Peptide nanowires for coordination and signal transduction of peroxidase biosensors to carbon nanotube electrode arrays

A strategy of metallizing peptides to serve as conduits of electronic signals that bridge between a redox enzyme and a carbon-nanotube electrode has been developed with enhanced results. In conjunction, a protocol to link the biological elements to the tips of carbon nanotubes has been developed to optimize contact and geometry between the redox enzyme and the carbon nanotube electrode array. A peptide nanowire of 33 amino acids, comprised of a leucine zipper motif, was mutated to bind divalent metals, conferring conductivity into the peptide. Reaction between a thiolate of the peptide with the sulfenic acid of the NADH peroxidase enzyme formed a peptide-enzyme assembly that are fully primed to transduce electrons out of the enzyme active site to an electrode. Scanning electron microscopy shows immobilization and linking of the assembly specifically to the tips of carbon nanotube electrodes, as designed. Isothermal titration calorimetry and mass spectrometry indicate a binding stoichiometry of at least three metals bound per peptide strand. Overall, these results highlight the gain that can be achieved when the signal tranducing units of a biosensor are aligned through directed peptide chemistry.     

Wide dynamic range phase-sensitive surface plasmon resonance biosensor based on measuring the modulation harmonics

In this study, a novel phase-sensitive surface plasmon resonance (SPR) setup, based on temporal modulation of a pumping beam by a photoelastic modulator, and subsequent extraction of phase information at the second and the third harmonics of the modulation frequency, has been developed to study biomolecular interactions on SPR-supporting gold. We demonstrated that the design setup provides ultra-high phase sensitivity, together with a wide dynamic range of measurements. In particular, the proposed scheme was used to study real-time interaction of biotin-protein and streptavidin-BSA complexes. We have found that the proposed technique has a detection limit as high as 2.89 x 10(-7) in terms of refractive index units (RIU). In terms of biosensing performance, a detection sensitivity of 1.3 nM from the streptavidin-maleimide/thiolated BSA complex binding reaction has also been demonstrated.     

Dual electrochemical determination of glucose and insulin using enzyme and ferrocene microcapsules

Dual electrochemical determination of glucose and insulin has been developed, based on enzymatic reaction and immunoassay with utilization of ferrocene microcapsules, respectively. Glucose was determined through electrochemical oxidation of formed product, hydrogen peroxide, by the action of glucose oxidase (GOx). The layer-by-layer (LbL) films on the ferrocene microcrystal followed by anti-insulin antibody sensitization were employed for the biolabled ferrocene microcapsules production. The antibody sensitized ferrocene microcapsules worked as a probe in the proposed system. The microcapsules provided a higher signal generating molecule to antibody (S/P) ratio of 4.52x10(6) to 12.4x10(6). Microcapsules with different antibody loads (388-1070 antibody molecules per capsule) were subjected to a solid-phase immunoassay for the detection of insulin. The microcapsule having 1030 anti-insulin antibody molecules per capsule demonstrated good performance for insulin determination. The calibration curve for insulin had a linear range of 10(-10) to 10(-7) g mL(-1) with R(2)=0.990, 3.9% R.S.D. The limit of detection for insulin was 10 pg mL(-1) of 100 microL sample (equivalent to 10(-12)g of insulin). The determination range for the glucose was 0.5 and 40 mM with R(2)=0.996 and 4.1% R.S.D.     

Aromatase inhibitors--gene discovery

Microarray analysis of tumour RNA is an extremely powerful tool which allows global gene expression to be measured. When used in combination with neoadjuvant treatment protocols in which therapy is given with the primary tumour within the breast, sequential biopsies may be analysed and results correlated with clinical and pathological response. In the present study, a neoadjuvant protocol has been used, administering the third generation inhibitor, letrozole, for 3 months and subjecting RNA extracted from biopsies taken before and after 10–14 days of treatment to microarray analysis. The objectives were to discover: (i) genes that change with estrogen deprivation (the only known biological effect of letrozole is to inhibit aromatase activity and reduce endogenous estrogens in postmenopausal women) and (ii) genes whose basal, on treatment or change in expression differ between tumours which are either responsive or resistant to treatment (so that predictive indices of response/resistance may be developed).

Early changes in gene expression were identified by comparing paired tumour core biopsies taken before and after 14 days treatment in 58 patients using three different approaches based on frequency of changes, magnitude of changes and SAM analysis. All three approaches showed a greater number of genes were down-regulated than up-regulated. Merging of the data produced a total of 143 genes which were subject to gene ontology and cluster analysis. The ontology of the 91 down-regulated genes showed that they were functionally associated with cell cycle progression, particularly mitosis. In contrast, up-regulated genes were associated with organ development and extra-cellular matrix turnover and regulation.

Clinical response was assessable in 52 patients; 37 (71%) tumours were classified as clinical responders (>50% reduction in volume at 3 months). Microarray analysis of pre- and 14-day biopsies identified 291 covariates (84 baselines, 72 14-day and 135 changes) highly predictive of response status. A similarity matrix using the covariates showed responding tumours have a similar genetic profile which was dissimilar to non-responding cancers whereas non-responsive cases were distinctive from each other. Changed genes predicting for response showed no concordance with those changed significantly by treatment in the overall group.     

