhesion processes generated by the engagement of CD99 between various types of immune cells. Here we report that the CD99 gene encodes two distinct proteins with opposite functions in the LFA-1/intercellular adhesion molecule 1 (ICAM-1)-mediated cell adhesion process. The two forms of the CD99 protein are produced by alternative splicing of the CD99 gene transcript. The major form induced homotypic adhesion of the human B lymphoblastoid cell line IM-9, whereas the minor, truncated form inhibited the adhesion process. Activation of the major form of CD99 with anti-CD99 monoclonal antibodies induced rapid aggregation of IM-9 cells, which was blocked by the addition of mAbs to LFA-1 or intracellular adhesion molecule 1. Overexpression of the minor truncated form of CD99 markedly down-regulated the expression of LFA-1. The two forms of CD99 are differentially expressed in most human cells tested and are highly conserved in monkey. Taken together, these observations suggest that the two forms of CD99 function in vivo in both positive and negative regulation of LFA-1-mediated adhesion of lymphocytes during an immune response.

Rapid evolution in a conserved gene family. Evolution of the actin gene family in the sea urchin genus Heliocidaris and related genera.

Camarodont sea urchins possess a rapidly evolving actin gene family whose members are expressed in distinct cell lineages in a developmentally regulated fashion. Evolutionary changes in the actin gene family of echinoids include alterations in number of family members, site of expression, and gene linkage, and a dichotomy between rapidly and slowly evolving isoform-specific 3' untranslated regions. We present sequence comparisons and an analysis of the actin gene family in two congeneric sea urchins that develop in radically different modes, Heliocidaris erythrogramma and H. tuberculata. The sequences of several actin genes from the related species Lytechinus variegatus are also presented. We compare the features of the Heliocidaris and Lytechinus actin genes to those of the the actin gene families of other closely related sea urchins and discuss the nature of the evolutionary changes among sea urchin actins and their relationship to developmental mode.

Thymocytes positively select thymocytes in human system.

We previously demonstrated the expression of MHC class II molecules in a significant percentage of human fetal and postnatal thymocytes. These results, at that time, raised the question as to whether the MHC class II molecules on immature thymocytes could actively be involved in the selection of immature T cells. We have developed a human reaggregate culture system to address this issue. Surprisingly, despite the fact that thymic epithelial cells (TECs) have been shown to be a major selecting cell type of positive selection, we were clearly able to see the involvement of MHC class II+ thymocytes during selection process through T-T interaction. In addition, maturation to single positive (SP) cells occurred only in the presence of MHC class II molecules and immature thymocytes were found to be arrested at the double positive (DP) stage of differentiation by blocking of TCR recognition of MHC class II molecules. All these results strongly suggest that human MHC class II+ thymocytes actively participate in the selection of the TCR repertoire, for which TCR recognition of peptide/MHC class II may be an initial determining step.

Postelectrophoresis capillary scanning method for DNA sequencing.

A postelectrophoresis capillary scanning method for increasing the throughput of DNA sequencing has been developed. The method features a spatially and temporally separated arrangement between capillary gel electrophoresis separation of DNA sequencing fragments and the visualization of the separation pattern. Fluroescently labeled DNA sequencing fragments of pBluescript SK(-) were produced in enzymatic chain-termination reactions and separated by capillary gel electrophoresis using a 110 cm fused silica capillary with a transparent polymer coating. A partial sequence from the DNA was then determined by repeated scanning of the capillary at a rate of 8.39 cm/s, with changing excitation wavelengths and spectral filter sets.

DN4: a novel cell surface molecule on cortical epithelial cells of the human thymus.

A novel cell surface molecule, DN4, defined by an mAb raised against human thymic epithelial cells, showed a specificity for epithelial cells of the thymic cortex. This antigen was not expressed at detectable levels on any other types of tissues in the human body except for the thymus and bone marrow. Immunohistochemical analysis revealed that the reactivity of anti-DN4 mAb was restricted to the thymic cortex, and the antigen-expressing cells were arranged in a reticular network with long processes between thymocytes. The cellular nature of DN4-positive cells was identified as cortical epithelial cells, as DN4 was expressed in a subpopulation of freshly prepared thymic stromal cells which contain a large amount of keratin and expression of DN4 was strictly confined to the cortical area within the thymus on immunohistochemical analysis of frozen tissues. Immunofluorescence and flow cytometric analysis revealed that a subpopulation of bone marrow cells was also positive for DN4 (20%). The large blasts of normal bone marrow cells were clearly labeled with anti-DN4 mAb, in contrast to small-sized bone marrow cells. This finding suggests that DN4 seems to be transiently expressed in certain blastic stages during the differentiation of bone marrow cells. Immunoprecipitation of 125I-labeled cell lysates from THP-1 and U937 cell lines with anti-DN4 mAb yielded a single chain glycoprotein with an approximate size of 80-85 kd. There was a reduction in apparent molecular weight of approximately 40 kd in the immunoprecipitation of cell lysates after endoglycosidase F treatment. Thus, DN4 seemed to have a considerable amount of carbohydrate group. DN4 appears to be a novel cortical epithelial cell antigen of the human thymus, and although the role of this molecule has not been well established experimentally, the possibility can be suggested that the DN4 molecule might be involved in the positive selection of thymocytes which occurs predominantly in the thymic cortical area.

Structure and evolution of CyI cytoplasmic actin-encoding genes in the indirect- and direct-developing sea urchins Heliocidaris tuberculata and Heliocidaris erythrogramma.

The CyI cytoplasmic actin-encoding genes of Heliocidaris erythrogramma (He), a direct-developing sea urchin, and H. tuberculata, an indirect developer, were isolated and compared to the homologous CyI gene of another indirect developer, Strongylocentrotus purpuratus. Comparisons show that despite the differences in development, the actin gene structures and sequences are highly similar. The coding and 3' untranslated regions are conserved. The 5' He regulatory region has an inserted repeat element, but is otherwise similar to its homologues in the arrangement of presumptive transcription control elements.

Patient callback program: a quality improvement, customer service, and marketing tool.

The authors developed, implemented, and evaluated a callback program in which hospital patients are contacted three weeks after discharge to resolve clinical or service concerns. Of the more than 2,000 patients contacted during the initial pilot test, 6% said they had a clinical concern and were promptly directed to a physician's office. A randomized/controlled study comparing a control group of patients (who were not called) to an experimental group (called) shows that several satisfaction measures increased positively within the experimental group. The authors conclude that the Patient Callback Program contributes to more effective clinical care and to perceptions of higher customer service.

Rapid DNA sequencing based upon single molecule detection.

We are developing a laser-based technique for the rapid sequencing of 40-kb or larger fragments of DNA at a rate of 100 to 1000 bases per second. The approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA fragment into a flowing sample stream, and detection of individual fluorescently labeled bases as they are cleaved from the DNA fragment by an exonuclease. The ability to sequence large fragments of DNA will significantly reduce the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information.

Application of two-step laser mass spectrometry to cosmogeochemistry: direct analysis of meteorites.

Polycyclic aromatic hydrocarbons in C1, C2, and C3 carbonaceous chondrites and in some ordinary chondrites have been directly analyzed by two-step laser desorption/ laser multiphoton ionization mass spectrometry, a selective and sensitive method requiring only milligram samples. At the ionization wavelength of 266 nanometers, parent ion peaks of polycyclic aromatic hydrocarbons dominate the mass spectra. Quantitative analysis is possible; as an example, the concentration of phenanthrene in the Murchison meteorite was determined to be 5.0 parts per million.