Anti-inflammatory property of the urinary metabolites of nobiletin in mouse

Nobiletin, a major component of polymethoxyflavones in citrus fruits, has a broad spectrum of health beneficial properties including anti-inflammatory and anti-carcinogenic activities. The metabolite identification of nobiletin in mouse urine has concluded that it undergoes mono-demethylation (3'- and 4'-demethylnobiletin) and di-demethylation (3',4'-didemethylnobiletin) metabolic pathway. Biological screening of nobiletin and its metabolites has revealed that the metabolites possess more potent anti-inflammatory activity than their parent compound. Therefore, this letter reports the identification of nobiletin metabolites and their anti-inflammatory activity against LPS-induced NO production and iNOS, COX-2 protein expression in RAW264.7 macrophage.     

Modification of cap group in delta-lactam-based histone deacetylase (HDAC) inhibitors

Novel delta-lactam-based HDAC inhibitors which have various substituted benzyl, bi-aromatic cap groups were prepared using ring closure metathesis reaction, and evaluated their HDAC inhibitory activities and anti-proliferative effects. Among prepared analogues, 11m and 11o have very strong HDAC enzymatic inhibition and showed the most potent growth inhibitory activity to five human tumor cell lines including PC-3, ACHN, NUGC-3, HCT-15, and MBA-MB-231 tumor cell lines. Compounds 11m and 11o also showed good tumor growth inhibition of MDA-MB-231 cells in in vivo xenograft model. Structure-activity relationship study using docking model explained the significance of hydrophobic aromatic cap groups for their in vitro activities.     

Phosphorylation of HuR by Chk2 regulates SIRT1 expression

The RNA binding protein HuR regulates the stability of many target mRNAs. Here, we report that HuR associated with the 3' untranslated region of the mRNA encoding the longevity and stress-response protein SIRT1, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. Unexpectedly, oxidative stress triggered the dissociation of the [HuR-SIRT1 mRNA] complex, in turn promoting SIRT1 mRNA decay, reducing SIRT1 abundance, and lowering cell survival. The cell cycle checkpoint kinase Chk2 was activated by H(2)O(2), interacted with HuR, and was predicted to phosphorylate HuR at residues S88, S100, and T118. Mutation of these residues revealed a complex pattern of HuR binding, with S100 appearing to be important for [HuR-SIRT1 mRNA] dissociation after H(2)O(2). Our findings demonstrate that HuR regulates SIRT1 expression, underscore functional links between the two stress-response proteins, and implicate Chk2 in these processes.     

Exogenous nitric oxide stimulated collagen type I expression and TGF-beta1 production in keloid fibroblasts by a cGMP-dependent manner.

Keloids arise from the aberrant wound healing process and nitric oxide (NO) plays an important role in the inflammation stage of wound healing. In order to better define the potential effect of NO/cGMP signal pathway in the keloid pathogenesis, the enhancing effect of exogenous NO (released from NO donor) on collagen expression in the keloid fibroblast (KF) as well as on the induction of collagen type I protein and TGF-beta1 expression in the KF were studied in this investigation. The DETA NONOate, an NO donor, was added to the KF, as the exogenous NO, to release NO in the culture medium. The expression of collagens was then determined by assaying the total soluble collagens and collagen type I in the KF. The cellular concentration of cGMP was measured by EIA in the KF. Exogenous NO was found to enhance the expression of collagens and elevate the cellular levels of cGMP. Moreover, to evaluate the effect of the elevated cellular cGMP levels on the expression of collagen and TGF-beta1, both cGMP and TGF-beta1 were measured by ELISA. The inhibitors for phosphodiesterase (PDE), such as IBMX (3-isobutyl-1-methylxanthine), Vinpocetine, EHNA, Milrinone and Zapriast, which have been reported to reduce the ability of PDE and subsequently produce an increase of cellular cGMP, induce the production of autocrine TGF-beta1 as well as the synthesis of collagen in the KF. In this investigation, the inhibition of the PDE enzyme activity was observed to enhance the effect on the collagen synthesis, and was induced by exogenous NO. Taken together, these results have suggested that the NO/cGMP pathway could positively influence the progression of keloid formation, via the TGF-beta1 expression in the KF.     

Production and biological activity of laidlomycin, anti-MRSA/VRE antibiotic from Streptomyces sp. CS684

Culture broth of a streptomycete isolate, Streptomyces sp. CS684 showed antibacterial activity on methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE). Among purified substances from the organism, CSU-1, which is active against MRSA and VRE, is a C37H62O12Na (M+, 721.3875), and identified as laidlomycin. The anti-MRSA and anti-VRE activity of CSU-1 was stronger than oxacillin and vancomycin. Phylogenetic analysis showed that strain CS684 is very similar to Streptomyces ardus NRRL 2817T, whereas the ability of Streptomyces sp. CS684 to produce laidlomycin was shown to be unique.     

Identification of genes differentially expressed in RAW264.7 cells infected by Salmonella typhimurium using PCR method

Salmonella typhimurium, causing mouse typhoid, infects hosts such as macrophage cells, and proliferates in intracellular vacuoles causing infected cells to trigger numerous genes to respond against the infection. In this study, we tried to identify such genes in RAW264.7 cells by using the PCR screening method with degenerate primers. Fourteen genes were found to be differentially expressed after a 4 h infection in which the expression of 8 genes increased while expression of the others decreased. Most of the genes were involved in proinflammatory responses such as cytokines production and cell death. The mutation in msbB gene encoding the myristoyl transferase in lipid A of lipopolysaccharide (LPS) resulted in much lower toxicity to the inoculated animals. We compared the expression of the identified genes in wild-type and msbB-mutated S. typhimurium infections and found that Lyzs encoding lysozyme type M was differentially expressed. This gene is quite likely to be related to bacterial survival in the host cells.